Aflatoxin 2014 My

FOOD SCIENCE AND TECHNOLOGY
AFLATOXINS
FOOD SOURCES, OCCURRENCE
AND TOXICOLOGICAL EFFECTS
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AFLATOXINS
FOOD SOURCES, OCCURRENCE
AND TOXICOLOGICAL EFFECTS
ADINA G. FAULKNER
EDITOR
New York
Copyright 2014 by Nova Science Publishers, Inc.
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CONTENTS
Preface vii
Chapter 1 Bio-Prevalence, Determination and Reduction
of Aflatoxin B1 in Cereals 1
Jelka Pleadin, Ksenija Markov, Jadranka Frece,
Ana Vuli and Nina Peri
Chapter 2 Aflatoxin Occurrence 35
Elham Esmaeilishirazifard and Tajalli Keshavarz
Chapter 3 Aflatoxins in Food and Feed: Contamination
Exposure, Toxicology and Control 63
Marta Herrera, Antonio Herrera and Agustn Ario
Chapter 4 Immunosuppressive Actions of Aflatoxin
and Its Role in Disease Susceptibility 91
Johanna C. Bruneau, Orla Hayden,
Christine E. Loscher and Richard OKennedy
Chapter 5 Aflatoxins Hazards and Regulations Impacts
on Brazil Nuts Trade 107
Otniel Freita-Silva, Renata Galhardo Borguini
and Armando Venncio
vi Contents
Chapter 6 Polymorphisms of DNA Repair Genes

and Toxicological Effects of Aflatoxin
B1 Exposure 125
Xi-Dai Long, Jin-Guang Yao, Qian Yang,
Cen-Han Huang, Pinhu Liao, Le-Gen Nong,
Yu-Jin Tang, Xiao-Ying Huang, Chao Wang,
Xue-Ming Wu, Bing-Chen Huang, Fu-Zhi Ban,
Li-Xia Zeng, Yun Ma, Bo Zhai, Jian-Jun Zhang,
Feng Xue, Cai-Xia Lu and Qiang Xia
Chapter 7 Incidence of Aspergillus Section Flavi
and Interrelated Mycoflora in Peanut
Agroecosystems in Argentina 157
Mara Alejandra Passone, Andrea Nesci,
Anala Montemarani and Miriam Etcheverry
Chapter 8 Toxicological Effects, Risk Assessment and
Legislation for Aflatoxins 191
Marina Goumenou, Dimosthenis Axiotis,
Marilena Trantallidi, Dionysios Vynias,
Ioannis Tsakiris, Athanasios Alegakis,
Josef Dumanov and Aristidis Tsatsakis
Chapter 9 Food Sources and Occurrence of Aflatoxins:
The Experience in Greece 233
Ioannis N. Tsakiris, Elisavet Maria Renieri,
Maria Vlachou, Eleftheria Theodoropoulou,
Marina Goumenou and Aristides M. Tsatsakis
Chapter 10 Aflatoxins As Serious Threats to Economy and Health 259
Lipika Sharma, Bhawana Srivastava, Shelly Rana,
Anand Sagar and N. K. Dubey
Index 287
PREFACE
Progress in understanding the biology of Aspergillus has greatly improved

with the new techniques in genome sequencing and the developed molecular
tools that enable rapid genetic analysis of individual genes. Particularly, the
genetics of aflatoxin synthesis is regarded as a model to gain insight into
fungal secondary metabolism. This compilation discusses topics that include
the prevalence of aflatoxin B1 in cereals; contamination exposure, toxicology
and control of aflatoxins in food and feed; immunosuppressive actions of
aflatoxin; hazards and regulations; toxicological effects, risk assessment and
legislation for aflatoxins; and the threat aflatoxins have on the economy and
health.
Chapter 1 - Moulds of Aspergillus genus are among the most important
causes of food and feed spoilage and can produce mycotoxins as toxic
secondary metabolites when under adverse conditions. Aflatoxins are a group
of mycotoxins that commonly contaminate maize and groundnuts, and are
categorized by the International Agency for Research on Cancer under Class
1A human carcinogens. From the food safety standpoint, one of the most
important mycotoxins is aflatoxin B1 (AFB1). Due to its potent carcinogenic,
teratogenic and mutagenic effects dependent on the level and length of
exposure, the presence of this contaminant in food and feed should be kept as
low as achievable. In order to investigate the occurrence of AFB1, determine
its concentrations and explore the possibility of its reduction using different
methods, samples of maize, wheat, barley and oat were collected from
different cultivation fields during a three-year period. The immunoassay
(ELISA) as a screening method and high performance liquid chromatography
tandem mass spectrometry (LC-MS/MS) as a confirmatory method were used
to determine AFB1 concentrations. Maize contamination seen with AFB1
viii Adina G. Faulkner
concentrations higher than permitted was associated with climate conditions

established in the period of concern, which was extremely warm and dry, and
might had favored mould production and AFB1 formation. Substantial to
almost absolute AFB1 reduction in the maize samples was achieved using
gamma radiation. A strong antifungal effect was also obtained upon the use of
essential oils and lactic acid bacteria as biological AFB1-reduction alternatives.
As the presence of AFB1 in cereals could be dangerous for human and animal
health, in order to prevent its harmful effects and huge economic problems, the
prevention of formation of this contaminant and consistent control over it are
of major interest. Based on these substantiated grounds, possibilities of
implementing new methods of AFB1 determination and reduction within the
frame of safe food production are virtually countless.
Chapter 2 - Toxigenic fungi in crops have been divided historically into
two groups, field and storage fungi. Mycotoxins are produced by toxigenic
fungi at the fields and in the storage. Although many compounds are termed as
mycotoxin, there are only five agriculturally-important fungal toxins:
deoxynivalenol, zearalenone, ochratoxin A, fumonisin and aflatoxin.
Penicillium and Aspergillus species are the most important storage fungi.
However, they can also invade stressed plants in the field. The main
mycotoxins produced by Aspergillus species are aflatoxins, citrinin and
patulin. The word aflatoxin comes from Aspergillus flavus toxin, based on
the fact that A. flavus and A. parasiticus are the predominant species
responsible for aflatoxin contamination of crops prior to harvest or during
storage. Aflatoxins B1, B2, G1, and G2 are the four major isolated aflatoxins
from food and feed commodities.
A. flavus and A. parasiticus have distinct affinity for nuts and oilseeds
including peanuts, maize and cotton seed. Cereals are a general substrate for
growth of A. flavus but, unlike nuts, small grain cereal spoilage by A. flavus is
the result of poor handling. Moreover, aflatoxin M1 as a milk contaminant has
potential risk for animal and human health. The character of the aflatoxin
problem varies by region. For instance, aflatoxin accumulation in stored maize
in subtropical Asia has risen rapidly in post-harvest conditions whereas in the
US, the issue is pre-harvest condition of maize. Therefore, the exposure to
aflatoxins differs between countries particularly due to different diets. Food
contamination with Aspergillus is associated with warm and dry climates.
However, in variable environmental conditions, the aflatoxin contamination
may differ from one year to another at the same location.
Preface ix
tools that enable rapid genetic analysis of individual genes. Particularly, the
genetics of aflatoxin synthesis is regarded as a model to gain insight into
fungal secondary metabolism. Well-designed research on production of the
aflatoxin precursor sterigmatocystin with the genetic model A. nidulans, has
contributed greatly to our knowledge of the aflatoxin pathway and the global
regulatory mechanisms. According to the recent studies, fungal pathogenesis is
related to lipid-mediated fungal-host crosstalk, suggesting that secondary
metabolism may be controlled by oxylipins at the transition level. Also, some
oxylipins have been reported to be engaged in the signalling mechanism like
quorum sensing responses in Aspergillus. Quorum sensing molecules and their
genes which are responsible for intra and inter kingdom communications could
be applied in the future aflatoxin bio-control strategies.
Chapter 3 - Aflatoxins (AFs) are secondary metabolites produced by
various fungal species of the genus Aspergillus such as Aspergillus flavus and
Aspergillus parasiticus. The most important compounds are aflatoxins B1, B2,
G1 and G2, as well as two metabolic products secreted in milk, M1 and M2.
The worldwide occurrence of aflatoxins contamination in raw agricultural
products has been well documented; such contamination occurs in a variety of
food and feed, such as cereals, nuts, dried fruits, spices and also in milk as a
consequence of the ingestion of contaminated feed. However, pistachios,
peanuts and corn are the most frequently contaminated food items reported in
the Rapid Alert System for Food and Feed (RASFF) of the European Union.
The occurrence of aflatoxins is mainly affected by environmental factors such
as climatic conditions, geographic location, agricultural practices, and
susceptibility of the products to fungal growth during harvest, storage and
processing. High contamination levels of aflatoxins are mainly associated with
post-harvest growth of Aspergillus moulds in poorly stored commodities.
Aflatoxins can cause adverse effects to the health of animals and humans.
These toxins have been reported to be associated with acute liver damage,
liver cirrhosis, induction of tumors and teratogenic effects. Aflatoxin B1
(AFB1) is usually predominant and the most toxic among aflatoxins because it
is responsible for hepatocarcinoma in animals and strongly associated with the
incidence of liver cancer in humans. AFB1 is a genotoxic and mutagenic
chemical, and it has been classified by the International Agency of Research
on Cancer (IARC) as human carcinogen (group 1). The toxic effects of the
ingestion of aflatoxins in both humans and animals depend on several factors
including intake levels, duration of exposure, metabolism and defense
mechanisms, and individual susceptibility. Aflatoxins affect not only the
health of humans and animals but also the economics of agriculture and food.
x Adina G. Faulkner
Because of the multiple adverse health effects to humans and animals

caused by aflatoxin consumption, many nations worldwide have regulatory
standards on aflatoxin in food and feed. The European Union (EU) regulation
on aflatoxins in foodstuffs is among the strictest in the world (Commission
Regulation (EC) n 1881/2006 and successive amendments). Maximum
contents of aflatoxins in feeds are also established by Commission Regulation
(EU) n 574/2011 on undesirable substances in animal feed.
Throughout the world there are many advisory bodies concerned with
food safety, including the World Health Organization (WHO), the Food and
Agriculture Organization of the United Nations (FAO), the Codex
Alimentarius Joint Expert Committee for Food Additives and Contaminants
(JECFA), and many others, which regularly assess the risk from mycotoxins,
advise on controls to reduce consumer exposure and establish different
regulations for these toxins in different countries.
Chapter 4 - Aflatoxins are secondary metabolites produced by fungi of the
Aspergillus species. They occur as contaminants in a variety of food and feed
stuffs that have been infected with the producing fungi. Aflatoxin exposure is
known to cause a number of acute and chronic effects in both humans and
animals, including immunosuppression, liver and other cancers, and failure of
vaccination regimens. The immunomodulatory effects of the aflatoxins have
been shown to affect cell-mediated immunity more than humoral immunity. In
particular, aflatoxin exposure modulates secretion of inflammatory cytokines
and phagocytic function. Decreases in phagocytosis and inflammation
observed following aflatoxin exposure may reduce the effectiveness of the
host immune response to infection, thereby increasing susceptibility to
infection in individuals exposed to these toxins. The aim of this chapter is to
summarise the immunomodulatory effects of aflatoxin exposure in order to
better understand its potential immunosuppressive effects in humans and
animals. The relationship between these immunosuppressive actions and
susceptibility to infection will also be discussed.
Chapter 5 - Brazil nut is an important non-timber forest product produced
in Amazon region. This nut is used as food with high value in the international
market, due to its high nutritional and flavor characteristic and to their
association with environmental conservation and alleviation of poor people
living from Amazonia. Annually, several hundred tons of Brazil nuts are
produced in Brazil. However, they are susceptible to aflatoxins (AF)
contamination. Because of the detection of unacceptable level of AF in Brazil
nuts consignments arriving in European Union ports, in 2003, special
conditions were imposed on Brazil nuts entering the European Union,
Preface xi
decreasing the acceptable levels of AF. In 2010, the European Union revised
AF regulation on nuts; these new limits are more adequate when considering
the complexity of Brazil nut chain and the low risk related to its low
consumption. This chapter points data on the occurrence of AF in Brazil nuts,
as reported by the Rapid Alert System for Food and Feed (RASFF), and
evaluates the efforts made by all sectors involved in the agribusiness of Brazil
nuts, in Brazil, in order to contribute to protection of both domestic and
international consumers from possible health hazard caused by AF.
Chapter 6 - Aflatoxin B1 (AFB1) is an important genic toxin produced by
the moulds Aspergillus parasiticus and Aspergillus flavus. AFB1 is
metabolized by cytochrome P450 enzymes to its reactive form, AFB1-8,9-
epoxide (AFB1-epoxide), which covalently binds to DNA and induces DNA
damage. DNA damage induced by AFB1, if not repaired, may cause such
genic tox toxicological Effects as DNA adducts formation, gene mutations and
hepatocellular carcinoma (HCC). During the repair process of DNA damage
produced by AFB1, DNA repair genes play a central role, because their
function determines DNA repair capacity. In this study, the authors
investigated the association between seven polymorphisms (including rs25487,
rs861539, rs7003908, rs28383151, rs3734091, rs13181, and rs2228001) in
DNA repair genes XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and
XRCC3, and toxicological effects of AFB1 using a hospital-based case-control
study. Toxicological effects of AFB1 were analyzed by means of the levels of
AFB1-DNA adducts, the mutant frequency of TP53 gene, and the risk of
AFB1-related HCC. The authors found that the mutants of XPC, XRCC4,
XRCC1, XRCC4, XPD, XRCC7, and XRCC3 had higher AFB1-DNA adducts
levels, compared with the wilds of these genes (3.276 vs 3.640 mol/mol DNA
for rs25487, 2.990 vs 3.897 mol/mol DNA for rs861539, 2.879 vs 3.550
mol/mol DNA for rs7003908, 3.308 vs 3.721 mol/mol DNA for
rs28383151, 3.229 vs 3.654 mol/mol DNA for rs3734091, 2.926 vs 4.062
mol/mol DNA for rs13181, and 3.083 vs 3.666 mol/mol DNA for
rs2228001, respectively). Furthermore, increasing risk of TP53 gene mutation
and HCC was also observed in these with the mutants of DNA repair genes.
These results suggested that polymorphisms of DNA repair genes might
modify the toxicological effects of AFB.
Chapter 7 - Studies in typical and new Argentinean peanut areas showed
that toxigenic Aspergillus section Flavi strains are widely distributed in soils
and seeds, with high probability of being transferred to the storage ecosystem.
Mycological analyses of soil showed that Aspergillus section Flavi population
were present in the two areas at similar counts (3.2x102 cfu g-1). Within this
xii Adina G. Faulkner
section, two fungal species were frequently isolated with isolation percentages
of 73 and 90% for A. flavus and of 27 and 9% for A. parasiticus in soil
samples from traditional and new areas, respectively. The percentages of the
different A. flavus phenotypes from both peanut-growing areas showed that L
strains were recovered in the highest percentage and represented 59 and 88%
of the isolates with variable ability to produce aflatoxins (AFs). Peanut kernels
collected at harvest time from different localities of Crdoba and Formosa
provinces showed A. flavus and A. parasiticus contamination. The 42.8 and
70% were classified as type L and the percentages of aflatoxigenic A. flavus
strains were 68.6 and 80.0% in samples from traditional and recent peanut-
growing areas, respectively. Highly toxigenic A. flavus S strains were isolated
with major frequency from soil and kernel samples coming from traditional
peanut-growing area. Aflatoxin contamination was detected in peanut kernels
from typical peanut growing area. Harvested peanut were stored during 5
months in three storage systems (big bags, wagons of conditioning and drying
and stockpiled warehouse) and mycological population succession was
analyzed. Fungal isolation was greater from pod (95%) than from kernel
tissues. The most common fungi identified included Penicillium, Aspergillus,
Eurotium and Fusarium spp. Within Aspergillus genus, the section Flavi had
the greatest mean counts of 1.4x104, 9.4x102, 5.2x102 cfu g-1 for big bags,
wagon and warehouse, respectively. A. flavus and A. parasiticus strains with
variable ability to produce AFs were isolated from peanut kernels stored in the
three systems at all sampling periods in the order of 1.5x102, 2.3x102 and 4.5
cfu g-1, respectively. .A. flavus S and L strains contributed to silo community
toxigenicity during all storage period. Total AF levels ranging from 1.1 to
200.4 ng g-1 were registered in peanuts conditioned at the higher aW values
(0.940.84 aW) and stored in big bags. Despite the water stress conditions
registered in the stockpiled warehouse throughout the storage period, AFB1
levels ranging between 2.9 and 69.1 ng g-1 were registered from the third
sampling.
Therefore, the interaction between biological and abiotic factors and
substrate may promote the Aspergillus contamination and the subsequent AF
accumulation in peanut from sowing to storage, highlighting the need to
promote good practices in order to avoid the risk of these metabolites
contamination in peanut food chain.
Chapter 8 - Aflatoxins are toxic metabolites produced by the fungus
Aspergillus. The main representatives are aflatoxins B1, B2, G1, G2. Their
occurrence in food like nuts, cereals and cereal-derived products is a result of
fungal contamination before harvest and during storage. Milk can also be
Preface xiii
contaminated by aflatoxin M1 (main metabolite of B1) as a result of animals

exposure to feed contaminated by the aflatoxin B1.
Aflatoxins manifest acute and chronic toxicity. Evidence of acute
aflatoxicosis in humans involving a range of symptoms from vomiting to death
has been reported mainly in Third World Countries. In relation to chronic
toxicity aflatoxins are well known for their genotoxic and carcinogenic
properties while recent studies evident a series of other possible effects like
reprotoxicity, impaired growth in children, intestinal functions, chronic fatigue
syndrome, compromise immunity and interfere with protein metabolism and
multiple micronutrients that are critical to health.
The critical step for aflatoxins risk assessment is the estimation of the real
exposure. For this reason a number of surveys are conducted globally using
tools like biomarkers of exposure and modeling. In addition new parameters
like the climate change are now taken into consideration in order to predict
possible current and future changes of exposure to aflatoxins. As aflatoxins are
compounds of natural origin and their presence in food cannot be totally
eliminated the risk management is based on keeping the total exposure as low
as reasonably achievable taking into account the social-economic impact of
crop and livestock losses. Exposure reduction is achieved mainly by reducing
the number of highly contaminated foods reaching the market by regulatory
control but also applying detoxification strategies. According to the EU
regulatory framework minimization of the exposure to aflatoxins is based on
setting maximum levels of aflatoxins in different foodstuffs (4 10 g/kg total
aflatoxins) and feed (EC/1881/2006, Directive 2002/32/EC). Products
exceeding the maximum levels should not be placed on the EU market.
Methods of sampling and analysis for the official control of aflatoxins, are also
set (EC/401/2006) in order to ensure common sampling criteria to the same
products and that certain performance criteria are fulfilled. The United States
Food and Drug Administration (FDA) has established the action levels for
aflatoxin present in food to the 20 g/kg (0.5 g/kg for milk) and up to 300
g/kg for feed. Finally an action level of 10 g/kg total aflatoxins is also used
from Japan authorities.
Chapter 9 - This paper presents a review of the occurrence of aflatoxins in
different food commodities in Greece, based both on results represented in
literature as well as results derived from monitoring programs of the Center of
Toxicology Science & Research, Medical School, University of Crete.
Aflatoxins, can pose a severe threat to food safety, since they are characterized
carcinogenic to humans, IARC Group 1. They may be formed or developed in
any stage of the agricultural production (primary production, processing and
xiv Adina G. Faulkner
storage) as a result of transitional weather conditions or of poor storage.

Studies, monitoring programs and surveys, which have been carried out in
Greece, are mainly focused in milk and dairy products. In this context, several
studies have been conducted in animal feeds as well, since there is notable
evidence that they are potential sources of aflatoxins in milk production.
Additionally, both black and green olives have been examined for possible
contamination by aflatoxins, due to the fact that they are damaged during
harvest and processing and thus providing a substrate for aflatoxin
development. Finally, a limited number of studies investigate the presence of
aflatoxins in different processed products like breakfast cereals. The above
foodstuffs have been studied on account of their high nutritional value and the
fact that they are consumed by different population groups. Results indicate
that residue levels of aflatoxins which are presented in fresh as well as
processed agricultural products, do not pose any considerable risk for the
Greek population groups. The most important factors influencing the levels of
aflatoxins in major agricultural products appear to be the growing and
cultivation techniques, as well as the food safety parameters during harvesting,
storage and processing. An additional issue, which seems to raise concern
internationally, is the fact that climate change in combination with
modifications in the cultivation techniques may affect the frequency and
severity of aflatoxin residues in agricultural products.
Chapter 10 - This review deals with the aflatoxins especially with their
food sources, wide occurrence and toxicological effects on animals and
humans. Aflatoxins are highly oxygenated, heterocyclic, difuranocoumarin
compounds and are an important group of mycotoxins produced by the fungi.
There are almost 20 different types of aflatoxins identified till now; among
these AFB1 is considered to be the most toxic. Aflatoxins persist to some
extent in food even after the inactivation of the fungi by food processing
methods, such as ultra-high temperature products, due to their significant
chemical stability. Aflatoxins can affect a wide range of commodities
including cereals, oilseeds, spices, and tree nuts as well as milk, meat, and
dried fruits. Twenty-five percent of the worlds crops are affected with
mycotoxins. On a worldwide scale, the aflatoxins are found in stored food
commodities and oil seeds. Some of the foods on which aflatoxin producing
fungi grow well include cereals (maize, sorghum, pearl millet, rice, wheat,
corn, oats, barley), oilseeds (peanut, soybean, sunflower, cotton), spices (chile
peppers, black pepper, coriander, turmeric, ginger), and tree nuts (almond,
pistachio, walnut, coconuts), sweet potatoes, potatoes, sesame, cacao beans,
almonds, etc., which on consumption pose health hazards to animals, including
Preface xv
aquaculture species of fish, and humans. Food commodities affected by

aflatoxins are also susceptible to other types of mycotoxins and multiple
mycotoxins can co-exist in the same commodity. Various cereals affected by
aflatoxins are also susceptible to contamination by fumonisins, trichothecenes
(especially deoxynivalenol), zearalenone, ochratoxin A and ergot alkaloids.
More than 5 billion people in developing countries worldwide are at risk
of chronic exposure to naturally occurring aflatoxins through contaminated
foods. Aflatoxin is a potent liver toxin causing hepatocarcinogenesis,
hepatocellular hyperplasia, hepatic necrosis, cirrhosis, biliary hyperplasia, and
acute liver damage in affected animals. Effects of aflatoxins in animals depend
on age, dose and length of exposure, species, breed and nutritional status of the
animal. Health effects occur in fish, companion animals, livestock, poultry and
humans because aflatoxins are potent hepatotoxins, immunosuppressants,
mutagens, carcinogens and teratogens. Aflatoxin B1 has been shown to cause
significant morphological alterations along with reduced phagocytic potential
in chicken and turkey macrophages. Aflatoxin- B1 exposure to chicken
embryos causes significant suppression in macrophage phagocytic potential in
chicks after hatch. Aflatoxin intercalates into DNA and alkylates the DNA
bases through its epoxide moiety resulting in liver cancer. Other effects
include mutagenic and teratogenic effects. Exposure of biological systems to
harmful levels of aflatoxin results in the formation of epoxide, which reacts
with proteins and DNA leading to DNA-adducts, thus causing liver cancer.
The primary target of aflatoxins is the hepatic system. Acute effects include
hemorrhagic necrosis of the liver and bile duct proliferation while chronic
effects include hepatocellular carcinoma (HCC). HCC is the sixth most
prevalent cancer worldwide with a higher incidence rate within developing
countries. Preliminary evidence suggests that there may be an interaction
between chronic aflatoxin exposure and malnutrition, immunosuppression,
impaired growth, and diseases such as malaria and HIV/AIDS. Outbreaks of
acute aflatoxin poisoning are a recurrent public health problem. The discussion
of this problem and its remedies must be held in the context of the associated
question of food insufficiency and more general economic challenges in
developing countries. Aflatoxin constitutes a serious health concern to the
entire food chain, necessitating a multidisciplinary approach to analysis,
action, and solution.
In: Aflatoxins ISBN: 978-1-63117-298-4
Editor: Adina G. Faulkner 2014 Nova Science Publishers, Inc.
Chapter 1
BIO-PREVALENCE, DETERMINATION
AND REDUCTION OF AFLATOXIN B1
IN CEREALS
Jelka Pleadin1,, Ksenija Markov2, Jadranka Frece2,

Ana Vuli1 and Nina Peri1
1
Croatian Veterinary Institute,
Laboratory for Analytical Chemistry, Zagreb, Croatia
2
Faculty of Food Technology and Biotechnology,
Zagreb, Croatia
ABSTRACT
Moulds of Aspergillus genus are among the most important causes of
food and feed spoilage and can produce mycotoxins as toxic secondary
metabolites when under adverse conditions. Aflatoxins are a group of
mycotoxins that commonly contaminate maize and groundnuts, and are
categorized by the International Agency for Research on Cancer under
Class 1A human carcinogens. From the food safety standpoint, one of the
most important mycotoxins is aflatoxin B1 (AFB1). Due to its potent
carcinogenic, teratogenic and mutagenic effects dependent on the level
and length of exposure, the presence of this contaminant in food and feed
should be kept as low as achievable. In order to investigate the
occurrence of AFB1, determine its concentrations and explore the
Corresponding Author: Tel: +38516123626; Fax: +38516123670; E-mail: pleadin@veinst.hr.

2 Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.
possibility of its reduction using different methods, samples of maize,

wheat, barley and oat were collected from different cultivation fields
during a three-year period. The immunoassay (ELISA) as a screening
method and high performance liquid chromatography tandem mass
spectrometry (LC-MS/MS) as a confirmatory method were used to
determine AFB1 concentrations. Maize contamination seen with AFB1
concentrations higher than permitted was associated with climate
conditions established in the period of concern, which was extremely
warm and dry, and might had favored mould production and AFB 1
formation. Substantial to almost absolute AFB1 reduction in the maize
samples was achieved using gamma radiation. A strong antifungal effect
was also obtained upon the use of essential oils and lactic acid bacteria as
biological AFB1-reduction alternatives. As the presence of AFB1 in
cereals could be dangerous for human and animal health, in order to
prevent its harmful effects and huge economic problems, the prevention
of formation of this contaminant and consistent control over it are of
major interest. Based on these substantiated grounds, possibilities of
implementing new methods of AFB1 determination and reduction within
the frame of safe food production are virtually countless.
1. INTRODUCTION
Cereal grains may become contaminated by moulds that produce
mycotoxins as toxic chemical compounds while in the field and during
storage. This group of compounds represents a significant food safety issue
and poses as a risk to health and wellbeing of humans and animals. Food and
feed contamination with mycotoxins, as toxins of frequent incidence in
agricultural goods, has a negative impact on economies of the affected regions,
especially in the developing countries where harvest and post-harvest
techniques of mould growth prevention are not adequately implemented
(Rustom, 1997).
Cereals such as maize, wheat, barley and oat represent a significant part of
not only human, but also animal diet, and play a role in industrial food & feed
processing. Cereal grains balance the nutrition by virtue of providing a low-fat
diet that has a number of advantages, especially when whole-grain foodstuffs
are consumed. However, grains are a common source of contaminants,
especially mycotoxins, which, under favorable temperature and humidity
conditions, may produce mycotoxins before and/or during harvest, handling,
shipment and storage. The most important mycotoxins are aflatoxins B1, B2,
G1 and G2, fumonisin B1, T-2 toxin, zearalenone, ochratoxin A and
Bio-Prevalence, Determination and Reduction of Aflatoxin B1 3
deoxynivalenol. Maize and maize products are known to be prone to

contamination by fungi that produce secondary metabolites such as aflatoxins
(Groopman and Donahue, 1988).
Among food & feed contaminants, aflatoxins are of current concern and
have received a great deal of attention during the last three decades. They were
first heavily researched and truly understood after the death of more than
100,000 young turkeys on poultry farms in England that was found to be
related to the consumption of Brazilian peanut meal (Goldblatt, 1969).
Aflatoxins are known to be produced by two species of Aspergillus genus,
specifically Aspergillus flavus and Aspergillus parasiticus, and represent
highly toxic, mutagenic, teratogenic and carcinogenic compounds that exhibit
an immunosuppressive activity, causing both acute and chronic toxicity in
humans and animals (Eaton and Gallagher, 1994; Massey et al., 1995; EFSA,
2004; Meggs, 2009). Among them, aflatoxin B1 (AFB1) is the most potent
liver carcinogen known in mammals, and is classified by the International
Agency for Research on Cancer (IARC) as Group 1 carcinogen (IARC, 1993).
Factors that promote fungal infection and AFB1 production are inoculum
availability, weather conditions and pest infestation during crop growth,
maturation, harvesting and storage (Lopez-Garcia and Park, 1998). Generally
speaking, crops stored for more than a few days become a potential target for
mould growth and mycotoxin formation (Turner et al., 2009).
In general, mycotoxins, aflatoxins included, are stable compounds not
destroyed during most of the food processing operations, which might lead to
the contamination of cereals and their final products. However, aflatoxin
presence can sometimes be reduced by making improvements in farming
practices, such as providing better storage conditions or using modified seeds,
or by making improvements in manufacturing processes.
Due to the fact that aflatoxins represent the type of mycotoxins most
commonly found in cereals, many studies have attempted to define multiple
aspects of contamination of human food and animal feed chains, and still do
so, so that the topic is a very hot one. Such a contamination is often
unavoidable and still poses as a serious problem associated with important
agricultural goods, which emphasizes the need for suitable processing capable
of inactivating the toxin. Maize, as the most widely grown crop extensively
used for animal feeding and human consumption, represents a particular
problem. Due to its nutritional value, a high percentage of the world maize
production is destined for animal feeding.
The European Food Safety Authority document (EFSA, 2013), prepared
based on analytical data on four aflatoxins (B1, B2, G1, and G2) recovered in
food samples collected between 2007 and 2012, reports that the collection of
data on the occurrence of aflatoxins in relevant foodstuffs should be continued
in order to gather a representative number of samples in different food
categories; in addition, the document draws attention to the need for
harmonizing the reporting formats across the European countries.
This chapter presents the results of AFB1 determination in four types of
commonly used cereals intended for food and feed, collected during a three-
year period from different cultivation fields, as well as the results of an
investigation into the possibilities of contamination reduction and/or
avoidance. For the sake of AFB1 determination, the immunoassay (ELISA) as
a screening method and high performance liquid chromatography tandem mass
spectrometry (LC-MS/MS) as a confirmatory method were used. Gamma
radiation and essential oils & lactic acid bacteria, on the other hand, were used
to investigate the possibilities of AFB1 reduction in contaminated maize
samples.
1.1. Exposure to AFB1 through the Food Chain
The Food and Agriculture Organization (FAO) estimated that 25% of the
world food-intended crops are contaminated with mycotoxins, and that
aflatoxins, as the most toxic among them, are the trickiest to deal with because
of their widespread occurrence in maize, peanuts and its products, cottonseed,
chilies, peppers, pistachio nuts and some other foodstuffs (Scholthof, 2003).
Contaminated feed also represents the main source of AFB1 infestation in farm
animals, which get to be contaminated through parasites living on plants even
prior to harvesting or on stored harvested crops (Huwing et al., 2001; Gareis
and Wolff, 2000). As fodder, cereals and seeds used for dairy cattle feeding
are inevitably in contact with yeasts and filamentous fungi, contamination of
these raw materials frequently occurs already in the field. AFB1 contamination
can also occur during harvesting, transport and storage of cereals and their
products, as well as due to post-harvest mishandling that can lead to rapid feed
spoilage (Alonso et al., 2011).
In animals intended for meat production that had consumed contaminated
feed, the ingestion of AFB1 leads to substantial degradation of meat quality
(Bonomi et al., 1994). Cattle exposure to mycotoxins generally occurs through
the consumption of contaminated feed. Nelson et al.. (1993) described
mycotoxicoses arising on the grounds of exposure to mycotoxin-contaminated
rations. Ruminants, such as cattle and sheep, are generally more resistant to
mycotoxins than most animals, especially pigs, as ruminal microbial

population plays a role in detoxification process. This assumption is based on
the finding that rumen flora is able to convert a number of mycotoxins into
metabolites that are less potent or even biologically inactive at common
exposure levels (Kiessling et al., 1984). The first identified source of
mycotoxins in ruminant diets was the contamination of feed concentrates with
aflatoxins. AFB1 occurs in many typical energy-rich concentrates such as grain
maize, sorghum, pearl millet, rice, soybean products, peanuts, sunflower &
cotton seeds, palm kernels and copra (Vargas et al., 2001; Abbas et al., 2002;
Attala et al., 2003).
Humans are exposed to AFB1 either directly through the consumption of
contaminated food or indirectly through the consumption of animal products
(i.e. milk and eggs) coming from animals that had consumed contaminated
feed (Rustom, 1997; Bennett and Klich, 2009; Markov et al., 2013). Since it
was first observed that dairy animals consuming feeds contaminated with
AFB1 excrete aflatoxin M1 (AFM1) in their milk, studies have established that
variations in carry-over rates are significant both at high- and low-level AFB1
feed contamination (Prandini et al., 2009).
1.1.1. AFB1-Related Effects Seen in Humans and Animals

Although animal species may vary in their susceptibility to aflatoxins,
toxic effects of the latter, known as aflatoxicoses, can generally be divided into
acute and chronic, based on some determinants such as the duration and level
of exposure, entry route, environmental factors, age, health, nutritional status,
and other factors such as stressors affecting the animal (Leeson et al., 1995;
FDA, 2002). In case of humans, exposure to AFB1 occurs mainly through the
consumption of contaminated food such as corn, peanuts, sorghum, copra and
rice, cashew, hazel, peanuts, walnuts, pistachios and almonds (Busby and
Wogan, 1984; Abdel-Gawad and Zohri, 1993; Mahoney and Rodriguez,
1996). AFB1 also exhibits its toxicity through the metabolite AFM1, which
was first determined in human urine while elucidating the etiology of liver
cancer caused by AFB1 (Campbell et al., 1970). It has been reported that about
1.3 to 1.5% of the ingested AFB1 converts into AFM1 that gets to be excreted
in human urine (Zhu et al., 1987).
As the contamination of foodstuffs and feedstock with aflatoxigenic
moulds and their toxins is very common, toxic effects of AFB1 on animal
health are encountered worldwide (FAO, 2004). Many animal species such as
turkeys, ducklings, rainbow trout, guinea pigs, rabbits, rats and dogs show
high susceptibility to aflatoxins. AFB1 can cause liver and other cancers in
humans and livestock; this has been well established in several animal species
including rodents, nonhuman primates and fish, the first symptoms thereby
being a lack of appetite and weight loss (Busby and Wogan, 1984; Eaton and
Gallagher, 1994). Several research reports have agreed that AFB1 is more
toxic for young animals (IARC, 1993, Vainio et al., 1994). It has been
observed in many parts of the world that AFB1 poses a major etiological factor
in the development of hepatocellular carcinoma in individuals infected with
hepatitis B virus (Wild and Hall, 2000). Particularly high incidences of AFB1
contamination have been seen in tropical and subtropical regions, where warm
and humid weather provides for conditions optimal for mould growth.
Chronic ingestion of AFB1 causes various adverse effects such as
increased susceptibility to diseases, loss of reproductive performance and, in
case of dairy cattle, a decrease in quantity and quality of milk production.
Animal exposure to AFB1 leads to a decrease in feed consumption or even to
feed refusal, as well as to the reduction in nutrients absorption, metabolic
impairments, decreases in protein synthesis, and endocrine and immune
system suppression. Acute intoxication is often fatal for both humans and
livestock. In poultry and livestock, severe and sudden anorexia, convulsions,
feed refusal, weight loss, discolored liver, reduced egg production, reduced
energy conversion rate and milk contamination can be encountered. On top of
that, the consumed feed loses its common nutritional value and efficiency,
leading to reduced livestock growth rates (Waliyar et al., 2007).
1.1.2. Conditions under Which AFB1 Gets to Be Produced in Cereals

Accumulation of mycotoxins before and after cereal harvesting largely
reflects actual climate conditions. Fusarium toxins are known to be produced
during cereal harvesting under high moisture conditions (Munkvold and
Desjardins, 1997), whereas pre-harvest aflatoxin contamination of crops,
including peanuts (Dorner, 2008) and maize (Payne, 1998), is associated with
high temperatures, insect-mediated damage and prolonged drought. Chronic
contamination occurs in warm, humid, tropical, and subtropical maize-
growing environments (Widstrom, 1996). The degree of moisture mostly
depends on the water content available at the harvesting point, but also on the
frequency and extensiveness of drying, aerating, and turning of the grain
before and during storage, and the respiration of insects and microorganisms
harbored by stored grain (Bryden, 2012). Since Aspergillus can tolerate lesser
water activity than Fusarium, these contaminations may occur both pre- and
post-harvesting, whereas Fusarium contamination is more specific to the pre-
harvesting period (Abramson, 1998). Stored cereals may become infested with
fungi and insects; such an infestation is also affected by climatic factors such
as temperature and humidity, geographical location, type of storage container,
and handling & transport procedures (Chelkowski, 1991; Jayas et al., 1995).
Climate changes can alter the dynamics of insect populations that facilitate
fungal crop infections (Wu et al., 2011).
Earlier studies have pointed toward significant dependence of AFB1
occurrence on country or region in which the cereals are grown, as well as on
high AFB1 concentrations found in maize, peanuts, tree nuts, rice and
cottonseed (Rustom, 1997; Reddy et al., 2009). It has been pointed out that the
growth of A. flavus and the production of aflatoxins in various biological
materials are influenced by many factors including the type of substrate, its
moisture content, culpable fungal species, presence of minerals, relative
humidity of the surroundings, temperature, and physical damage of the kernels
(Viquez et al., 1994). It has been shown that the type of mould and its conidial
concentration, as well as maize moisture content, play critical interactive roles
in the initiation of mould infestation, spoilage and AFB1 production in maize
(Oyebanji and Efiuvwevwere, 1999).
Limitation of AFB1 occurrence in crops before harvest can be achieved
through the reduction of drought and temperatures, weed control, insect
damage reduction, effective harvesting techniques and Aspergillus spore
reduction in soil by virtue of crop turnover. Genetic engineering and the
development of hybrids resistant to Aspergillus spp infection (Widstrom,
1996) may offer new ways of limiting pre-harvest aflatoxin contamination of
certain crops. Post-harvesting control of AFB1-susceptible crops can be
achieved through the control of factors that affect fungal growth, e.g. water
activity, temperature, gas atmospheres, and through the use of insecticides or
food preservatives. The prime concern relative of the storage of grains and
nuts should be to maintain water activity below the limit favoring fungal
growth (which is achievable by virtue of moisture control) (IARC, 2002). The
risk of kernel damaging and consequent AFB1 production can be reduced by
harvesting solely grains having the moisture of around 24% (Prandini et al.,
2009).
1.1.3. The Occurrence of AFB1 in Feed

In many cases, the levels of AFB1 naturally occurring in feeds intended
for dairy cows have been shown to exceed the regulation limits. The contents
of feed intended for milking cows slightly vary dependent on the season and
geographical area; some 10% of feed is commonly intended for these
purposes. Rye, oats, mocha, wheat and sorghum are selected on dairy farms
based on the acreage and selected pasture; the use of commercial pelleted feed
is not uncommon either (Alonso et al., 2011).
Given the fact that in geographical regions having a tropical or sub-
tropical climate the risk of AFB1 contamination has generally been
acknowledged as high, monitoring of feed ingredients for the presence of
AFB1 has been focused on imported feeds such as extracted copra, peanut
cake, sunflower cakes, corn gluten, rice bran, cottonseed, palm kernel and soy
beans, which seem to be the major carriers of AFB1. In some countries,
contamination levels above legal limits were linked to high contamination of
locally grown maize that was used as animal feed (EFSA, 2004).
In different countries AFB1 has been found to be a contaminant of dairy,
cottonseed, barley, soy bean, pellet wheat, peanut shells, corn silage and
sorghum silage (Decastelli et al., 2007; Sassahara et al., 2005). Certain cases
pointed toward an outbreak of acute aflatoxicosis, with high levels of AFB1
observed in maize stored under high humidity conditions (Lewis, 2005). As
for dairy cattle, the problem does not end with animal diseases or production
losses, since AFB1 presence in feed leads to the presence of its metabolic
product AFM1 in milk and dairy products, possibly affecting human health as
well (Boudra et al., 2007; Veldman et al., 1992).
1.2. Current EU Legislation
Since the discovery of aflatoxins in the 1960s, regulations have been

enforced in many countries so as to protect the consumers from harmful
effects of these toxins that may contaminate both foodstuffs and feedstuffs.
Various factors play a role in defining permissible mycotoxin levels. These
include evidence-based data underpinning the risk assessment, such as the
availability of toxicological data, food consumption data, data on the level and
distribution of mycotoxins in goods intended for human and animal
consumption, and data on analytical methodology. Economic factors such as
commercial and trade interests and food safety issues also have an impact
(FAO/WHO, 2008). Compared to other regions of the world, the European
Union (EU) has the most extensive and most detailed regulations governing
AFB1 presence in various types of food and feed. Also, many of the EU
candidate member states have mycotoxin presence-governing regulations
covering the topic as much in depth as the regulations currently in force across
the EU itself.
Methods of sampling and analysis used within the frame of the official
mycotoxin control, AFB1 included, are laid down under the Commission
Regulation No 401/2006, amended by the Commission Regulation (EU) No
178/2010. This ensures that the same sampling criteria are applied for the
same products by the competent authorities throughout the EU and that certain
performance criteria, such as recovery and precision, are fulfilled. Maximum
permitted levels (MPLs) of aflatoxins in food, including those of AFB1 and
total aflatoxins, are laid down under the Commission Regulation (EC) No
1881/2006, amended by the Commission Regulation (EU) No 165/2010.
Legal limits for AFB1 in feedstuffs currently adopted by the EU member
states and set under the Commission Directive 2003/100/EC that amends the
Directive 2002/32/EC, are substantially different from those in other countries
that have enforced AFB1 MPLs for animal feeding stuffs. As AFB1 is a
genotoxic carcinogen and a strong acute toxin that affects various animal
species, it is the only individual mycotoxin whose MPLs are set under the
Directive. Some countries have a number of limits, often dictated by the
destination of the feedstuff. From the human healths point of view, the most
stringent criteria apply to feedstuffs intended for dairy cattle because of
AFB1s conversion into AFM1 that takes place in milk and dairy products
(MPL= 5 g/kg across the EU).
1.3. Analytical Methods of AFB1 Determination
For the purpose of AFB1 determination, different screening and

confirmatory analytical methods are used. Most of these analytical methods
have to be performed using the appropriate cleanup procedures, except
perhaps for the immunological assay called the ELISA, with which this step
might be skipped. The development of semi-quantitative ELISA as a screening
method was a major step forward in the development of rapid, repeatable and
sensitive assays suitable for AFB1 determination. Gaur et al. (1981) produced
and characterized AFB2-antiserum, equally specific for AFB2 and AFB1, and
used it within the ELISA for AFB1 quantification.
In the recent years, the ELISA has been described to have advantages over
other methods when it comes to AFB1 determination; these advantages mostly
lie within its rapidity, high specificity, simplicity of use, low cost and the use
of safe reagents (Pestka, 1994; Zheng et al., 2005; Goryacheva et al., 2007,
Ayejuyo et al., 2011). Commercially available ELISA kits suitable for the
detection of mycotoxins are based on the competitive assay format that uses
either a primary antibody specific for the target molecule or an enzyme

conjugate and the required target. The advantage of micro-titer plate-based
immunoassays lies within the fact that these can be used to analyze a large
number of samples simultaneously. The complex formed on the occasion then
interacts with a chromogenic substrate to give a measurable result. The
disadvantage of the ELISA mostly comes as a result of its limited detection
range consequential to the narrow scope of the antibodies sensitivity (Turner
et al., 2009).
Other methods for AFB1 quantification require sophisticated laboratory
equipment, including high performance liquid chromatography (HPLC), gas
chromatography (GC), liquid chromatography/mass spectrometry (LC/MS) or
gas chromatography/mass spectrometry (GC/MS) (Xiang et al., 2006; Krska et
al., 2008; Rahmani et al., 2009; Stephard et al., 2011).
HPLC has a high efficiency, sensitivity and resolution (Herzallah, 2009;
Peiwu et al., 2011). Modern analysis of components heavily relies on HPLC
that employs various adsorbents depending on physical and chemical structure
of different components. The most commonly used HPLC detectors are
fluorescence detectors (FLDs). In order to widen the detection limits, HPLC is
used in combination with mass spectrometry (MS) (Li et al., 2009). MS
represents a method that allows for a highly accurate and specific detection of
AFB1, with limiting factors such as high cost of equipment, complex
laboratory requirements, and limitations in the type of solvents used for
extraction and separation (Turner et al., 2009).
1.4. Methods of AFB1 Reduction
As the presence of moulds and/or mycotoxins in food can be dangerous

for human health and represents a huge economic problem, one has all the
reasons to allow for the implementation of new methods providing for a safe
food production. Methods of control can be classified into two categories: (1)
prevention of mould contamination and growth, and (2) detoxification of
contaminated products (Riley and Norred, 1999; Mishra and Das, 2003). The
prevention of mould growth can be achieved either through pre- or post-
harvesting strategies. The applied AFB1-reduction procedure must effectively
inactivate or remove the toxin, maintaining at the same time both nutritional
and technological properties of the product, while not generating reactive toxic
products (Lpez-Garca and Park, 1998). These methods can be divided into
chemical, biological and physical (Kabar et al., 2006). Investigation into the
methods of AFB1 inactivation in contaminated food and feed has revealed that
pre-harvest contamination can be reduced by virtue of proper curing, drying,
sorting and storage, all of the aforementioned limiting the growth of
aflatoxigenic fungi. However, the implementation of unique, totipotent method
of aflatoxin reduction, capable of effectively performing in any given
biological material, is virtually impossible.
The efficiency of methods of AFB1 inactivation depends on many
parameters such as the nature of food and feed, their moisture content and
composition, and the level of contamination. Some studies have attempted to
achieve detoxification of, or toxin inactivation in, AFB1-contaminated
feedstuff using gamma irradiation, thermal inactivation, physical separation,
microbial degradation and different chemical treatments (Piva et al., 1995;
Rustom, 1997).
1.4.1. Biological Reduction

Many microorganisms including bacteria, yeasts and acid-producing
moulds can metabolize and inactivate AFB1, Flavobacterium aurantiacum
being the most active among them. AFB1 production is also inhibited by lactic
acid bacteria, Bacillus subtilis and many moulds. As shown in the fermenting
industry settings, aflatoxins do not degrade during fermentation, but have been
proven absent from alcohol fraction after distillation. Aflatoxins are usually
concentrated in spent grains. When contaminated products are used for
fermentation, it is important to determine the end-use of the contaminated by-
products. A specific compound found to be a good decontaminating agent is
usually more biologically- and cost-efficient if added directly. Literature has
revealed that true efficacy of biological methods demonstrating effective
decontaminating properties is usually dependent on specific compounds
produced by selected microorganisms (Waliyar et al., 2007), as well as on the
competition for nutrients required for toxin production, space competition and
the production of anti-aflatoxigenic metabolites coming from coexisting
microorganisms. Studies have suggested that certain fungi, including A.
parasiticus, degrade AFB1, possibly through fungal peroxidases (Lpez-
Garcia and Park 1998).
1.4.2. Physical Reduction

Inactivation of AFB1 using physical methods involves extraction with
solvents, adsorption, and heat- or irradiation-based inactivation. AFB1 levels
can be reduced in stored goods using physical procedures such as color
sorting, density flotation, blanching and roasting.
Despite the debate on safety of irradiated foods, food irradiation is

becoming a technique of a commercial-scale application, employed so as to
render food products sterile (Diehl, 1990). Gamma radiation as a sterilizing
treatment with a high penetrating power passes through materials without
leaving any residues and causes a direct damage to cell DNA through
ionization, inducing mutations and cell killing. There exist a number of reports
on increased, decreased or even unaffected production of mycotoxins after
irradiation of fungi under various conditions. The Joint FAO/IAEA/WHO
Expert Committee pointed out that the irradiation of any food up to the
average total dose of 10 kGy poses no toxicological hazard and no special
nutritional or microbiological problem (WHO, 1991; Mariotti et al., 2011). In
light of the foregoing, in 1999 the European Community authorized this dose
as the maximum total radiation dose allowed to be absorbed by irradiated food
on average.
1.4.3. Chemical Reduction

The use of chemicals to inactivate, bind or remove aflatoxins has been
studied extensively using different chemicals such as propionic acid,
ammonia, copper sulfate, benzoic acid, urea, citric acid and some other
chemicals capable of reacting with aflatoxins (Gowda et al., 2004). These
chemicals convert AFB1 to less toxic and less mutagenic compounds including
acids, bases, oxidizing agents, bisulfites and gases. Where approved, AFB1
levels in goods destined for animal feeds can be reduced by agents such as
adsorbent clays, as well as by ammonization. The main purpose of
ammonization is the elimination of AFB1 from feed intended for dairy cows
(IARC, 2002). As for the chemical methods of AFB1 reduction, they have
generally been labeled as impractical as they call for drastic conditions in
terms of temperature and pressure, as well as unsafe because of toxic residues,
and unfavorable since leading to degradation of nutritional, sensory and
functional properties of the product (Rustom, 1997). To date, chemical
methods have been approved only for the reduction of AFB1 in animal feed.
Techniques other than the use of chemical sorbents and ammonization have
achieved reduction in AFB1 bioavailability that comes as a result of hydrated
sodium calcium aluminosilicate binding (Phillips et al., 1988). Ammonization
is the only chemical inactivation process that has been shown to efficiently
destroy AFB1 in cottonseed and cottonseed meal, peanuts and peanut meal,
and maize (Park et al., 1988; Park and Price, 2001).
2. SURVEY OF AFB1 BIO-PREVALENCE IN CEREALS

2.1. Samples under Study
In order to investigate the bio-prevalence, i.e. the occurrence of AFB1 in

cereals, a total of 792 samples of maize (388), wheat (155), barley (148) and
oat (101) were collected during a three-year period (2010-2012) from different
fields situated in northwestern and eastern part of Croatia. Sampling and
preparation of the test samples were performed in line with ISO 6497:2002
and ISO 6498:1998, respectively.
Determination of moisture content in the sampled materials was
performed as well. Prepared test portions were ground into a fine powder
having a particle size of 1.0 mm using an analytical mill (Cylotec 1093,
Tecator, Sweden) and stored at +4 C prior to AFB1 analysis that made use of
ELISA and LC-MS/MS methods.
2.2. Implementation of the ELISA Method
2.2.1. Validation of the ELISA Method

Validation parameters of the ELISA method were determined using
control maize and wheat samples. AFB1 standards employed with the
validation of analytical methods were provided by Sigma-Aldrich Chemie
GmbH (Steinheim, Germany).
The limit of detection (LOD), calculated from the mean value of ten
determinations of blank maize and wheat samples plus three standard
deviations, was 1 g/kg in both cases. The recovery rate was determined at
four different levels (2, 5, 10 and 50 g/kg) by virtue of spiking the control
maize and wheat samples with the prepared standard mycotoxin working
solution (100 g/L) adopted for in-house use (six replicates per concentration
level per day). For the determination of intermediate precision, the same steps
were repeated on two additional occasions by two independent analysts within
a three-month period and under the same analytical conditions.
Validation results (given in Table 1) proved the applied ELISA method to
be efficient and suitable for the determination of AFB1 in cereals under
consideration.
Table 1. Results of the ELISA method validation
Spiked Intermediate
Mean recovery CV CV
Material concentration precision
(%) (%) (%)
(g/kg) (%)
2 85.4 6.1 88.5 8.4
5 90.7 5.7 93.2 7.3
Maize
10 92.2 6.3 93.6 7.1
50 95.5 4.9 95.9 6.7
2 86.7 4.6 82.6 6.7
5 88.5 5.8 88.9 7.7
Wheat
10 93.6 7.4 94.6 8.2
50 96.8 6.8 95.2 8.8
2.2.2. Employment of the ELISA Method
2.2.2.1. Sample Preparation

Samples were prepared using 5 g of the homogenized sample
supplemented with 25 mL of 70%- methanol and shaken vigorously head-
over-head on a shaker for three minutes. The extract was then filtrated
(Whatman, black ribbon); in the further course, 1 mL of the obtained filtrate
was diluted with the appropriate volume of deionized water. When calculating
the final AFB1 concentration in the analyzed sample, the applied dilution
factor was duly taken into account.
2.2.2.2. ELISA Assay

All study samples were first analyzed for AFB1 concentration using the
ELISA method that made use of AFB1 ELISA Ridascreen kits provided by R-
Biopharm (Darmstadt, Germany). ELISA was also used after the
implementation of AFB1 reduction methods. Each kit contains a micro-titer
plate with 96 wells coated with antibodies against AFB1, aqueous solutions of
AFB1 standard (0, 1, 5, 10, 20, and 50 g/L), peroxidase-conjugated AFB1,
substrate/chromogen (urea peroxide), a stop-reagent (1 N-sulfuric acid), and
the washing buffer (10 mM-phosphate buffer, pH=7.4). All other chemicals
used for the analysis were of an analytical grade. The ELISA assay employed
with the determination of AFB1 in the analyzed cereals, was performed in full
line with the kit manufacturers instructions, and made use of an auto-analyzer
ChemWell 2910 (Awareness Technology, Inc, USA). The obtained AFB1
concentrations were calculated from a six-point calibration curve and

corrected for recovery.
2.3. The Implementation of LC-MS/MS Method
2.3.1. LC-MS/MS Validation

LC-MS/MS validation was carried out according to the Commission
Decision 2002/657/EC using an alternative approach of matrix-comprehensive
in-house factorial design validation. The software used for the factorial design
and calculation was InterVal Plus (quo data, Gesellschaft fr
Qualittsmanagement und Statistik GmbH, Dresden, Germany). Within the
frame of the validation process, decision limit (CC), detection capability
(CC), precision, recovery, repeatability, in-house reproducibility, matrix
effects, specificity and ruggedness of the method were studied. The validation
process started with the factorial design (Table 2).
For the determination of AFB1 in cereals, 8 runs, each at 6 concentration
levels, were done within 8 days using different factor combinations. In total,
48 measurements were performed. Within each run, blank samples were
fortified at six concentration levels: 2.5, 5, 7.5, 15, 30, and 60 g/kg. In
addition, a blank matrix sample, blank reagent sample and a fortified matrix
sample were included into each run. For maize, CC and CC of 5.86 g/kg
and 6.70 g/kg were determined, respectively.
Validation results observed with maize (Table 3), as also the results of
other validation parameters determined with both cereals under study, proved
LC-MS/MS suitable for AFB1 determination.
Table 2. Factors of interest and their levels used for the determination
of AFB1 in cereals
Factor Level
Operator Analyst 1 / Analyst 2
Cereal Maize / Barley
Extraction 2h / 3h
Storage of extracts 24 hours, +4 C before injection/
(injection solution) without
RC filter Producer 1- Agilent Technologies/
Producer 2 - Phenomenex
Table 3. Repeatability (sr), in-house reproducibility (sWR) and recovery

established for LC-MS/MS used for the analysis of AFB1 in maize
Spiked AFB1
sr sWR Recovery
concentration RSD (%) RSD (%)
(g/kg) (g/kg) (%)
(g/kg)
2.5 0.43 17.2 0.43 17.3 101.1
5.0 0.44 8.9 0.44 8.9 100.5
7.5 0.47 6.2 0.47 6.2 100.3
15 0.57 3.8 0.57 3.8 100.1
30 0.86 2.9 0.88 2.9 100.0
60 1.56 2.6 1.63 2.7 99.9
2.3.2. LC-MS/MS Implementation
2.3.2.1. Sample Preparation

To 25 g of the sample, a 100 mL of the extraction solution
(ACN/H2O=80/20) were added. The mixture was shaken for 2 hours on a
horizontal shaker and afterwards filtrated through the Whatman black ribbon
filter paper. One mL of the obtained filtrate was diluted with 3 mL of ultrapure
water, mixed and filtrated through 0.45m-RC filter. Forty L of the sample
were injected into the HPLC system.
2.3.2.2. Conditions under Which LC-MS/MS Was Implemented

LC-MS/MS method was used to confirm the presence of AFB1 in the
samples in which this mycotoxin was initially determined at levels higher than
MPLs using the ELISA method (that is to say, in the maize samples only).
The HPLC (LC) system (1260 Infinity, Agilent Technologies, Santa Clara,
USA) consisted of a degasser, a binary pump, an auto-sampler and a column
compartment, and was coupled with a 6410 QQQ-mass spectrometer (MS)
(Agilent Technologies, Santa Clara, USA). HPLC separation was performed
on XBridge BEH C18 columns (150x4.6, particle size 2.5 m) at the flow rate
of 0.80 mL/min and the temperature of +40 C. The mobile phase consisted of
the constituent A (0.1%-formic acid dissolved in water) and the constituent B
(acetonitrile). A gradient elution program was employed as follows: 0-3 min:
90%-A, 18 min: 10%- A, 18.1 min: 90%-A, with the post-run time of 4 min
and the injection volume of 40 L. The conditions under which the mass
spectrometry was performed were as follows: electro-spray ionization, positive
polarity, capillary voltage of 6 kV, source temperature of +350 C, nebulizer
operating pressure of 45 psi, and the gas flow rate of 9 L/min. The mass
spectrometer was operated in the multiple reaction monitoring mode, the

protonated molecular AFB1 ion at m/z = 313 being the precursor ion. Two
product ions at m/z = 285 and m/z = 241 were monitored. The quantification
was performed during the most intense transition (m/z 313 285) by virtue
of extrapolation from six-point calibration curves.
2.4. AFB1 Concentrations Determined in Cereals
Statistical analysis of data on AFB1 concentrations obtained by the two

methods, was performed using the Statistica Software Ver. 10.0 (StatSoft Inc.
1984-2011, USA), with the statistical significance set at 95%-level (p=0.05).
AFB1 presence detected using ELISA was confirmed by virtue of LC-MS/MS,
indicating a high concordance between these two methods when employed to
the effect of AFB1 determination.
The results of AFB1 analyses per each investigated cereal harvested during
2010-2012 period on different fields, together with the determined number
(No) and percentage of positive samples, the average (mean), as well as
minimal and maximal concentrations and the accompanying standard
deviations (SDs) obtained within this investigation, are summarized in
Table 4.
Table 4. Concentrations of AFB1 in cereals harvested during 2010-2012

period on different fields
Percentage Mean of
No. of No. of
of positive positive SD Mine Maxf
Cereal total positive
samples samplesb (g/kg) (g/kg) (g/kg)
samples samplesa
(%) (g/kg)
Maize 388 63 16.2 18.5c 20.3 1.9 97.5
Wheat 155 11 7.1 2.2d 1.0 1.1 3.0
Barley 148 8 5.4 1.5d 0.5 1.2 2.4
Oat 101 2 2.0 1.1 0.1 0.9 1.2
a
AFB1 is detected (>LOD).
b
Mean AFB1 concentrations determined using ELISA and LC-MS/MS.
c
In 32/25 maize samples, AFB 1 concentrations were higher than MRLs applicable to
food / feed.
d
In 2 wheat/1 barley sample, AFB1 concentrations were slightly higher than MRL
applicable to food.
e
Minimal AFB1 concentration determined among the positive samples.
f
Maximal AFB1 concentration determined among the positive samples.
Among the investigated cereals, maize was proven to be most

contaminated, with AFB1 determined in 16.2% of samples, as compared to
7.1% AFB1-positive wheat, 5.4% AFB1-positive barley, and 2.0% AFB1-
positive oat samples. Taking into account the contamination level of AFB1 in
cereal samples detected in this research, and given the MPL for cereals
intended for foodstuffs, which is 2 g/kg for all cereals except for maize (to
which the MPL of 5 g/kg applies), it can be concluded that 32 maize samples
(8.2%), 2 wheat samples (1.3%) and 1 barley sample (0.7%) had AFB1
concentrations over the MPL, whereas all oat samples met the stipulated value.
Comparing the obtained AFB1 level to the MPL of 20 g/kg, applicable to all
cereals intended for feed, it can be concluded that levels higher than MPL
were determined in 25 maize samples (6.4%), whereas all wheat, barley and
oat samples had satisfied the given criterion.
The maximal AFB1 level detected in the maize samples was 97.5 g/kg,
which is around 5 times higher than allowed for feed and even 20 times higher
than allowed for food. The lowest number of positive samples and the lowest
average concentration of AFB1 were observed with oat, AFB1 thereby being
determined in only two samples at concentrations approximating to, or being
slightly higher than, the ELISA limit of detection. In general, AFB1 levels
higher than maximally allowed were exclusively found in the maize samples
of 2012 genus, sampled mostly from fields in the eastern part of the country,
i.e. the part known to have the largest grain production and the most developed
farming in Croatia. The results of the analysis of variance (ANOVA) revealed
statistically significant differences (p<0.05) in AFB1 concentrations between
various types of samples under investigation (significantly higher in maize),
but no differences (p>0.05) either in AFB1 concentrations determined across
the same cereal group (barley, wheat or oat), or between the sampling regions,
except for maize under any given scenario.
Given the fact that elevated mycotoxin concentrations are usually
associated with humidity and temperature as the factors most critical for
mould formation and thus also mycotoxin production (Pleadin et al., 2013), the
explanation of the results of this investigation could also be sought among
these factors. In conclusion, such a high cereal contamination, especially that
of maize, could likely be associated with climate conditions established in the
investigated regions in the period of concern, which was extremely warm and
dry (data obtained from the Croatian Meteorological and Hydrological
Institute), which might had favored mould production and AFB1 formation.
Therefore, an inadequate food/feed control could result in the consequent
contamination of food and feed, which is even more worrying should one bear
in mind that the affected region is famous for its production of cereals,
particularly that of maize, and its wide-scale use of the latter.
3. INVESTIGATION INTO THE POSSIBILITIES OF AFB1

REDUCTION IN MAIZE
3.1. Reduction of AFB1 Using Gamma Radiation
The use of gamma () radiation to inactive aflatoxins has already been

investigated on some other materials; the results have shown that fungi that
produce AFB1 can successfully be deactivated in paper, wood and soil using
irradiation doses ranging from 6 to 15 kGy (da Silva et al., 2006). It has also
been observed that doses higher than 10 kGy inhibit seed germination (Chiou
et al., 1990). Aziz et al. (1997) reported that the dose required for the complete
inhibition of fungi in different food and feed range from 4 to 6 kGy. After
gamma irradiation with the dose of 1 and later on of 10 kGy, the toxicity of a
peanut meal contaminated with AFB1 was reduced by 75% and 100%,
respectively (Temcharoen and Thilly, 1982).
The presence of water plays an important role in ray-based AFB1
destruction, since the radiolysis of water leads to the formation of highly
reactive free radicals. These radicals can readily attack AFB1 at the terminal
furan ring, yielding the material of lower biological activity (Rustom, 1997).
Van Dyck et al. (1982) established the mutagenic activity of AFB1 in an
aqueous solution (5 g/mL water) to be reduced by 34%, 44%, 74% and 100%
after the exposure to 2.5, 5, 10, and 20 kGy -rays, respectively. The dose of
10 kGy completely inactivated AFB1, and destroyed 95% of AFG1 in
dimethyl-sulphoxide-water (1:9, v/v) solution (Mutluer and Erkoc, 1987).
AFB1 degradation in range from 37% to 100% was observed after the addition
of 1 mL of 5%-hydrogen peroxide to an aqueous AFB1 solution (50 g/mL)
under 2 kGy -irradiation.
As the prevention of pathogenic fungi growth and the production of AFB1
in agricultural goods represents a very important issue, this study included the
investigation into possibilities of reducing AFB1 detected in maize samples
using -irradiation at the doses of 5 and 10 kGy (which were applied to 25
maize samples containing AFB1 in concentrations over MPLs set for feed).
The radiation source was the 60Co -irradiation chamber situated at Rudjer
Boskovic Institute, Zagreb, Croatia.
Table 5. Concentrations of AFB1 in maize before and after -irradiation
Range of Number of Mean AFB1 level Dose of 5 kGy Dose of 10 kGy

AFB1 level in samplesa before irradiation
Mean AFB1 Reduction Mean AFB1 Reduction
maize (g/kg) (g/kg)
concentration (g/kg) (%) concentration (g/kg) (%)
20-40 4 28.1 n.d.b 100 n.d. b 100
40-60 6 53.1 8.71 83.6 1.87 96.5
60-80 6 67.6 15.3 77.4 5.01 92.6
80-100 9 93.0 32.5 65.1 16.4 82.4
a
Maize samples in which AFB1 concentrations were higher than MPL set for feed (20 g/kg).
b
After maize samples irradiation, AFB1 was not detected.
The exposure time was calculated based on the natural decay rate (the half-
life) of the source and the location of the sample. The absorbed dose was
measured using a dosimeter. The results obtained in our earlier preliminary
studies showed that the dose of 2, 3 and 5 kGy can effectively stop the
germination of aflatoxicogenic mould spores both in vitro and in situ
(unpublished data). After -irradiation with the doses of 5 and 10 kGy, AFB1
level in the contaminated maize samples was determined using the ELISA
method, as described earlier. The mean reduction of AFB1 achieved in the
contaminated maize samples under this investigation using -radiation doses
of 5 kGy and 10 kGy, ranged from 65.1% to 100%, and from 82.4% to 100%,
respectively. As can be seen from the obtained results, gamma irradiation
yielded a significant AFB1 reduction with both applied doses, especially with
that of 10 kGy. It was also observed that the level of AFB1 reduction depends
on the level of maize contamination, i.e. the higher the level of maize
contamination, the lower the rate of AFB1 reduction, irrespective of the
radiation dose applied (Table 5).
3.2. The Reduction of AFB1 Using Essential Oils and Lactic Acid
Bacteria
A novel way of reducing the proliferation of microorganisms and/or the

production of their toxins is the use of essential oils. These oils are natural
products extracted from plant materials, which have been proven to inhibit a
wide range of food-spoiling microorganisms and the Aspergillus (Bluma et al.,
2005). Essential oils applied to that effect insofar have shown a significant
impact on AFB1 accumulation, their ultimate effect thereby being dependent
on water activity, AFB1 concentration, and the length of incubation (Bluma
and Etcheverry, 2008). In the study by Bluma et al. (2009), the effects of
essential oils added to maize grains, in terms of their influence on mould
growth rate, lag phase and AFB1 accumulation by Aspergillus section Flavi,
were evaluated under different water activity conditions. The results showed
that essential oils do influence the lag phase length and the mould growth rate,
their efficacy thereby being dependent mainly on their concentration and water
activity of the substrate; a significant impact on AFB1 accumulation was
demonstrated as well.
For the purpose of this investigation, the essential oils extracted from wild
thyme, cinnamon, sage, lavender, and rosemary were used to examine the
potential of controlling the aflatoxigenic fungi A. parasiticus 2999, A. flavus
305, A. niger 388 and their AFB1 production. Essential oils obtained from a
local pharmacy were dissolved in 96 % (by volume) - ethanol (Kemika,
Croatia) to the final concentration of 100 L/mL. The inhibition of mould
colonies growth was determined on a PDA supplemented with an essential
oil. The results showed that the growth and survival of food/feed-spoiling and
AFB1-producing Aspergillus species can be controlled using essential oils,
particularly that of wild thyme and cinnamon, which were the most effective
in their inhibiting action. In the descending order of efficiency, these were
followed by lavender, sage and rosemary essential oils. Wild thyme essential
oil inhibited mould growth by about 85%, while cinnamon essential oil
completely (100%) inhibited the growth of all tested moulds (Table 6).
Soliman and Badeaa (2002) reported a complete inhibition of A. flavus, A.
parasiticus and A. ochraceus by thyme and cinnamon essential oils added in
concentrations lower than 500 mg/kg. In their research, inhibitory effects of
essential oils or their components on mould growth were proportional to their
concentration in the cultivation medium. It has been suggested that the mode
of antifungal activity of essential oils could include their attack on the fungal
cell wall and the retraction of hyphal cytoplasm, ultimately resulting in the
myceliums death. Montes-Belmont and Carvajal (1998) investigated the
effect of eleven plant essential oils used for the protection of maize against A.
flavus and found that the essential oils of cinnamon (C. zeylanicum),
peppermint (Mentha piperita), basil (Ocimum basilicum), thyme (Thymus
vulgaris), oregano (Origanum vulgare), flavoring herb epazote (Teloxys
ambrosiodes) and clove (Syzygium aromaticum) caused a total inhibition of
fungal development in maize kernels.
In this investigation, the verification of AFB1 production was performed
after 21 days of mould incubation in the YES broth (yeast extract 2%, sucrose
20%, and distilled water up to 1 L) into which essential oils were added in pre-
defined concentrations. The results showed that only cinnamon oil completely
inhibited the production of AFB1 in all tested moulds (Table 6). The addition
of wild thyme essential oil significantly inhibited AFB1 production (about
75%) by A. parasiticus 2999, A. flavus 305 and A. niger 388. Approximately
68% of AFB1 production inhibition was attained by the addition of lavender
essential oil. Rosemary and sage essential oils showed similar results, their
addition inhibiting from 45 to 57% of the toxin production. The obtained
results are in agreement with the data published by Atanda et al. (2007). These
authors showed that essential oils of the aforementioned plant species can
reduce the concentration of the produced AFB1 by about 90%.
Table 6. Inhibitory effects (%) of essential oils on mould growth

and AFB1 production
Inhibition (%)
Moulds/ AFB1
Wild thyme Cinnamon Lavender Sage Rosemary
A. parasiticus 2999 87 100 61 47 25
AFB1 77 100 70 62 48
A. flavus 305 89 100 72 53 27
AFB1 80 100 68 58 43
A. niger 388 81 100 68 58 42
AFB1 74 100 65 51 43
The results presented in this section suggest that wild thyme, cinnamon
and lavender essential oils could be efficiently used against fungi growth and
AFB1 production in food and feed during the storage period.
Several lactic acid bacteria have been found to be able to bind AFB1 in
vitro and in vivo, their efficiency dependent on the bacterial strain. The
inhibition of AFB1 accumulation was not related to the pH-decrease, but rather
to the occurrence of low molecular weight metabolite produced by the lactic
acid bacteria at the beginning of the exponential growth phase (Dali et al.,
2010). The investigation by El-Nezami et al. (1998) showed that probiotic
strains such as Lb. rhamnosus GG and Lb. rhamnosus LC-705 are very
efficient in removing AFB1, with more than 80% of the toxin trapped in a 20
g/mL solution (Haskard et al., 1998). It has also been shown that other
organisms such as Saccharomyces cerevisiae have the potential to bind AFB1
(Baptista et al., 2004) and are most efficient in AFB1 quenching (Bueno et al.,
2006). In order to investigate the possibility of AFB1 reduction, several
bacterial strains of lactic acid bacteria (LAB), originally isolated from the
traditional Croatian fermented milk and meat products, were tested for their
ability to bind aflatoxins. Lactobacillus delbrueckii S1, Lactococcus lactis
subsp. lactis SA1, L. plantarum B and L. plantarum A1 were isolated from
milk products, while L. plantarum 1K, Leuconostoc mesenteroides K5, Lactoc.
lactis subsp. lactis 5K1 and L. acidophilus K6 were isolated from meat
products and stored in the Collection of Microorganisms kept by the
Laboratory of General Microbiology and Food Microbiology of the Faculty of
Food Technology and Biotechnology, University of Zagreb (Croatia). Lactic
acid bacteria were cultivated in 5 mL of the de Mann-Rogosa-Sharpe (MRS)
broth at +37 C for 24 h. Bacterial growth was determined using MRS agar
plates after a 24 hour- incubation at +37 C by virtue of traditional plate
counting (CFU/mL). Ten mL of the MRS broth were inoculated with 10%-
inoculums of each bacterial strain and artificially contaminated with AFB1 in
the final concentration of 5 g/mL. The bacteria and AFB1 introduced into the
MRS broth were incubated (at +37oC) for 48 h. After centrifugation (3,500 x g
for 10 min), the sample supernatants were collected at 12-, 24-, and 48-h time
points. The unbound AFB1 was quantified using the ELISA method.
Many studies have suggested that significantly different binding abilities
of the LABs can be attributed to different cell wall structures. In our study,
L. plantarum A strain (isolated from cow cheese) exhibited a weaker binding
ability (25.1 to 34.3%) than L. plantarum B (isolated from sheep cheese) in
spite of their equal genetic structure, which could be explained by differences
in their biological activities (Peltonen et al., 2001). Among eight LAB strains,
L. delbrueckii S1 and L. plantarum 1K appeared to be the most efficient
binders of AFB1, removing approximately 70% of the latter from the liquid
media after 0 hours of incubation, which implies that the binding process runs
swiftly. The inter-strain differences in AFB1 binding can probably be
explained by different bacterial cell wall and cell casing structure. AFB1 is
bound to LAB surface components; it appears that this binding involves a
number of components (Haskard et al., 2001). In summary, the obtained
results clearly show that probiotic strains L. delbrueckii S1, L. plantarum B, L.
plantarum 1K and Leuco. mesenteroides K5 bind over 50% of AFB1 present in
the MRS broth after a 48-h incubation (Table 7).
Table 7. AFB1 binding by lactic acid bacteria
AFB1 bound SDa (%)

Incubation period (h)
LAB 0b 12 24 48
L. delbrueckii S1 67.80.5 48.30.6 53.20.3 59.11.3
Lactoc. lactis subsp. lactis 21.60.2 18.10.3 27.51.1 28.20.5
SA1
L. plantarum A 25.10.2 21.10.4 30.12.1 34.31.3
L. plantarum B 29.71.6 45.30.5 50.10.5 56.60.5
L. plantarum 1K 78.30.6 51.60.6 60.10.4 71.30.7
Leuco. mesenteroides K5 47.20.5 31.30.6 43.20.5 51.30.8
L. acidophilus K6 22.10.4 18.40.4 29.20.6 32.31.1
Lactoc. lactis subsp. lactis 5K1 19.80.8 16.30.2 25.50.6 27.20.5
a
The results are expressed as the average values SDs obtained with triple assays.
b
0-h sample collected after centrifugation.
CONCLUSION
The highest level of cereal AFB1 contamination was observed with maize
in comparison to wheat, barley and oat (in which the lowest AFB1 levels were
observed). Radiation-based technology could be used as an effective method
of mould growth & development prevention and the reduction of AFB1 in food
and feed. The results pointed towards the possibility of essential oils usage as
an alternative method of AFB1 reduction in agro-industries. Lactic acid
bacteria, characterized as functional cultures and proven to bind mycotoxins,
could also be used for human and animal protection against harmful effects of
mycotoxins. The toxicity of AFB1 and its seemingly unavoidable occurrence
in cereals later used as food and feed components, make the prevention and
detoxification of this mycotoxin the most challenging toxicological problem
that needs further studying and the establishment of an effective control using
screening and confirmatory analytical methods, so as to arrive at accurate
detection and prevention strategies.
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Chapter 2
AFLATOXIN OCCURRENCE
Elham Esmaeilishirazifard and Tajalli Keshavarz

Faculty of Science and Technology, University of Westminster, London
ABSTRACT
Toxigenic fungi in crops have been divided historically into two
groups, field and storage fungi. Mycotoxins are produced by toxigenic
fungi at the fields and in the storage. Although many compounds are
termed as mycotoxin, there are only five agriculturally-important
fungal toxins: deoxynivalenol, zearalenone, ochratoxin A, fumonisin and
aflatoxin. Penicillium and Aspergillus species are the most important
storage fungi. However, they can also invade stressed plants in the field.
The main mycotoxins produced by Aspergillus species are aflatoxins,
citrinin and patulin. The word aflatoxin comes from Aspergillus flavus
toxin, based on the fact that A. flavus and A. parasiticus are the
predominant species responsible for aflatoxin contamination of crops
prior to harvest or during storage. Aflatoxins B1, B2, G1, and G2 are the
four major isolated aflatoxins from food and feed commodities.
A. flavus and A. parasiticus have distinct affinity for nuts and
oilseeds including peanuts, maize and cotton seed. Cereals are a general
substrate for growth of A. flavus but, unlike nuts, small grain cereal
spoilage by A. flavus is the result of poor handling. Moreover, aflatoxin
M1 as a milk contaminant has potential risk for animal and human health.
The character of the aflatoxin problem varies by region. For instance,
aflatoxin accumulation in stored maize in subtropical Asia has risen
rapidly in post-harvest conditions whereas in the US, the issue is pre-
harvest condition of maize. Therefore, the exposure to aflatoxins differs
36 Elham Esmaeilishirazifard and Tajalli Keshavarz
between countries particularly due to different diets. Food contamination

with Aspergillus is associated with warm and dry climates. However, in
variable environmental conditions, the aflatoxin contamination may differ
from one year to another at the same location.
Progress in understanding the biology of Aspergillus has greatly
improved with the new techniques in genome sequencing and the
developed molecular tools that enable rapid genetic analysis of individual
genes. Particularly, the genetics of aflatoxin synthesis is regarded as a
model to gain insight into fungal secondary metabolism. Well-designed
research on production of the aflatoxin precursor sterigmatocystin with
the genetic model A. nidulans, has contributed greatly to our knowledge
of the aflatoxin pathway and the global regulatory mechanisms.
According to the recent studies, fungal pathogenesis is related to lipid-
mediated fungal-host crosstalk, suggesting that secondary metabolism
may be controlled by oxylipins at the transition level. Also, some
oxylipins have been reported to be engaged in the signalling mechanism
like quorum sensing responses in Aspergillus. Quorum sensing molecules
and their genes which are responsible for intra and inter kingdom
communications could be applied in the future aflatoxin bio-control
strategies.
TOXIGENIC FUNGI AND THEIR MYCOTOXINS

Toxigenic fungi in crops have been divided historically into two groups.
The first group called field fungi invade the crops and produce their
mycotoxins before harvest. The second group, cause post-harvest diseases, and
are known as storage fungi. The preliminary source of these fungi, in both
cases, is the environment field. Invasion by fungi in pre-harvest diseases is
regulated mainly by host-fungus and other biological interactions (e.g.
insects), while fungal post-harvest diseases are governed by factors which act
as nutrients, physical conditions (temperature, moisture) and biotic agents
(insects, competitive interference). Therefore, it has been suggested that
toxigenic fungi could be classified into four types; a) Plant pathogens such as
F. graminearum, b) Mycotoxin producers on stressed plants, such as F.
moniliforme and Aspergillus flavus, c) Colonisers, e.g. A. flavus, that colonise
plants for subsequent mycotoxin infection after harvest, and d) Fungi living in
the soil or decomposing plants e.g. Penicillium verrucosum and Aspergillus
ochraceous. These fungi inoculate the developing kernels of the crops in the
field and proliferate in storage under favourable conditions (Miller, 1995).
Aflatoxin Occurrence 37
Although many compounds are referred to as mycotoxin, there are only

five agriculturally-important fungal toxins: deoxynivalenol, zearalenone,
ochratoxin A, fumonisin and aflatoxin. Mycotoxins reproduced by Fusarium
include fumonisins, deoxynivalenol and zearalenone. Although Penicillium
and Aspergillus species are storage fungi, they can also invade stressed plants
in the field as well. Penicillium species produce ochratoxins, citrinin and
patulin. Aspergillus species produce aflatoxins, citrinin and patulin. A. flavus
and A. parasiticus are the predominant species responsible for aflatoxin
contamination of crops prior to harvest or during storage. The word aflatoxin
comes from Aspergillus flavus toxin (IARC, 1993; Gokmen et al., 2005;
Miller, 1995; Sinha & Sinha, 1992; Yu et al., 2004b).
Aflatoxins are considered as the most important mycotoxins because of
their occurrence, toxicological effects and impact on human well-being and
crop trade (Gnonlonfin et al., 2013). A variety of soil inhabiting Aspergillus
strains are aflatoxin producers. These include A. flavus, A. parasiticus, A.
nomius (Wilson et al., 2002), A. pseudotamarii (Ito et al., 2001), and A.
bombycis (Peterson et al., 2001).
Aflatoxins found in food are classified as B1, B2, G1, and G2. 'B' and 'G'
refer to the blue and green fluorescent colours produced by these toxins under
UV light during the thin layer chromatography plate visualization; the
subscript numbers 1 and 2 indicate major and minor compounds, respectively.
When aflatoxins B1 and B2 are ingested by lactating cows, a proportion (about
1.5%) is hydroxylated and excreted in the milk as aflatoxins M1 and M2.
These two compounds have lower toxicity than the parent molecules but this
toxicity would be significant due to the widespread consumption of cows' milk
by infants. Because of their high toxicity, low levels of aflatoxins have been
set and regulated in foods and feeds by many countries. Aflatoxin M1 has been
detected in human breast milk from Victoria, Australia and Thailand as well as
in raw milk from cows and water buffaloes in Iran at high concentrations
(Rahimi et al., 2010; Lanyasunya et al., 2005; Pitt, 2000).
Two Aspergillus species, A. flavus and A. pseudotamarii, produce only B
aflatoxins. They are unable to synthesize G aflatoxins due to deletion (0.8- to
1.5-kb) in the aflatoxin biosynthesis 28-gene cluster (Ehrlich et al., 2004). The
other aflatoxigenic species including A. nomius, A. parasiticus and A.
bombycis produce all four aflatoxins (El-Nezami et al. 1995).
Since the discovery of aflatoxins, A. flavus has become the most widely
reported food-borne microorganism. This reflects its importance in health care
and economy (Pitt, 2000). A. flavus is a ubiquitous and morphologically
complex species including two groups based on its sclerotia size: L strains
(Group I) with sclerotia >400 mm in diameter and S strains (Group II) with
sclerotia <400 mm in diameter (Cotty, 1989). Both A. flavus strains produce
aflatoxins B1 and B2, but A. flavus S strains can also produce aflatoxins G1
and G2. S strains are geographically distributed worldwide but rare in the
United States (Tran-Dinh, et al., 1999). The sexual stage of A. flavus has been
identified as Petromyces where ascospores are found to develop within
sclerotia (Horn, et al., 2009a). A. parasiticus is an important plant pathogen
and produces B and G aflatoxins. Although its sexual stage also belongs to
Petromyces sp., its host specificity is generally limited to ground crops
whereas A. flavus infects a wide range of plant hosts (Horn, et al., 2009b).
Some species belonging to section Ochraceorosei including A. ochraceoroseus
and A. rambellii have been reported to produce aflatoxin (Cary et al., 2005 and
2009). Moreover, several other Aspergilli produce aflatoxin precursors, such
as sterigmatocystin and o-methylsterigmatocystin, which have similar
biological properties to aflatoxin (Brown, et al., 1996).
Aflatoxins are thermo-stable. So they may contaminate the dairy products
and fermented food despite pasteurization and sterilization. It has been
reported recently that aflatoxin M1 (hydroxylated metabolites of aflatoxin B1)
contamination in milk is a potential risk for animal and human health (Prandini
et al., 2009). The occurrence of aflatoxin M1 in raw milk depends on the
climatic conditions. Milk contamination by this mycotoxin was notably
affected during dry periods. In this study, raw milk samples contained
aflatoxin M1 since the food provided to cows was probably contaminated with
the toxin. This contamination occurred particularly during the dry period with
<8.0 mm rainfall and moderate temperatures. Under these climatic conditions,
as the cattle are usually kept in confinement, there is a need for supplementary
feedstuff. It appears that the additional feed was contaminated. However, in
the rainy period, when the animals are usually free to roam on pasture land, the
risk of contamination decreased (Picinin et al., 2013).
A recent report suggests that certain food and food ingredients in African
countries are highly susceptible to contamination by several mycotoxins.
Among these, maize is the main source of fumonisin, deoxynivalenol and
zearalenone while groundnuts are the main source of contamination by
aflatoxin and ochratoxin A. Aflatoxin (primarily aflatoxin B1) exerts toxic
effects on humans, pose the major threat as a potential risk factor for many
human diseases in Cameroon. This threat is likely to be more profound if
aflatoxins co-exist with other mycotoxins (Abia et al., 2013).
DIFFERENT CROPS AND AFLATOXIN CONTAMINATION

A. flavus and A. parasiticus have a distinct affinity for nuts and oilseeds.
Peanuts, maize and cotton seed are the three most important crops affected by
these Aspergillus species. Initial investigation assumed that invasion by
Aspergillus was mainly due to inadequate drying or inappropriate storage.
These factors are important in the occurrence of aflatoxins in the humid tropics
(Pitt, 2000). However, in temperate climate, these crops get infected by A.
flavus before harvest. Invasion of peanuts occurs as a result of drought stress
(Cole et al., 1982). Preharvest invasion in maize rely partly on insect damage
to cobs, but the fungus can also invade the silks of the developing ears
(Lillehoj et al., 1980). Most other nuts are also susceptible to the inoculation
(Pitt & Hocking, 1997). Cereals are a general substrate for growth of A. flavus
but, unlike nuts and oilseeds, small grain cereal spoilage by A. flavus is the
result of poor handling. Aflatoxin levels in small grains are rarely significant
but aflatoxin contamination of spices by A. flavus could be at high levels
(Lillehoj et al., 1980).
AFLATOXINS IN CROPS AND ITS DISTRIBUTION

IN DIFFERENT COUNTRIES
Aflatoxin is mainly a problem in maize because this crop is colonized in

the field depending on environmental conditions. Of the other grains, rice is an
important dietary source of aflatoxin with poor storage in tropical and
subtropical areas (Miller, 1995).
The character of the aflatoxin problem varies by region. In the US, the
issue is pre-harvest contamination of maize as the storage systems are good
(Payne, 1992). In tropical countries, such as Thailand and the Philippines,
storage of maize is an additional, substantial problem (Siriacha et al., 1991;
Quitco, 1991). As notable attention has been paid to the management of
aflatoxin in food/feed by systems which help to detect contamination and
perform better storage, it is generally agreed that a reduction in the levels of A.
flavus inoculum would help to reduce final aflatoxin contamination in storage
(Payne, 1992).
Extensive attention has been paid to the ecology of the aflatoxin-
producing fungi. In the US, Mexico and South America, A. flavus and A.
parasiticus infect corn, although A. parasiticus is relatively uncommon
(Payne, 1992). In corn-belt of the US, corn contamination with A. flavus

occurs in two fundamental ways: a) contamination of the silks with airborne
conidia, and b) colonisation of damaged kernels. In either case, drought-,
nutrient-, or temperature-stressed plants are more susceptible to colonisation
by A. flavus and accumulation of aflatoxin (Diener et al., 1987). In the US, A.
flavus conidia do not overwinter in soil, but sclerotia can survive for several
years in soil. A. flavus sclerotia in the plant debris provide a source of inocula
for the subsequent crop. At the time of silk emergence in the plant, the
survived sclerotia germinate and produce abundant conidia (Wicklow &
Wilson, 1986). This fungus also endures as mycelia in plant debris left in the
field during the winter. A. flavus has been isolated from maize silks in all
stages of plant growth, but principally when pollen is present. The fungus does
not enter the kernel from contaminated silks, but it acts as a source of
inoculum for the further colonisation of the base of the kernels (Payne, 1992;
Wicklow, 1994). A. flavus can then enter the kernels through the attachment
point of the kernel to the rachis. Once there, the fungus spreads throughout the
rachis entering additional kernels (Smart et al., 1990). While colonisation of
maize silk is a common phenomenon, such colonisation plays an important
role in contaminating insects associated with the developing ear not leading to
kernel colonisation in the field (Payne, 1992).
As an experiment, inoculated corn seeds with spores of A. flavus were
planted in a Missouri field. It was observed that compared to the control (non-
contaminated corn seeds), there was a reduction in the germination of seeds.
Furthermore, it was found that the infestation was systemic through the leaf,
stem and roots. However, some differences were found in the spread of
contamination among the tissues, suggesting that the fungus started to spread
through the meristem (Kelly & Wallin, 1987). The occurrence of A. flavus was
investigated in vegetative tissues of cotton plants. The contaminated mature
cotton bolls were collected with their subtending stems and peduncles. The
bolls were inoculated through the carpel wall 30 days after anthesis were
allowed to mature in the field. A high percentage (78 percent) of the naturally
contaminated cotton bolls with A. flavus in the seed had the fungus present in
the stem and peduncle whereas only 31 percent of these contaminated bolls
with no fungus in the seed had A. flavus in the stem. As this difference was
significant (p=0.001), a positive relationship between seed infection and
stem/peduncle infection was suggested. Moreover, it was found that all bolls
inoculated through the carpel wall had fungus in the seed but only 11 percent
of the stem sections were infected, indicating that the fungus did not grow
downward from the boll into the supporting stem. That result was supported
following further experiments on inoculated cotton seedling of cotyledonary

leaf scar. Fungal isolation from flower buds, developing bolls and stem in the
upper parts of the plant but not from roots or stem below the cotyledonary
node indicated that the fungus did not readily move downward through the
plant (Klich et al., 1986).
The ecology of A. flavus in subtropical regions of Asia is different from
that described above regarding crop contamination with Aspergillus in the US.
In Asia, A. parasiticus does not appear to occur in maize (Pitt et al., 1993;
Siriacha et al., 1991). As a result, there is an absence of aflatoxin G from
human samples collected in Asia (IARC, 1993). In the Philippines, maize
inoculated with A. flavus resulted in an infestation of 10% of the ears (Ilag,
1975). Such a high rate of infestation from conidial inoculation has not been
reported in the US in similar studies (Payne, 1992; Wicklow, 1989).
Aflatoxin accumulation in stored maize in subtropical Asia has risen
rapidly in post-harvest conditions (Kawashima et al., 1993; Quitco, 1991). In
contrast to the US corn-belt, A. flavus conidia are common in soil samples
from maize-growing regions of Thailand throughout the year, i.e. in both the
rainy and dry seasons. This important difference seems to lead to variation in
colonisation of different parts of the plants: in Thailand, some internal plant
tissues were contaminated by A. flavus from silking whereas in the US the
exterior of tassels and silks were always contaminated (Siriacha, 1991;
Siriacha et al., 1991).
In the studies carried out in Thailand, it was found that the silk internal
tissues were contaminated from silk emergence (Siriacha, 1991). This was also
reported in the US (Payne, 1992). However, as mentioned above, under the
conditions observed at the corn-belt, this contamination does not appear to be
the primary route for infestation of the developing kernels but rather acts as a
source for producing inoculum for further contamination. So this fungus
spreads to the adjacent kernels through cracks in the outer layers of the kernel
and through the inoculation of the wounds in rachis. Typically, in the US corn-
belt, these contaminations from infested kernels are distributed randomly in
the ears after wound inoculation. The contaminated kernels as the sites of
initial infection have very high aflatoxin contents (Smart et al., 1990). On the
other hand, in the Thailand studies, the contaminated kernels contained little
aflatoxin. Also, A. flavus was found commonly as an internal contaminant of
the husk. A. flavus infestation of silks, husks and kernels peaked as the tissues
senesced or developed to maturity in the absence of significant internal
contamination of the rachis by this fungus (Siriacha, 1991).
Table 1. Food contamination by aflatoxins in different countries
Countries Commodity Contamination rate

Australia Peanut 381 g/Kg
Straw/silage 17 g/Kg
Bangladesh Maize 33 g/Kg
Groundnut 65 g/Kg
Botswana Peanut To 64 g/Kg
Peanut butter 0.3-23 g/Kg
Brazil Corn 0.2-129 ppb
Peanuts and products 43-1099 ppb
China Corn 9-1396 ppb
Egypt Hazelnut 25-175 ppb
Soybean 5-35 ppb
Walnut 15-25 ppb
Gambia Ground nut source 162 ppb
Ghana Kernels 5.7-22168
India Pistachio nuts 15 to 259 g/Kg
Dry slices of quince 96 to 8164 g/Kg
Maize >30 ppb
Peanut 275 g/Kg
Malaysia Wheat flour 25.6-289 g/Kg
Peanut 1-378 g/Kg
Maize 106 g/Kg
Nepal Peanut, Corn flakes >30 ppb
Peanut butter, Vegetable
oli
Nigeria Yam chips 4-186 g/Kg
Pre harvest maize 3-138 g/Kg
Shelled melon 5-20 g/Kg
Corn and corn based 25-770 g/Kg
snacks
Philippines Rice bran and rice hull 0.27-11 g/Kg
Corn 130 g/Kg
Senegal Peanut 40 ppb
Sudan Peanut butter and peanut 87.4-197.3 g/Kg
Thailand Corn 73 g/Kg
Peanut oil 102 g/Kg
Turkey Red pepper 1.1-97.5 ppb
Adopted from: Farombi, 2006; Binder et al., 2007.
The occurrence of toxigenic fungi in medicinal plants in Brazil has already

been reported by several investigations. One investigation evaluated 91
samples of medicinal plants from 65 different species. A. flavus was the
dominant isolated species (58 isolates; 23.39%). Among these, 16-27.6% was
able to produce aflatoxin B1 or B1 and B2 (Bugno, et al., 2006). Aquino and
colleagues (2010) analysed the samples of plants including Boldo (Peumus
boldus), green tea (Camellia sinensis), Espinheira-Santa (Maytenus ilicifolia),
and Senna (Cassia angustifolia). Except for three samples of P. boldus and
two samples of C. sinensis, all the samples presented fungal contamination,
with 75% above the limit established by the World Health Organization for the
Total Fungal Count of 103 UFC/g (World Health Organization, 1998; Aquino
et al., 2010). The other survey identified eight A. flavus isolates in chamomile
(Matricaria recutita), two of which were aflatoxin producers (B1 and B2). The
total fungal count reached values over 105 UFC/g in these plants (Prado et al.,
2009). In Argentina, researchers detected 52% of the genus Aspergillus in 56
species of medicinal plants. A. flavus and A. parasiticus were the prevalent
species, 50% among the 40 aflatoxin-producer isolates (Rizzo, et al., 2004).
Exposure to aflatoxins differs between countries particularly due to
consumption of different diets. A summary of various commodities and
aflatoxin contamination rates in different countries is presented in Table 1
(Farombi, 2006; Binder et al., 2007).
ENVIRONMENTAL FACTORS IN AFLATOXIN OCCURRENCE

Food contamination with Aspergillus, and its produced aflatoxins in
general, are associated with warm and dry climates (Hell et al., 2003). Several
mathematical models on climatic risk of Aspergillus species growing and their
in situ production of aflatoxins have been published (Chauhan et al., 2008,
Pitt, 1993). However, it has also been reported that aflatoxin contamination
may considerably differ from one year to another year at the same location due
to variable environmental conditions in different growing seasons and also
diverse (inconstant) management practices (Hell et al., 2003). For instances,
growing crops consecutively in the same field increases the risk of toxin
contamination year after year (Hennigen et al., 2000).
The influence of temperature on the growth of A. flavus and A.
parasiticus, and their aflatoxin production has been studied in different
commodities using artificial media. In one study, the optimum temperature for
aflatoxin production by A. flavus was defined at 25C on ground nuts, while
the optimum temperature for A. parasiticus production of aflatoxin was 25-
30C. Also, this study showed a change in the proportions of aflatoxin B1 and
G1 produced by A. parasiticus, with a reduction in aflatoxin G1 as
temperatures increased (Diener & Davis, 1967). Molina and Giannuzzi (2002)
with using laboratory media and mathematical modelling found that optimum
temperatures for aflatoxin production by A. parasiticus were 27.8C and
27.3C at pH 5.9 and 5.5, respectively. The optimum temperature for aflatoxin
production by A. bombycis and A. nomius was 25C (Peterson et al., 2001).
The optimum water activity (aw) for growth of A. flavus is indicated as
0.996, with the minimum supporting growth at 0.80-0.82. At higher water
activities (0.98-0.99), aflatoxins are produced in greater amount but toxin
production apparently ceases at or near aw 0.85 (Gqaleni et al., 1997; Northolt
et al., 1977). It is also reported that more than 70% of high moisture grains
(>18%) are infected with A. flavus with a positive correlation between the rate
of infection and aflatoxin development. Toxin contamination is directly
correlated with the moisture content of crops (Mora & Lacey, 1997).
According to an investigation on medicinal plants, no aflatoxin was detected
with water activity below 0.81 and temperatures of 25 2C, 30 2C and 40
2C. Similar observation was made when the water activity was over 0.81
and temperature below 10 2C (Kulshrestha et al., 2008).
One study concluded that locations with both dry and hot climates have a
higher probability of aflatoxin risk compared with locations having either dry
or hot conditions alone (Gnonlonfin et al., 2013). On the other hand, other
studies have shown that the relations between climate and toxin development
are complex. Climate influences contamination partly by direct effects on the
causative fungi. As the climate changes, complex fungal communities develop.
This includes changes in quantity of aflatoxin-producers as well as the
alterations to fungal community structure. Fluctuations in climate also
influence predisposition of hosts to the insects that wound the plant. This
increases the case for fungal contamination (Chauhan et al., 2008; Setamou et
al., 1998; Hell et al., 2003; Cotty & Jaime-Garcia, 2007).
Favourable conditions of temperature and water activity are crucial for
mycotoxigenic fungi. In general, the countries with cool or temperate climates
may become more liable to aflatoxins when the temperature increases. An
example is Italy during recent years (FAO, 2000). In particular, tropical
countries may become too inhospitable for fungal growth and mycotoxin
production. Countries which afford to control the storage environment may be
able to avoid postharvest contamination but at high additional cost. The lack of
awareness about the link between food safety and climate change could be
problematic in terms of aflatoxin contamination especially in Africa

(Gnonlonfin et al., 2013).
Aflatoxin production is an aerobic process. Production of aflatoxin by A.
flavus cultures grown on a groundnuts medium in oxygene-depleted
atmosphere is lower than under normal conditions (Diener & Davis, 1967;
Dobson & Sweeney, 1998). A maximum yield of 212 mg of aflatoxin per litre
of fluid culture was produced at an aeration rate of 9 L/min while a
considerable reduction in aflatoxin occurred at lower aeration rates (Heathcote
& Hibber, 1978). Diener and Davis (1967) investigated the effects of different
levels of the normal atmospheric gases, carbon dioxide, oxygen and nitrogen
on aflatoxin production under conditions of varying temperature and humidity.
It was found that aflatoxin production in sound mature peanut kernels
decreased with increasing concentrations of carbon dioxide from 0.03% to
100%. Reducing the oxygen concentration decreased aflatoxin production.
Another study reported that a significant decrease in mycotoxin production
resulted when the oxygen was reduced from 5% to 1% regardless of the
carbon dioxide concentration, so that storage under reduced oxygen or in a
modified atmosphere could be one of the options to reduce aflatoxin
biosynthesis (Magan & Aldred, 2007).
AFLATOXIN GENE CLUSTER

Aflatoxins are polyketide synthases (PKS) -derived mycotoxins,
synthesized from a large cluster near one telomere of chromosome 3 of A.
flavus (C Figure 1, Amaike & keller, 2009) (secondary metabolite cluster 54 in
the SMURF chromosome map). Mapping of A. parasiticus and A. flavus
genomic DNA has established that the genes in the aflatoxin biosynthetic
pathway are clustered. In general, the aflatoxin gene cluster in A. parasiticus
and A. flavus consists of 25 genes, approximately 70 kb. This cluster is of
critical importance since its genetics has been a model for better understanding
of fungal secondary metabolism. So it has been subject to many reviews
(Georgianna and Payne, 2009). The aflatoxin cluster is conserved to varying
degrees in several fungi including A. parasiticus, A. ochraceoroseus,
Emericella astellata, A. flavus var. parvisclerotigenus, Aspergillus toxicarius,
A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis, Emericella
venezuelensis, A. rambellii, A. nidulans, A. oryzae, A. sojae, and related fungi
such as Dothistroma. However, only some Aspergillus species produce
aflatoxin (Amaike & Keller, 2011). The specific pathway regulatory gene,
aflR, is located in the cluster. A Zn(II)2Cys6 type transcriptional factor

encoded by aflR regulates expression of the aflatoxin/sterigmatocystin
biosynthetic genes. Over-expressed aflR results in increasing aflatoxin
production and up-regulates other biosynthetic genes in the aflatoxin
biosynthesis pathway. While, in aflR deletants, aflatoxin/sterigmatocystin
biosynthetic genes, and their products, are not expressed. AflR is able to bind
the consensus motif 5-TCGN5CGR-3 found in the promoter regions of many
aflatoxin and sterigmatocystin genes (Fernandes et al., 1998). A second
binding site 5- TTAGGCCTAA is reported as important in autoregulation of
aflR transcript in A. flavus and A. parasiticus (Chang et al., 1995). Expressed
divergently from aflR is aflS (formerly termed aflJ), whose product also
regulates aflatoxin production through binding and activating AflR in A.
parasiticus and A. flavus and presumably other Aspergilli (Du et al., 2007).
tools that enable rapid genetic analysis of individual genes within the genome.
Particularly, the genetics of aflatoxin synthesis is regarded as a model for
better understanding of fungal secondary metabolism through its role in the
identification of the secondary metabolite clusters in chromatin regulation of
such clusters through histone modifications (Amaike & Keller, 2009).
REGULATION OF AFLATOXIN BIOSYNTHESIS

Aflatoxin represents a classic polyketide produced by species of
Aspergillus (Duran et al., 2007). So aflatoxins are synthesized by polyketide
metabolic pathway. As noted above, aflR located in the cluster is the specific
pathway regulatory gene and encodes a zincfinger DNA-binding protein which
is required for transcriptional activation of most of the aflatoxin structural
genes (Bhatnagar et al., 2003; Yu et al., 2004a).
Well-designed research on production of the aflatoxin precursor
sterigmatocystin with the genetic model A. nidulans, has contributed greatly to
our understanding of the aflatoxin pathway and the global regulatory
mechanisms (Georgianna & Pyne, 2009). In some fungi distantly related to A.
flavus and A. parasiticus, sterigmatocystin is the final metabolite rather than a
precursor for aflatoxin. The biosynthetic and regulatory genes of
sterigmatocystin production in A. nidulans are clustered and also homologous
to those genes of aflatoxin production in A. flavus and A. parasiticus.
However, the organization of the genes in the A. nidulans cluster is different
from that in A. parasiticus and A. flavus (C Figure 1, Yu et al., 2004a) (Yu et

al., 2004a).
The biosynthesis of aflatoxin has many levels of regulation. Some of them
are almost specific to the pathway while others present a more global
regulation of secondary metabolism. Also many environmental factors control
aflatoxin biosynthesis, including light (Calvo et al., 2002), carbon source,
temperature, and pH (OBrian et al., 2007; Price et al., 2005). The
physiological state of the fungus is also a factor that affects aflatoxin
biosynthesis. It is predictable that many of the factors which regulate the
biosynthesis of aflatoxin, also regulate the synthesis of other secondary
metabolites such as proteins VeA and LaeA (Duran et al., 2007).
Two genes, aflR and aflS are in the aflatoxin cluster and they are located
divergently adjacent to each other. These genes are involved in the regulation
of aflatoxin/ sterigmatocystin gene expression (Chang et al., 1993; Price et al.,
2006). Despite clear differences in the sequence of AflR between A. nidulans
and A. flavus, its function is conserved. AflR from A. flavus is able to regulate
expression of the sterigmatocystin cluster in an A. nidulans which is an aflR
deletion strain (Yu et al., 1996). The genes aflS and aflR are divergently
transcribed, but have independent promoters. Although, the intergenic region
between them is short, they may share binding sites for transcription factors or
other regulatory elements (Ehrlich & Cotty, 2002). The exact role of AflS in
aflatoxin biosynthesis is not clear (Georgianna & Payne, 2009).
According to some studies, AflR is sufficient to initiate gene transcription
of early, mid, and late genes in the pathway, and that AflS enhances the
transcription of early and mid aflatoxin pathway genes. It has been suggested
that AflSs roles are as diverse as assisting in transport of pathway
intermediates to the interaction of AflS with AflR for altered aflatoxin
pathway transcription. According to the observation of binding AflS to AflR,
it has been indicated that AflS modulates aflatoxin expression through its
interaction with AflR (Chang, 2003).
Although the role for aflatoxin in the ecology of the fungus is not known,
biosynthesis of this mycotoxin is tightly regulated by environmental and
developmental cues. The complete signalling network for processes regulating
aflatoxin biosynthesis is unclear, but components of this network have been
distinguished (Yu & Keller, 2005). Several environmental and cultural
conditions modulate aflatoxin biosynthesis including light, temperature, pH,
nitrogen, carbon source and metals (Calvo et al., 2004; Luchese & Harrigan,
1993). As it is important to determine the role of aflatoxin in the ecology of
the producing organism, an understanding of how these factors impact
aflatoxin biosynthesis is critical and it may identify target sites for control of
aflatoxin formation. Unfortunately, the regulatory networks involved in
sensing and transmitting environmental and nutritional stimuli are not well
understood (Georgianna & Payne, 2009). One study has examined the effect of
four cultural and environmental conditions on gene transcription in the
aflatoxin pathway. It has been found that temperature have the most profound
effect followed by pH, nitrogen source, and then carbon source (Price et al.,
2005). Other researcher surveyed temperature and water activity in relation to
secondary metabolism genes in several fungal species, including the aflatoxin
cluster in A. parasiticus. Under suboptimal growth conditions, intermediate
environmental stress to the organism was most favourable for production of
mycotoxins (Schmidt-Heydt et al., 2008). Calvo and colleges (2004) indicated
that light affects the transcription of several genes, including aflatoxin gene
cluster and genes putatively involved in the development of sclerotia in A.
flavus (Calvo et al., 2004).
GLOBAL SECONDARY METABOLITES REGULATORS

As mentioned in the previous section, the biosynthesis of aflatoxin
involves many levels of regulation. Some are almost specific to the aflatoxin
biosynthesis pathway like aflR and aflS regulatory genes, whereas others
present a more global regulation of secondary metabolism including LaeA.
While investigating the expression of deficient aflR, a novel protein termed
LaeA (Bok & Keller, 2004) was detected in A. nidulans. In this study, a
mutagenesis screen was used to identify sterigmatocystin production mutants
(Butchko et al., 1999). Therefore, LaeA has been shown to be a global
regulator of secondary metabolism in Aspergilli and other filamentous fungi,
including Penicillium spp. and Fusarium fujikuroi. LaeA is a member of a
heterotrimeric nuclear complex, termed the Velvet complex, with two other
proteins, VeA and VelB. The Velvet complex is also conserved in filamentous
fungi (Yu et al., 2008; Xing et al., 2010). LaeA (and possibly the Velvet
complex) is hypothesized to activate secondary metabolite clusters through
histone modifications. Consequently, loss of laeA lead to accumulation of
heterochromatic marks in the sterigmatocystin gene cluster (Reyes-Dominguez
et al., 2010).
Moreover, Velvet complex is appeared as a transcriptional complex
regulating both sporulation and secondary metabolism in A. nidulans. Also
proteins LaeA and VeA are important in A. flavus colonisation and aflatoxin
contamination of seed crops. Both proteins are required for virulence. Null
mutants produce fewer conidia and less aflatoxin in seed. In addition, these
mutants are impaired in lipid degradation of host cells (Amaike & Keller,
2009; Bayrum et al., 2008).
LaeA, encodes a putative methyltransferase that affects the expression of
secondary metabolite genes in different clusters including aflatoxins,
sterigmatocystin, penicillin, emericellamide, terrequinone, gliotoxin, and
lovastatin (Bok & Keller, 2004). Well conserved LaeA in numerous fungi has
suggested that it has significant evolutionary functions in fungal physiology
(Chang et al., 2012). How LaeA regulates the expression of secondary
metabolite biosynthesis genes is still not well understood. LaeA was known as
a protein that was thought to be crucial for expression of aflR, the gene
encodes the transcriptional regulator of genes in the sterigmatocystin cluster in
A. nidulans (Bok & Keller, 2004) and the aflatoxin cluster in A. flavus (Kale et
al., 2008). In another investigation to find further characterization of LaeAs
function, A. flavus laeA deletion strains have been able to express low levels of
aflR, but are unable to produce aflatoxins, although they have produced small
amounts of an early precursor metabolite such as noranthrone (Chang et al.,
2012).
GENES RELATED TO ASPERGILLUS-HOST CROSSTALK

According to the recent studies, fungal pathogenesis is related to lipid-
mediated fungal-host crosstalk. As previously described, A. flavus
preferentially colonizes oilseed crops. These crops, as well as this fungus and
other Aspergilli, contain high levels of the unsaturated fatty acids including
linoleic (18:2) and oleic acid (18:1). These fatty acids are substrates for
oxygenases. Oxylipins, derived from oxygenases, are a class of oxygenated,
unsaturated fatty acids involved in signalling pathways in different kingdoms
such as fungi (filamentous fungi, yeasts and oomycetes), plants and animals.
Considerable attention has been paid to the fungal oxylipins where one fungal
oxylipins, a precocious sexual inducer (psi factor), was discovered as
extracellular signals to regulate asexual and sexual spore development
(Champe et al., 1987). Oxylipins are encoded by ppo (psi producing
oxygenase in fungi) and lox (lipoxygenases of plants, animals, and fungi),
could regulate sclerotia and conidia production and secondary metabolism
such as aflatoxin in A. flavus (Calvo et al., 1999; Tsitsigiannis et al., 2004).
Further studies confirm a global regulatory role for ppo genes in natural
product biosynthesis. For example, long chain unsaturated fatty acid mutants
in A. nidulans and the field pathogens A. parasiticus and A. flavus with
oxylipin defects negatively affected sterigmatocystin and aflatoxin production
at the level of gene regulation (Maggio-Hall et al., 2005), suggesting that
secondary metabolism may be controlled by Ppo-derived oxylipins at the
transcription level (Christensen and Kolomiets, 2011).
A. flavus contains four dioxygenases including PpoA, PpoB, PpoC, and
PpoD, as well as one lipoxygenase such as LoxA (Horowitz et al., 2009). The
fungal oxylipin structure has similarities to plant and mammalian oxylipins.
This resemblance has partly explained the oxylipin driven cross-signalling
observed in Aspergillus-host (Tsitsigiannis et al., 2004; Brodhagen et al.,
2008). It has been found that A. flavus ppoA and ppoC mutants produce less
conidia but more sclerotia, whereas the ppoD mutant shows the opposite
phenotype (Horowitz et al., 2009). A knockdown mutant of all four
dioxygenases and lox represented high levels of aflatoxin and sclerotial
production (Tsitsigiannis et al., 2004). Intercellular communications with
regards to the lox and ppo expression in both plants and Aspergilli have not
revealed an exact role for any oxylipin in cross-kingdom communication but
have shown the importance of this entire system in the Aspergillus-host
interaction (Table 2) (Amaike & Keller, 2011).
Table 2. Synopsis of major virulent factors in A. flavus
Fungal morphology
Gene Conidiaa Sclerotiab Aflatoxin
ppoA NA NA NA
ppoB Decreased Increased Slight increase
ppoC Increased Decreased Decrease on seed
lox Decreased Increased Slight increase
ppoA/B/D (IRT2) Decrease NA Slight increase
ppoA/B/C/D Decreased Increased Not done
ppoA/B/D/lox Decreased Increased Not done
ppoA/B/C/D/lox (IRT4) Decreased Increased Increased
veA Decreased No production Decreased
laeA Increased No production Decreased
a,b
The data indicate the relative differences in conidial, sclerotial, and aflatoxin
production compared to the wild type, NRRL3357.
Adopted from Amaike & Keller, 2011.
QUORUM SENSING MOLECULES AND SECONDARY

METABOLITE PRODUCTION IN ASPERGILLUS
Signalling mechanisms that control physiological and morphological
responses based on cell density are called quorum sensing. This phenomenon
is very common in bacteria and has also been reported in fungi, especially
yeasts. Among filamentous fungi, Aspergillus uses quorum regulation to affect
population dependant behaviour such as morphogenesis and secondary
metabolite production (Brown et al., 2009).
Several studies report the effect of certain types of quorum sensing
molecules, secreted from one group of fungi, on the growth, morphology,
sporulation (conidiation), apoptosis and metabolite production of the other
fungi. Oxylipins constitute a large family of oxidized fatty acids and
metabolites derived from them (Brodhun & Feussner, 2011). Linoleic acid-
derived oxylipins have been reported to be engaged in the quorum sensing
responses in Aspergillus cultures. A recent investigation on the impact of
linoleic acid on lovastatin (cholesterol-lowering drug) production in A. terreus
revealed that production of lovastatin was enhanced by up to 1.8-fold (Flavia
et al., 2010). Another study on A. terreus has shown the novel role for
butyrolacton I as a quorum sensing molecule. This molecule is a growth
phase-specific inducer of lovastatin production (Raina et al., 2012). In A.
nidulans, oxylipins are involved in cell density-dependent production of
asexual and sexual spores, as well as secondary metabolites such as penicillin
(Tsitsigiannis et al., 2005). Also, -heptalactone is a quorum sensing molecule
in A. nidulans that regulate growth and secondary metabolite (penicillin)
production (Williams et al., 2012). Moreover, in A. flavus the switch from
conidium to sclerotium and the production of aflatoxin are cell density
dependent and related to oxylipins such as linoleic, oleic and linolenic acid
(Brown et al., 2008 and 2009). Curiously, instances of small molecule
exchange between bacteria and eukaryotes have also been reported (Mullard,
2009).
According to Brown et al., (2009), the deletion of lox gene, Aflox, reduced
density-dependent development of both sclerotia and conidia significantly. It
has been shown that the lox mutant results in increasing the number of
sclerotia and decreasing the number of conidia at high cell density. All these
evidences indicate that LOX-derived metabolism may be crucial for shifting
sclerotia to conidia in a density dependent morphology. It has been shown that
at high population density, the PpoA, PpoC and Aflox products suppress
sclerotia formation but induce conidiation. At low population density, products

of PpoD apparently cause sclerotia production while they inhibit conidia
formation. All things considered, these result suggested that Ppo-and LOX-
derived oxylipins govern sexual and asexual reproduction, harmonize a
quorum sensing process and modulate density-dependent sporulation. The
survey on ppo mutants of A. flavus at high cell density illustrated the
suppression of aflatoxin production due to the ppoA, ppoC and lox mutants
effects. In the IRT4 strain (reduced in expression of all four Ppo and LOX),
there was a significant rise in aflatoxin production through all the cell density.
So the population density regulation was entirely lost. These findings
demonstrated the importance of oxygenase activity for density dependent
aflatoxin production and governing fungal-seed interactions (Brown et al.,
2009).
CONTROL OF AFLATOXIN OCCURRENCE

AND FUTURE DIRECTIONS
Aflatoxin contamination of crops remains a critical problem worldwide

with additional health threats in increasing numbers of A. flavusinduced
aspergillosis. Pre-harvest control of A. flavus has traditionally depended on
determining resistant crop-lines to help little protection under environmental
conditions (e.g., drought) which are favourable to aflatoxin contamination
(Campbell & White, 1995). In addition, irrigation is a key to avoid drought
stress (Payne, 1998). Efforts have also focused on identifying plant proteins
that are important for defence against A. flavus invasion, including pathogen-
related and drought-resistant proteins (Chen et al., 2010). Likewise, effort has
been focused on specific molecules like oxylipins which are as the
oxygenated, unsaturated fatty acids involved in signalling pathways in
different kingdoms such as fungi, plants and animals. These molecules could
regulate sclerotia and conidia production and secondary metabolism such as
aflatoxin in A. flavus. They may play important role in Aspergillus host
interaction which could apply to control aflatoxin production as a chemical
agent or its relevant gene to make a resistant transplant. Further identification
of the quorum sensing molecules and their relevant genes that are responsible
for interactions between bacterial cells, unicellular and mycelial fungal cells or
inter-kingdom communications (bacteria and fungi /plant and fungi) would be
of critical importance in future control strategies. Moreover, the understanding
of this fungus biology has progressed with the advent of the genome sequence
and improved molecular tools allowing rapid genetic analysis of individual
genes within the genome as well as specific regulators.
Other available control methods, such as optimal cultural practices (date
of planting and harvesting, choosing the cultivar as well as a suitable region)
have reduced but have not eliminated pre-harvest aflatoxin contamination in
susceptible crops. Furthermore, in recent years public concern over pesticide
residues in the environment, food and feed, has led to a limitation and
reduction of availability of some chemical fungicides commonly used to
control plant pathogens and post-harvest diseases. Consequently, alternative
methods for controlling these pathogens and diseases are needed. Therefore,
biological control or use of microbial fungicides may be an alternative strategy
to chemical fungicides. Identification of new antifungal, quorum sensing
peptide molecules from antagonistic bacteria like Bacilli, against A. flavus has
been investigated. This ongoing survey could lead to the development of
biotechnological strategies. These strategies would facilitate aflatoxin
contamination control as well as genetic engineering of plant resistance to
fungal invasion through the use of genes related to the bacterial antifungal
peptide molecules.
All these novel knowledge will contribute to the development of inhibitors
of aflatoxin, design of the bio-competitive Aspergillus strains, application of
biocontrol bacterial strains and improvement in host- resistance against fungal
invasion or toxin production.
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Chapter 3
AFLATOXINS IN FOOD AND FEED:

CONTAMINATION EXPOSURE,
TOXICOLOGY AND CONTROL
Marta Herrera, Antonio Herrera and Agustn Ario*

University of Zaragoza, Department of Animal Production
and Food Science, Veterinary Faculty, Zaragoza, Spain
ABSTRACT
Aflatoxins (AFs) are secondary metabolites produced by various
fungal species of the genus Aspergillus such as Aspergillus flavus and
Aspergillus parasiticus. The most important compounds are aflatoxins
B1, B2, G1 and G2, as well as two metabolic products secreted in milk,
M1 and M2.
The worldwide occurrence of aflatoxins contamination in raw
agricultural products has been well documented; such contamination
occurs in a variety of food and feed, such as cereals, nuts, dried fruits,
spices and also in milk as a consequence of the ingestion of contaminated
feed. However, pistachios, peanuts and corn are the most frequently
contaminated food items reported in the Rapid Alert System for Food and
Feed (RASFF) of the European Union. The occurrence of aflatoxins is
mainly affected by environmental factors such as climatic conditions,
*
Contact: aarino@unizar.es (A. Ario), University of Zaragoza, Department of Animal
Production and Food Science, Veterinary Faculty. c/Miguel Servet 177, 50013 Zaragoza,
Spain.
64 Marta Herrera, Antonio Herrera and Agustn Ario
geographic location, agricultural practices, and susceptibility of the

products to fungal growth during harvest, storage and processing. High
contamination levels of aflatoxins are mainly associated with post-harvest
growth of Aspergillus moulds in poorly stored commodities.
Aflatoxins can cause adverse effects to the health of animals and
humans. These toxins have been reported to be associated with acute liver
damage, liver cirrhosis, induction of tumors and teratogenic effects.
Aflatoxin B1 (AFB1) is usually predominant and the most toxic among
aflatoxins because it is responsible for hepatocarcinoma in animals and
strongly associated with the incidence of liver cancer in humans. AFB1 is
a genotoxic and mutagenic chemical, and it has been classified by the
International Agency of Research on Cancer (IARC) as human
carcinogen (group 1). The toxic effects of the ingestion of aflatoxins in
both humans and animals depend on several factors including intake
levels, duration of exposure, metabolism and defense mechanisms, and
individual susceptibility. Aflatoxins affect not only the health of humans
and animals but also the economics of agriculture and food.
Because of the multiple adverse health effects to humans and animals
caused by aflatoxin consumption, many nations worldwide have
regulatory standards on aflatoxin in food and feed. The European Union
(EU) regulation on aflatoxins in foodstuffs is among the strictest in the
world (Commission Regulation (EC) n 1881/2006 and successive
amendments). Maximum contents of aflatoxins in feeds are also
established by Commission Regulation (EU) n 574/2011 on undesirable
substances in animal feed.
Throughout the world there are many advisory bodies concerned
with food safety, including the World Health Organization (WHO), the
Food and Agriculture Organization of the United Nations (FAO), the
Codex Alimentarius Joint Expert Committee for Food Additives and
Contaminants (JECFA), and many others, which regularly assess the risk
from mycotoxins, advise on controls to reduce consumer exposure and
establish different regulations for these toxins in different countries.
1. INTRODUCTION
Aflatoxins (AFs) are secondary metabolites produced by various fungal
species of the genus Aspergillus, and have the highest toxicity among
mycotoxins due to its genotoxic, mutagenic and carcinogenic properties.
Chemically, these toxins are difuranocoumarin derivatives (Figure 1) produced
primarily by two species of Aspergillus fungus which are especially found in
areas with hot and humid climates. A. flavus is ubiquitous, favouring the aerial
parts of plants (leaves, flowers) and produces only B aflatoxins (aflatoxin B1
Aflatoxins in Food and Feed 65
and B2). A. parasiticus produces both B and G aflatoxins (AFB1, AFB2,

AFG1 and AFG2) and it is more adapted to a soil environment and has more
limited distribution. The highest risk of aflatoxin contamination is due to the
more frequent growth of A. flavus (Pittet, 1998). Other Aspergillus species
such us A. bombycis, A. ochraceoroseus, A. nomius, A. pseudotamarii, A.
tamarii, A. foetidus and A. oryzae are known to produce aflatoxins but their
toxicological significance is low (Goto et al., 1996; Klich et al., 2000; Peterson
et al., 2001; Rodrguez et al., 2012). Aflatoxins M1 and M2 are the
hydroxylated metabolites of aflatoxin B1 and B2, respectively, and may be
found in milk and milk products obtained from livestock that have ingested
contaminated feed (EFSA, 2007).
Figure 1. Chemical structures of the B and G aflatoxins and of aflatoxin M1 (EFSA,

2007).
These mycotoxins are found in food as a result of fungal contamination

both pre and postharvest, with the rate and degree of contamination dependent
on various factors such as temperature, humidity (and water activity), substrate
and storage conditions. Aflatoxins have been found in a variety of agricultural

commodities, but the most pronounced contamination has been encountered in
cereals such as corn and barley, nuts and oilseeds such as peanuts and
pistachios, dried fruits such as figs as well as spices. The main sources of
aflatoxins in feed are peanut, maize and cottonseed meal.
Exposure to aflatoxins in European countries is generally considered to
occur mainly from imported materials, but it is currently uncertain whether
future changes in climate would lead to increased aflatoxin contamination in
domestic food chain.
2. AFLATOXIN CONTAMINATION IN FOOD AND FEED

Aflatoxins were first identified in 1961 in animal feed responsible for the
deaths of 100,000 turkeys in the United Kingdom (Sargeant et al., 1961), also
affecting ducklings and young pheasants among other animal species. The
occurrence and production of aflatoxins differ geographically and climatically
as in years. Aflatoxin contamination can generally be sourced from countries
of origin with hot climates, poor hygienic conditions, mould growth and poor
storage conditions (Trucksess and Scott, 2008).
Aspergillus contamination and subsequent aflatoxin production can
happen in crops themselves, as with peanuts, sometimes assisted by insect
action, or it can occur during transport or storage as for example in cereals
(ICMSF, 1996). Aflatoxin contamination is also promoted by stress or damage
to the crop due to drought prior to harvest, insect activity, poor timing of
harvest, heavy rains at harvest and post-harvest, and inadequate drying of the
crop before storage. Humidity, temperature, and aeration during drying and
storage are also important factors.
The toxin can persist in food, even when the mould has disappeared. In
addition, the fact that these toxins have a great thermal stability is also a key
factor, enabling them to remain in some cooked foods, and meaning that
freezing has very little effect on their presence in foods.
The biosynthesis of aflatoxins in food depends on several environmental
factors (Table 1), such as water activity, temperature, pH, redox potential and
microbial competition.
Aflatoxins production can be found in a wide range of substrates due to
non-visible spoilage in the field (pre-harvest), storage or processing (post-
harvest). However, high contamination levels are mainly associated with post-
harvest production by Aspergillus moulds in poorly stored commodities. These
fungi grow and produce toxins during storage and are mainly influenced by
factors related to inadequate moisture and temperature, combined with long
storage in warehouses, which are conductive situations that can originate
potential toxigenic outbreaks (Dilkin, 2002). The most important factors that
help to predict the occurrence of aflatoxins in foodstuffs include weather
conditions (temperature, atmospheric humidity, drought), agronomical
practices in the field (crop rotation, crop residues removal, soil cultivation)
and internal factors of the food chain (drying and storage conditions).
Table 1. Limits of mould growth and aflatoxin production

by A. flavus and A. parasiticus (ICMSF, 1996)
Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus

Parameter
flavus parasiticus flavus parasiticus flavus parasiticus
GROWTH Minimum Optimum Maximum
Temperature
10-12 12 33 32 43 42
(C)
Water activity 0.8 0.80-0.83 0.98 0.99 >0.99 >0.99
pH 2 2 5-8 5-8 >11 >11
AFLATOXIN
Minimum Optimum Maximum
PRODUCTION
Temperature
13 12 16-31 25 31-37 40
(C)
Water activity 0.82 0.86-0.87 0.95-0.99 0.95 >0.99 >0.99
pH - 2 - 6 - >8
Different studies developed in the European Union have reached a

consensus on the most important indicators for the risk of aflatoxins, based on
three stages in the food production chain. For cultivation stage the selected
indicators are: relative humidity, temperature, crop rotation, tillage practices
and water activity of seeds. For transportation and storage the following
factors are included: water activity, relative humidity, ventilation, temperature,
storage capacity and logistics and for the processing stage the indicators are
the fraction of grain used, the water activity of grains, implanted traceability
and system quality (Park and Bos, 2007).
Food legislation in the EU demands food businesses to be responsible for
the safety of the food and feed they sell, and not to place on the market unsafe
(including mycotoxin-contaminated) feed or food. These businesses are also
required to identify and review the risks associated with mycotoxins, and,
where practicable, put in place processes and controls to reduce these risks.
Since a zero-tolerance for mycotoxins in foods and feeds is not possible,
legislation has been set in terms of maximum contents for specific mycotoxins
in certain food and feed. These levels are set for mycotoxins with the greatest
health concern and are based on scientific advice. The aim of maximum
contents is to minimize human exposure and the risks of both acute and long-
term adverse health effects and to support international trade (Hwang et al.,
2004).
Table 2. Maximum contents of aflatoxins in foodstuffs

in the European Union
Foodstuffs Maximum levels (g/kg)

2.1. Aflatoxins B1 Sum of B1, M1
B2, G1 and
G2
2.1.1. Groundnuts (peanuts) and other oilseeds , to be 8,0 15,0
subjected to sorting, or other physical treatment,
before human consumption or use as an ingredient
in foodstuffs, with the exception of: groundnuts
(peanuts) and other oilseeds for crushing for refined
vegetable oil production
2.1.2. Almonds, pistachios and apricot kernels to be 12,0 15,0
subjected to sorting, or other physical treatment,
before human consumption or use as an
ingredient in foodstuffs
2.1.3. Hazelnuts and Brazil nuts, to be subjected to 8,0 15,0
sorting, or other physical treatment, before human
consumption or use as an ingredient in foodstuffs
2.1.4. Tree nuts, other than the tree nuts listed in 2.1.2 5,0 10,0
and 2.1.3, to be subjected to sorting, or other
physical treatment, before human consumption or
use as an ingredient in foodstuffs
2.1.5. Groundnuts (peanuts) and other oilseeds and 2,0 4,0
processed products thereof, intended for direct
human consumption or use as an ingredient in
foodstuffs, with the exception of: crude
vegetable oils destined for refining refined
vegetable oils
2.1.6. Almonds, pistachios and apricot kernels, intended 8,0 10,0
for direct human consumption or use as an
2.1.7. Hazelnuts and Brazil nuts, intended for direct 5,0 10,0
human consumption or use as an ingredient in
foodstuffs
2.1.8. Tree nuts, other than the tree nuts listed in 2.1.6 2,0 4,0
and 2.1.7, and processed products thereof, intended
for direct human consumption or use as an
Foodstuffs Maximum levels (g/kg)

2.1.9. Dried fruit, other than dried figs, to be subjected to 5,0 10,0
sorting, or other physical treatment, before human
Dried fruit, other than dried figs, and processed 2,0 4,0
2.1.10. products thereof, intended for direct human
All cereals and all products derived from cereals, 2,0 4,0
2.1.11. including processed cereal products, with the
exception of foodstuffs listed in 2.1.12, 2.1.15 and
2.1.17
Maize and rice to be subjected to sorting or other 5,0 10,0
2.1.12. physical treatment before human consumption or
use as an ingredient in foodstuffs
Raw milk, heat-treated milk and milk for the 0,05
2.1.13. manufacture of milk-based products
Following species of spices: 5,0 10,0
2.1.14. Capsicum spp. (dried fruits thereof, whole or
ground, including chillies, chilli powder, cayenne
and paprika)
Piper spp. (fruits thereof, including white and black
pepper)
Myristica fragrans (nutmeg)
Zingiber officinale (ginger)
Curcuma longa (turmeric)
Mixtures of spices containing one or more of the
above- mentioned spices
Processed cereal-based foods and baby foods for 0,10
2.1.15. infants and young children
Infant formulae and follow-on formulae, including 0,025
2.1.16. infant milk and follow-on milk
Dietary foods for special medical purposes 0,10 0,025
2.1.17. intended specifically for infants
Dried figs 6,0 10,0
2.1.18.
The foods most susceptible to aflatoxin contamination, and which are at

greater risk of exposure are included in Table 2 from Regulation (EC) n
1881/2006 (corn, rice, cereals in general, almonds, Brazil nuts, nutmeg,
hazelnuts, pistachios, peanuts and other oilseeds, dried fruits such as raisins or
figs, spices like paprika, nutmeg, turmeric or ginger). In the European Union,
maximum levels of aflatoxin B1, aflatoxin M1, and for the sum of aflatoxins
B1, B2, G1 and G2 in foodstuffs are laid down in above mentioned
Commission Regulation (EC) n 1881/2006, as amended by Commission
Regulation (EU) n 165/2010 and Commission Regulation (EU) n 1058/2012.
These maximum limits were established for certain food commodities based
on the principle of as low as reasonably achievable (ALARA) (EFSA, 2009).
By other hand, aflatoxin contamination in feeds is regulated by the
Commission Regulation n 574/2011 of 16 June 2011 on undesirable
substances in animal feed. This regulation states that the maximum content of
AFB1, related to a feeding stuff with a moisture content of 12%, varies from 5
to 20 g/kg .
The incidence of aflatoxins in domestic and imported food stuffs in the
European Union can be assessed using the data reported by the RASFF (Rapid
Alert System for Food and Feed). Mycotoxins, and especially aflatoxins, were
the hazardous category with the highest number of notifications in
commodities within EU in 2012 and in previous years as well (Table 3).
Table 3. RASFF notifications on mycotoxins in food and feed in the period

2003-2012 (RASFF, 2012)
Hazard 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Aflatoxins 762 839 946 801 705 902 638 649 585 484
Deoxynivalenol 10 4 3 2 11 4
Fumonisins 15 14 2 15 9 2 1 3 4 4
Ochratoxin A 26 27 42 54 30 20 27 34 35 32
Patulin 6 7 3
Zearalenone 1 6 2
Total
803 880 996 878 760 933 669 688 635 525
mycotoxins
The decrease in aflatoxins notification in 2012 can be explained by the

effectiveness of preventive measures and reinforced control of aflatoxins in
certain products such as pistachios and almonds from certain countries. The
most frequently affected food category was nuts, nut products and seeds with
more than 200 notifications in 2012 (Table 4).
In surveys and monitoring programs that have been carried out in several
countries attempting to obtain a general pattern of the extent of aflatoxin
contamination in nuts, pistachios showed the highest contamination incidence.
As a result, this situation not only causes uncertainty in consumer for buying
pistachios, but also leads to additional cost in the production and loss of
income for producers, distributors and other stakeholders.
The European Commission in order to control the presence of aflatoxins
in foodstuffs from different countries, published several regulations in 2009 as
regards the increased level of official controls on imports of certain feed and
food and imposes an increased frequency of controls and special conditions at
import on products from certain countries because of the presence of
aflatoxins (RASFF, 2012).
An extensive review of the levels of aflatoxins encountered in
commodities in North America, South America, Europe, Asia and Africa was
included in an early IARC monograph (IARC, 1993). Many years of research
have generated a great number of publications concerning aflatoxin
contamination in various products such as peanuts (Ding et al., 2012),
pistachios (Ario et al., 2009), chestnuts (Pietri et al., 2012), pepper (Set and
Erkmen, 2010), paprika (Shundo et al., 2009), corn (Wagacha and Muthomi,
2008) and chilli (Russell and Paterson, 2007) among others. In most surveys
and monitoring programs that have been carried out in several countries
attempting to obtain a general pattern of the extent of aflatoxin contamination
in foodstuffs, peanuts and pistachios have shown the highest contamination
incidence (Georgiadou et al., 2012).
Thus, aatoxin contamination of peanuts can occur in the field (pre-
harvest) when severe late-season drought stress occurs and during storage
(post-harvest) when improper conditions of moisture and temperature exist
(Cole et al., 1995; FAO, 2000). In China, during 2009, 1040 peanut samples
were analyzed and the incidence was 25%, one of them was contaminated with
720 g/kg of total aflatoxins (Ding et al., 2012). A survey carried out in Kenya
showed that 37% of the peanut samples exceeded the 10 g/kg regulatory limit
for aatoxin levels. Raw peanuts had the lowest levels of aatoxin, with 96%
having levels of less than 4 g/kg and only 4% having more than 10 g/kg.
The most aatoxin-contaminated products were peanut butter and spoilt
peanuts, with 69% and 75% respectively, exceeding 10 g/kg (Mutegi et al.,
2013). Aflatoxin levels of about 30 times higher than the legal limits (10
g/kg) have been reported in peanut butter given to school children in Eastern
Cape, South Africa (Wagacha and Muthomi, 2008).
Likewise, moulds of the genus Aspergillus frequently decay the kernel of
pistachio nuts. Pistachio nuts are among the commodities with the highest risk
of aflatoxin contamination due to more frequent growth of A. avus (Pittet,
1998; Freire et al., 2000). The serious problems occurring during post-harvest
handling and storage of pistachio nuts are mould spore contamination and
aflatoxins production which results in serious health hazards and economical
losses. Natural occurrence of aatoxins in pistachio nuts has been studied in
various countries. According to a report from Mexico, 2.2% of pistachio nut
samples showed aflatoxin contents higher than 20 g/kg (JECFA, 1998). In
Sweden, 9.5% pistachio nut samples contained AFB1 higher than 2 g/kg
(Thuvander et al., 2001). In the Netherlands, AFB1 was found in 17 of 29
pistachio nut samples with contamination levels ranging from 0.8 to 165 g/kg
(Scholten and Spanjer, 1996) and in Spain 50% of bulk pistachio nuts were
contaminated with AFB1 (Ario et al., 2009).
Table 4. RASFF notifications on mycotoxins by product category in 2012

(RASFF, 2012)
Product Aflatoxins Deoxynivalenol Fumonisins Ochratoxin A Zearalenone

category
Cereals and 17 4 4 6 3
bakery
products
Confectionery 7 1
Feed 79
Fruits and 137 19 1
vegetables
Herbs and 33 4
spices
Milk and milk 5
products
Nuts, nut 204

products ad
seeds
Prepared 2 2
dishes and
snacks
Total 484 4 4 32 4
A similar percentage of 50.5% was found for total aflatoxins and AFB1 in
95 samples of unpacked pistachio nuts with the contamination levels ranging
from 0.007 to 7.72 g/kg in Turkey (Set and Erkmen, 2010).
Other foodstuffs are also prone to fungal attack and subsequent aflatoxin
contamination. A total of 2183 cereals and cereal products collected around
Europe between 2007 and 2012 were available for occurrence data (EFSA,
2013). For cereals and their milling products, mean aflatoxin contents ranged
from 2.21 g/kg in unspecified grain milling products to 2.60 g/kg in oats,
while for processed cereal products the average concentrations varied from
0.45 g/kg in fine bakery wares and 1.87 in raw pasta (EFSA, 2013).
However, maize is probably the most sensitive cereal crop to Aspergillus

contamination and aflatoxin production worldwide. Muriuki and Siboe (1995)
reported 100% contamination incidence of three packed corn brands in Kenya
with aflatoxins B1 and B2 (0.4-2.0 g/kg).
The incidence of aatoxin B1 in chestnuts from Italy was 62.2 and 21.4%
in chestnut flour and dried chestnuts, respectively; in the same products, the
percentage of samples exceeding the value of 2.0 g/kg for aatoxin B1 was
24.3% and 7.1% (Pietri et al., 2012).
Twenty-seven aromatic herbs, 28 spices and 48 herbal infusions and
medical plants were analyzed for estimation of aflatoxins by high-performance
liquid chromatography (HPLC). Samples were randomly collected, from 2000
to 2005, from markets, shops and bonded warehouses in Italy. Of the 103
samples analyzed only 7 spices tested positive for aflatoxins: 5 chilli-peppers,
1 nutmeg and 1 cinnamon. Two samples contained the toxin at non-
permissible levels and none of the herbal samples were contaminated
(Romagnoli et al., 2007).
Also, a survey of aflatoxin contamination in 82 unpacked and packed
ground red pepper samples was conducted in Turkey from September 2008 to
February 2009. In unpacked ground red pepper the percentage of samples
exceeding maximum limits were 17.1% for total aflatoxins and 23.1 for
aflatoxin B1, respectively, while only one packed sample containing 89.99
g/kg was over the legal limit of AFB1 (Set and Erkmen, 2010).
Aflatoxin M1 is a metabolite of aflatoxin B1 that can occur in milk and
milk products from animals consuming feed contaminated with B aflatoxins
(Asi et al., 2012), which worldwide levels were reviewed by Galvano et al.
(1996). Fallah (2010) investigated the occurrence of AFM1 in 225 commercial
liquid milk samples composed of pasteurized milk (116 samples) and UHT
milk (109 samples).
AFM1 was detected in 67.1% samples, consisting of 83 pasteurized milk
samples at a mean of 52.8 ng/L (maximum 528.5 ng/L) and 68 UHT milk
samples at a mean of 46.4 ng/L (maximum 515.9 ng/L). In Pakistan, Iqbal et
al. (2011) analyzed a total of 178 milk samples (94 of buffalo and 84 of cow)
and reported that from Punjab about 46% of buffalos and 49% of cows milks
were contaminated with AFM1 as compared with 52% and 51% for milk
samples from NWFP, respectively. Overall, the mean AFM1 concentration
was 46 ng/L with a maximum of 350 ng/L.
Another set of 415 buffalos and cows milk samples (213 morning milks
and 202 evening milks) were analyzed and results revealed significant
differences between morning milks (mean of 43 ng/L) and evening milks (28
ng/L).
Aflatoxin contamination has been also reported in other products of
animal origin such as liver (Mahmoud et al., 2001), spiced hamburgers (Aziz
and Youssef, 1991), and poultry meat (Bintvihok et al., 2002, Hussain et al.,
2010).
2.1. Legislation Worldwide
Due to the significant health risks associated with the presence of

aflatoxins in food, many nations worldwide have regulatory standards on
aflatoxin in food and feed (Georgiadou et al., 2012). Of all mycotoxins
regulated worldwide, aflatoxin is the most regulated, and many countries
might have only legislation with limits for aflatoxins. It is important to note
that these standards vary greatly among countries, requiring harmonization to
remove the variability (Wu, 2008). For comparison, Tables 5 and 6 shows
aflatoxins regulations in different commodities in several countries, apart from
EU regulation already mentioned in Table 2. Currently, the Codex Committee
on Food Additives and Contaminants (CCFAC) has set two aflatoxin related
standards: one for peanuts destined for further processing (15 g/kg) and one
for aflatoxin M1 in milk (0.5 g/kg). The European Union (EU) has some of
the strictest standards for mycotoxins in food and feed in the world.
From a practical point of view, the best approach for the elimination of
mycotoxins from foods and feed is to prevent mould growth at all levels of
production, including harvesting, transport, and storage. Thus, the occurrence
of fungi and mycotoxins can be controlled by applying a number of preventive
measures both before and after harvest, including insect control, good
harvesting, drying, storage and good manufacturing practices. If mycotoxin
contamination has occurred, it is difficult to remove them from food because
they resist high temperatures.
Milling, food processing, and regulatory control of toxins to safety levels
can also have a positive impact on food safety (Trucksess and Diaz-Amigo,
2011).
Table 5. Maximum levels of aflatoxin B1, total aflatoxins and aflatoxin

M1 in foodstuffs (and feedstuffs) in several Asian countries (EMAN, 2013;
Romerlabs, 2013)
China
Maximum level (g/kg)
Commodity B1 Sum of M1
B1+B2+G1+G2
Corn and corn products, peanut and peanut products 20 - -
Rice, edible oil except corn and peanut oil) 10
Other grains, beans and fermented products 5
Infant food 5
Fresh milk - - 0.5
Dairy products - - 0.5
Aflatoxins in feed
Corn, peanut meal, cottonseed meal, rapeseed meal. 50 - -
Soybean meal. 30 - -
Complementary, complete and concentrated feeding 10 - -
stuffs for piglets
stuffs for fattening pigs
stuffs for young broilers, chicks.
stuffs for broilers, layers.
stuffs for young ducks, ducklings.
stuffs for ducks, layers.
stuffs for pigeons.
Supplementary feeding stuffs for dairy cattle. 10 - -
Supplementary feeding stuffs for beef cattle 50 - -
Singapore
Commodity
B1 Sum of B1+B2+G1+G2 M1
Food in general (mainly nuts, corns and their 5 5
products).
Milk and dairy products. 0.5
Infant formulae and follow-up formulae (ready-to- 0.5
consume).
Table 5. (Continued)
Indonesia
B1+B2+G1+G2
Corn and its products. 15 20 -
Peanut and its products. 15 20 -
Dairy products - Milk, milk drink product, fermented 0.5
milk, rennin hydrolysed milk products, concentrated milk
and its analog, cream and its related products, cheese and
analog products, dessert (pudding, yoghurt), whey and its
product.
Dried milk and related products. 5
Feed and corn (final products) 50
Malaysia
B1+B2+G1+G2
Groundnuts, almonds, hazelnuts, pistachios, Brazil nuts, - 15 -
shelled, for further processing.
Groundnuts, almonds, hazelnuts, pistachios, Brazil nuts, - 10 -
shelled, ready-to-eat.
Cereal-based food for infants and children. 0.1 - -
Milk. - - 0.5
Infant formula and follow-up formula (ready-to-drink). - - 0.02
5
Others. - 5 -
Japan
Commodity
Food-all Food B1 Sum of M1
B1+B2+G1+G2
- 10 -
Formula feed (for others). 20 - -
Formula feed (for suckling calf, dairy cattle, suckling 10 - -
pigs, starting chicks, starting broilers).
Korea
B1+B2+G1+G2
Grain, beans, peanut, nuts & their processed food 10 15 -
(grinding, cutting etc.).
Processed cereal products & processed bean product. 10 15 -
Korea
B1+B2+G1+G2
Nutmeg, turmeric, dried red pepper, dried red pepper, 10 15 -
dried paprika & spice products containing them.
Wheat flour, dried fruits. 10 15 -
Confectionaries (peanut of nut-containing food) 10 15 -
Processed corn products for popcorn 10 15 -
Soybean paste, red pepper paste, curry powder. 10 15 -
Meju (dried fermented soybeans). 10 15 -
Steamed rice. 10 15 -
Baby foods for infants and young children. 10 - -
Raw milk and milk before processing. 10 - 0.5
India
B1+B2+G1+G2
Wheat, maize, jawar (sorghum) and bajra (pearl millet), - 30 -
rice, whole and split pulse (dal) masur (lentil), whole
and split pulse urd (mung bean), whole and split pulse
moong (green gram), whole and split pulse chana
(gram), split pulse arhar (red gram), and other food
grain
Groundnut kernels (shelled) (peanuts); - 30 -
Milk - - 0.5
3. EXPOSURE AND TOXICOLOGY OF AFLATOXINS

Mycotoxicosis can be classified as acute or chronic. Acute toxicity
generally has a rapid onset and an obvious toxic response, while chronic
toxicity is characterized by low-dose exposure over a long time period (many
toxins are present in low amounts in daily food intake contributing to the risk
of cancer and other generally irreversible effects (Sforza et al., 2006). The
best-known mycotoxin episodes are manifestations related to acute effects (i.e.
turkey X disease); however, the main human and veterinary health hazard is
associated to chronic exposure (i.e. cancer induction). Diseases caused by
aflatoxin consumption are called aflatoxicosis. These toxins are highly toxic
secondary metabolic products of Aspergillus flavus and Aspergillus parasiticus
and exhibit acute and chronic toxicity including carcinogenic, mutagenic and
teratogenic effects on humans and most animals. Exposure to large doses of
aflatoxin (>6000 mg) may cause acute toxicity with lethal effects (Groopmann
and Kensler, 1999). In turn, the long-term ingestion of diets contaminated with
aflatoxin B1 has been associated with an increased risk of liver cancer.
Table 6. Maximum levels of total aflatoxins and aflatoxin M1

in foodstuffs (and feedstuffs) around the world
(Canadian Food Inspection Agency, 2013; EMAN, 2013;
Romerlabs, 2013)
Country Food Sum of M1

B1+B2+G1+G2 (g/kg)
(g/kg)
Australia/New Peanuts 15 -
Zealand Tree nuts
Canada Nut and nut products 15 -
Animal feeding stuffs 20
Codex, Peanuts, almonds, shelled Brazil nuts, 15 -
GCC (a), hazelnuts, pistachios intended for further
Nigeria processing
Nigeria Almonds, hazelnuts, pistachios, shelled 10 -
Brazil nuts, ready-to-eat
USA Brazil nuts, peanuts and peanut products, 20 -
pistachio products
Dairy products 0.5
Feedstuff ingredients 20
Cottonseed meal intended for beef cattle, 300 -
swine or mature poultry
Corn and peanut products intended for 100 -
breeding beef cattle, swine or mature
poultry
Corm and peanut products intended for 200 -
finishing swine of 100 lbs or more
Corn and peanut products intended for 300 -
finishing beef cattle.
South Africa Peanuts 15 -
Milk 0.05
Codex, GCC (a), Milk - 0.5
Kenya, USA
Argentina Milk, liquid including milk used in the - 0.5
manufacture of milk and milk products and
reconstituted milk
Milk, powder - 5
Mexico Milk - 0.05
(a) Members of GCC are Saudi Arabia, United Arab Emirates (UAE), Kuwait,
Bahrain, Oman, Yemen and Qatar.
Moreover, routine ingestion of aflatoxins may happen in countries where

populations are suffering starvation or where regulations are either not
enforced or non-existent, so that the incidence rates of liver cancer worldwide
are 2 to 10 times higher in developing countries than in developed ones
(Henry, 1999). The rank order of toxicity of aflatoxins is AFB1>
AFG1>AFB2>AFG2 (Erkmen and Bozoglu, 2008). AFB1, the most toxic and
widespread of aflatoxins, is a potent genotoxic carcinogen in laboratory
animals and there is strong evidence that it is a liver carcinogen in humans
(Shephard, 2008). Aflatoxins have been classified by the International Agency
for Research on Cancer (IARC) as a class 1 (human carcinogen) (IARC 1993,
2002). Consequently, a tolerable daily intake (TDI) was not set, but
contamination in food should be reduced to the lowest possible level.
In animals, aflatoxins cause liver damage and unspecific symptoms such
as decreased milk and egg production, reduced reproductivity and suppressed
immunity in animals consuming low dietary concentrations. The principal
target organ for aflatoxins is the liver. After the invasion of aflatoxins into the
liver, lipids infiltrate hepatocytes and lead to necrosis. The clinical signs in
acute toxicity include mainly gastrointestinal affections, inappetence, weight
loss, ascites, jaundice, decrease in milk and egg production, nervous
symptoms, bleeding, pulmonary edema and death. All species are susceptible
to aflatoxicosis, but outbreaks occur mostly in pigs and poultry followed by
sheep and cattle. Bovine species are generally less sensitive compared to non-
ruminants because aflatoxins B1 and B2 are turned into other components
(AFM1 and AFM2) by the rumen microbiota.
In humans, clinically, the main features of acute human aflatoxicosis are
edema of the legs and feet, abdominal pain and vomiting as well as liver
dysfunction, convulsions, gastrointestinal hemorrhage, hematemesis, fever,
diarrhea and coma. Fatty degeneration in the liver and kidneys, and cerebral
edema are the major findings in autopsy (Agag, 2004). Adult humans usually
have a high tolerance of aflatoxin, and, in the reported acute poisonings, it is
usually the children who die (Williams et al., 2004). The ingestion of 2-6
mg/day of aflatoxin for a month can cause acute hepatitis and death (Patten,
1981). Early symptoms of hepatotoxicity from aflatoxicosis can manifest as
anorexia, malaise and low-grade fever. Aflatoxicosis can progress to
potentially lethal acute hepatitis with vomiting, abdominal pain, hepatitis and
death (Etzel, 2002). Exposure to amounts less than 1000 g/kg have been
linked to aflatoxicosis in humans. Consuming approximately 5000 g/kg of
aflatoxin can cause acute aflatoxicosis leading to death (Chang et al., 2013).
The LD50 value of aflatoxin ranges from 0.3 to 10 mg/kg for most animal
species, and from 0.54 to 1.62 mg/kg for human beings (Wild and Gong,
2010). The individual susceptibility to aflatoxicosis depends on doses,
duration of exposure, species (according to their abilities to detoxify aflatoxins
by biochemical processes), age (young people are more susceptible than
elder), sex (levels of testosterone), weight, diet, immunologic status, and
exposure to infectious agents such as viral hepatitis or parasite infestation.
Consumption of sub-lethal quantities of aflatoxin for a long time can develop a
sub-acute (chronic) toxicity syndrome, which commonly includes moderate to
severe liver damage.
Although susceptibility of humans to aflatoxins is not well known,
epidemiological studies of human populations exposed to diets naturally
contaminated with aflatoxins, revealed an association between the high
incidence of liver cancer in Africa and elsewhere and dietary intake of
aflatoxins (Jaimez, 2000). It has been also reported that the risk of lung cancer
may increase among workers handling contaminated grain (Kelly et al., 1997).
For people who are infected with hepatitis B and C, which is common in
sub-Saharan Africa, aflatoxin consumption raises the risk of primary
hepatocellular carcinoma by more than ten-fold compared to either exposure
alone (Turner et al., 2000; Murphy et al., 2006). In addition, preliminary
evidence suggests that there may be an interaction between chronic mycotoxin
exposure and malnutrition, immuno-suppression, impaired growth, and
diseases such as malaria and HIV/AIDS (Gong et al., 2003, 2004).
3.1. Biomarkers
Each biochemical process results in derivatives (biomarkers) that have a

characteristic half-life within the body, and thus the exposure over a period of
days, weeks, or months can be studied. Thus, one of the best methods of
measuring human exposure to aflatoxins consists of the analysis of body fluids
for the presence of aflatoxin derivatives (Makarananda et al., 1998). For
epidemiologic studies, biomarkers of aflatoxins in urine and serum provide a
better estimate of dietary aflatoxin exposure than food analysis (Azziz-
Baumgartner et al., 2005; EFSA, 2007). Aflatoxin metabolites in urine reflect
recent exposure whereas the measurement of aflatoxin albumin adducts in
blood reflects exposure over a long-time period and hence is a more reliable
indicator of a persons chronic exposure (Groopman et al., 1994; Groopman
and Kensler 2005), given that the half-life in the body of aflatoxin-albumin
adducts is 30-60 days (EFSA, 2007). Thus, recent exposure to aflatoxin is
reflected in the urine as directly excreted aflatoxin M1 and other detoxification

products, but only a small fraction of the dose is excreted in this way.
Moreover, measurements of aflatoxins and its derivatives in urine have been
found to be highly variable from day to day, which reflects the wide variability
in the contamination of food samples, and, for this reason, the measurement of
aflatoxin M1 on a single day may not be a reliable indicator of chronic
exposure (Wild and Pisani, 1998).
Over 90% of West African sera were reported to contain detectable levels
of aflatoxin albumin (AF-alb) adducts, with exposure occurring throughout
life, including in utero and via breast milk (Turner et al., 2000; Wild et al.,
2000). Additionally, aflatoxin albumin (AF-alb) adducts were detected in 99%
of children in Benin and Togo (Gong et al., 2003).
However, the detection of aflatoxin metabolites or adducts in urine and
serum indicate exposure but do not necessarily equate to adverse health
effects. The evidence of contamination in market and food samples and the
human biomarker data show that, regardless of food preparation practices, the
human populations of developing countries are widely and significantly
exposed to aflatoxins, but usually at a level less than that needed for direct
acute illness and death. The data on the temperature conditions needed for
aflatoxin synthesis, the vulnerability of staple commodities to contamination,
the systems for food production, storage, and marketing, and the regulation
enforcement failures all indicate that there is high risk of chronic aflatoxin
exposure in developing countries. Population data from the FAO database
indicate that nearly 4,500,000 people live in this zone.
Economic pressures have created a double standard for allowable
contamination of commodities destined for human and animal consumption.
As a consequence of the successful regulation of aflatoxin in developed
countries, the human medical research literature is clearly focused on the
carcinogenic aspects of aflatoxin, which reflects the concerns of North
Americans and Europeans about the consequences of long-term cumulative
exposure, which is the only concern at the low concentrations of aflatoxins
that their food systems achieve (Williams et al., 2004).
CONCLUSION
Aflatoxins are produced by moulds that are especially found in areas with
hot, humid climates. They are most likely to contaminate tree nuts, ground
nuts, figs and other dried fruits, spices, crude vegetable oils, cocoa beans and
maize. Measures to apply Good Agricultural and Storage Practices by

producing countries are required in order to reduce incidents of highly
contaminated products being consumed. However, the possible aflatoxin
contamination of domestic foods should be kept under review, particularly in
the light of potential changes in climate.
Aflatoxin B1 was found to be the dominating aflatoxin in all foods. In the
European Union the highest total aflatoxin levels have been found in peanuts,
pistachios, and Brazil nuts, so improved pre-export controls are also required
to reduce incidents of highly contaminated products imported to the EU.
Aflatoxin B1 is clearly genotoxic and carcinogenic in a variety of animal
species. Increasing evidence demonstrates that aflatoxin B1 also has the
potential to affect the immune system, nutrition and growth. Because
aflatoxins are considered to be genotoxic and carcinogenic, it is not possible to
identify an intake without risk, and many countries worldwide introduced
regulations for these toxins at levels considered to be as low as reasonably
achievable. A number of epidemiological studies have shown clear
associations between aflatoxins exposure and incidence of hepatocellular
carcinoma in areas with high prevalence of hepatitis B and C, which is itself a
risk factor for liver cancer. A biomonitoring approach using validated
biomarkers would complement food analysis and consumption data in
providing information on prevalence and level of aflatoxin exposure
worldwide.
ACKNOWLEDGMENTS
This review chapter was supported by the Government of Aragn, Spain
(Grupo de Investigacin Consolidado A01) and the European Social Fund.
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Chapter 4
IMMUNOSUPPRESSIVE ACTIONS
OF AFLATOXIN AND ITS ROLE
IN DISEASE SUSCEPTIBILITY
Johanna C. Bruneau,*1 Orla Hayden,2

Christine E. Loscher2 and Richard OKennedy1,3
1
Applied Biochemistry Group, School of Biotechnology,
Dublin City University, Dublin, Ireland
2
Immunomodulation Group, School of Biotechnology,
Dublin City University, Dublin, Ireland
3
Biomedical Diagnostics Institute, Dublin City University, Dublin, Ireland
ABSTRACT
Aflatoxins are secondary metabolites produced by fungi of the
Aspergillus species. They occur as contaminants in a variety of food and
feed stuffs that have been infected with the producing fungi. Aflatoxin
exposure is known to cause a number of acute and chronic effects in both
humans and animals, including immunosuppression, liver and other
cancers, and failure of vaccination regimens. The immunomodulatory
effects of the aflatoxins have been shown to affect cell-mediated
immunity more than humoral immunity. In particular, aflatoxin exposure
*
Corresponding Author: Dr. Johanna Bruneau; Applied Biochemistry Group, School of
Biotechnology, Dublin City University, Dublin 9, Ireland. Email: johanna.bruneau@
gmail.com.
92 Johanna C. Bruneau, Orla Hayden, Christine E. Loscher et al.
modulates secretion of inflammatory cytokines and phagocytic function.

Decreases in phagocytosis and inflammation observed following
aflatoxin exposure may reduce the effectiveness of the host immune
response to infection, thereby increasing susceptibility to infection in
individuals exposed to these toxins. The aim of this chapter is to
summarise the immunomodulatory effects of aflatoxin exposure in order
to better understand its potential immunosuppressive effects in humans
and animals. The relationship between these immunosuppressive actions
and susceptibility to infection will also be discussed.
INTRODUCTION
Aflatoxins were first identified in the 1960s as the causative agent in
Turkey X disease in Britain. In this incident, thousands of turkey poults died
after consuming contaminated groundnut (peanut) meal (Spensley, 1963).
Since then, aflatoxin contamination has been identified in a number of
foodstuffs, including cereals (maize, wheat, sorghum, rice and millet), nuts
(peanuts, pistachios, walnuts, brazil and coconut) spices (chilli, turmeric,
paprika, black pepper, and ginger) dried fruit, and seeds (Pitt, 2000; Williams
et al., 2004).
Aflatoxins are produced by the fungal species Aspergillus as secondary
metabolites, therefore they are not necessary for the normal growth and
function of the fungus. Their production is regulated by environmental and
developmental signals such as light, temperature and pH (Georgianna and
Payne, 2008). Structurally, the aflatoxins belong to the coumarin family of
compounds, consisting of a dihydrofuran or tetrahydrofuran moiety fused to a
coumarin ring (Keating and O'Kennedy, 1997). While there are 17 related
aflatoxin isoforms and aflatoxin metabolites, only four of these (aflatoxin B1,
B2, G1 and G2) are the main food contaminants (Figure 1). Aflatoxin B1
(AFB1) and aflatoxin B2 (AFB2) are produced by A. flavus, while A.
parasiticus can produce all four isoforms (Ogundero, 1987; Creppy, 2002).
Aflatoxin M1 (AFM1) is the hydroxylated metabolite of AFB1 which can be
found in the milk, urine and feces of humans and animals that have consumed
contaminated food (Peraica et al., 1999; Creppy, 2002). AFB1, the
predominant isoform, is a potent hepatocarcinogen in humans. The naturally
occurring aflatoxins B1, B2, G1, and G2, including mixtures of isoforms, and
the metabolite aflatoxin M1 have been designated Group 1 carcinogens
(carcinogenic to humans) by the International Agency for Research on Cancer
(IARC) (IARC, 2002).
Immunosuppressive Actions of Aflatoxin and Its Role 93
Figure 1. Chemical Structure of Aflatoxins B1, B2, G1 and G2.
Once ingested, aflatoxin is metabolised by cytochrome P450 in the liver

(Figure 2) (McClean and Dutton, 1995; Turner et al., 1998). Cytochrome P450
converts aflatoxin into a highly reactive and mutagenic compound, AFB1-8,9-
epoxide. AFB1-8,9-epoxide forms a covalent bond with the N7 of guanine,
forming 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua)
(Bedard and Massey, 2006). This adduct causes a G T transversion that
results in DNA repair, lesions, and mutations, eventually leading to tumour
formation (Guengerich et al., 1998; Bennet and Klich, 2003). The formation of
AFB1-N7-Gua is directly proportional to the amount of AFB1 ingested.
Several human studies have used this correlation to investigate the relationship
between dietary exposure to AFB1 and hepatocellular carcinoma (HCC) by
measuring the amount of the AFB1-N7-Gua excreted in urine (Groopman et
al., 1992, Groopman et al., 1993; Groopman et al., 1996).
Aflatoxin exposure also causes toxic effects in addition to its carcinogenic
effects, which makes it a double hazard. The toxic effects of aflatoxin are
mediated by the AFB1-8,9-epoxide. The AFB1-8,9-epoxide undergoes rapid
hydrolysis to form AFB1-8,9-dihydrodiol. This product is relatively stable, but
undergoes a slow reaction to form AFB1-dialdehyde. The AFB1-dialdehyde
molecule binds to lysine residues on proteins (Guengerich, et al., 1998;
Guengerich et al., 2001). Aflatoxin-protein adducts may inhibit key cellular

functions, particularly if enzymes or signalling molecules are affected. It has
been demonstrated that AFB1 binds to a number of proteins, including albumin
(Wild et al., 2000), serine proteases (Cuccioloni et al., 2009), and histones
(Chih et al., 1993).
Figure 2. Metabolic Pathway of Aflatoxin B1. Hydroxylated metabolites include

Aflatoxin M1.
Aflatoxin has a positive association with hepatocellular carcinoma (HCC),

which is the fifth most common and the third most fatal cancer worldwide,
causing an estimated 500,000 deaths annually. Hepatitis B virus (HBV)
infection is endemic in areas where aflatoxin contamination of foodstuffs is
commonplace. Studies have shown that there is a synergistic relationship
between HBV infection, aflatoxin exposure, and the development of HCC
(Kew, 2003). Research has shown that a person is three times more likely to
develop HCC when they test positive for AFB1-N7-Gua, versus seven times
more likely when they are infected with HBV. However, when a person tests
positive for both AFB1-N7-Gua and HBV, they are 60 times more likely to
develop the disease (Smela, 2001; Smela, 2002). These studies provide
evidence to explain why HCC incidence is high in areas where HBV infection
and aflatoxin consumption are prevalent. For example, in Mozambique and

some provinces in China, HCC accounts for 65-75% of male and 30-55% of
female cancer fatalities, compared to 2% in the United States (Sell, 2003).
IMMUNOSUPPRESSIVE ACTION OF AFLATOXIN

The immunosuppressive effects of AFB1 have been studied in a number of
species both in vivo and in vitro. Many of these investigations have focused on
the effect of aflatoxin on macrophages. Macrophages are innate immune cells
which have an important role in the primary immune response and
maintenance of tissue homeostasis. Macrophages are derived from circulating
monocytes and can be found in virtually all tissues in the body, including the
lungs (peritoneal macrophages), liver (Kupffer cells) and the central nervous
system (microglia). These cells regulate the immune response and homeostasis
through the release of cytokines and chemokines. They also contribute to the
adaptive immune response by presenting foreign antigens to T and B cells
(Gordon and Taylor, 2005; Mosser and Edwards, 2008). Several groups have
investigated the effect of aflatoxin on murine-derived macrophages. Moon et
al. reported that murine peritoneal macrophages exposed to AFB1 in vitro
exhibited decreased production of the pro-inflammatory cytokines Tumour
Necrosis Factor (TNF)-, Interleukin (IL) -1 and IL-6. Reactive intermediates,
including nitric oxide (NO), hydrogen peroxide (H2O2) and superoxide anion
(O2-), which have potent anti-microbial activity, were also decreased (Moon et
al., 1999a). In agreement with these findings, murine peritoneal macrophages
exposed to AFB1 in vivo exhibited a significant decrease in the generation of
reactive intermediates (NO, H2O2, and O2-), as well as a significant decrease in
the phagocytic capability of the cells and a significant decrease in TNF-
production (Moon et al., 1999b). Results of an earlier study by Dugyala et al.
(1996) assessed the effect of LPS-induced cytokine production from murine
peritoneal macrophages exposed to AFB1 in vivo. AFB1 treatment increased
mRNA levels of the pro-inflammatory cytokines IL-1, IL-6 and TNF-,
however secretion levels of the same cytokines were significantly reduced.
This same study also found that AFB1 exposure in vivo had minimal effect on
cytokine expression and secretion from murine splenic lymphocytes stimulated
in vitro. Although there was a significant decrease in IL-2 mRNA, there was
no significant change in IL-2, IL-3 or IFN- secretion levels at any of the
doses tested (Dugyala and Sharma, 1996).
Studies in a mouse macrophage cell line have also demonstrated the

immunosuppressive capacity of aflatoxins. When the murine macrophage cell
line J774A.1 was pre-incubated with AFB1, AFB2 or AFG1, both singly and in
combination, altered secretion of key pro- and anti- inflammatory cytokines
was observed (Bruneau et al., 2012). In particular, AFB1 and AFB2
significantly decreased secretion of the anti- inflammatory cytokine IL-10,
which plays an important role in maintaining homeostasis (Saraiva and
OGarra, 2010). Interestingly, combination treatment of J774A.1 cells with
low doses of aflatoxin isoforms decreased the secretion of IL-12p40 even
though single isoform treatment did not, indicating that these molecules exert a
synergistic effect on cytokine secretion (Bruneau et al., 2012). Other
investigators also found that AFB1, AFB2 and their hydroxylated metabolites
AFM1 and AFM2, alone and in combination, reduced the production of
reactive oxygen intermediates (NO). In addition, they reported that the
cytotoxic activity of AFB1 in these cells is due to its effects on the cell cycle
rather than apoptotic pathways. In particular, AFB1 treatment of J774A.1
macrophages increased the proportion of cells in the S phase, with a
corresponding decrease in the number of cells in the G1/G0 phase (Bianco et
al., 2012).
Further investigations into the effect of AFB1 exposure on murine
macrophages have found that AFB1 treatment altered expression of CD14 on
the cell surface. CD14 is a cell surface protein which, along with Toll-like
receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2), form the
lipopolysaccharide (LPS) receptor complex. Binding of LPS, a component of
gram negative bacterial cell walls, to the receptor complex triggers an
inflammatory response, inducing secretion of inflammatory cytokines
(Pllson-McDermott and ONeill, 2004). Macrophages pre-treated with AFB1
followed by LPS stimulation showed a significant decrease in CD14
expression compared to cells treated with LPS only. AFB1 pre-treatment also
significantly increased the amount of CD14 released into the medium (Moon
and Pyo, 2000). Our study in a murine macrophage cell line found that
combination treatment with AFB1 and AFB2 decreased CD14 expression
levels (Bruneau et al., 2012). These results suggest that aflatoxin exposure can
decrease the ability of macrophages to mount an appropriate response to
infection.
The effect of AFB1 on immunological parameters has also been
investigated in pigs. Liu and colleagues (2002) assessed the effect of AFB1
treatment in vitro on primary swine alveolar macrophages. Their study found a
time and dose-dependent decrease in cellular viability following exposure to
AFB1. However, there was no change in TNF- or IL-1 mRNA levels

compared to controls in cells following LPS stimulation (Liu et al., 2002). A
study of whole blood samples taken from weaning piglets fed a diet containing
aflatoxin found a significant a decrease in IL-1 and TNF- mRNA
expression, and an increase in IL-10 mRNA expression. Th1 (IL-2) and Th2
(IL-4) cytokine mRNA expression was unaffected in these samples (Marin et
al., 2002). Meissonnier and colleagues (2008) undertook a similar
investigation into the effect of an AFB1-contaminated diet on immunological
parameters in piglets. Expression levels of TNF- , IL-1, IL-6, IFN- , and
IL-10 mRNA in spleen samples were significantly increased at the highest
dose tested compared to controls. There was also a significant decrease in
lymphocyte proliferation after stimulation with ovalbumin (OVA), indicating
that T cell activation may be impaired (Meissonnier et al., 2008).
Investigations into the immunological response in rats following AFB1
exposure have yielded varied results. One study examined the effect of chronic
or intermittent AFB1 dosing on isolated splenic lymphocytes. Hinton and
colleagues (2003) found that the percentage of T cells was significantly
increased with intermittent and chronic doses of AFB1 and the percentage of B
cells were significantly decreased compared to untreated control samples. The
effect of AFB1 treatment on cytokine secretion in this model depended on the
dosage time. There was a significant decrease in the production of IL-2, IL-1
and IL-6 with both chronic and intermittent dosage schedules at 8 weeks.
However, increased production of IL-1 and IL-6 was observed in both chronic
and intermittent dosage schedules at 12 weeks, while IL-2 production was
unaffected (Hinton et al., 2003). Another study examined the effect of AFB1
treatment in vivo and in vitro on rat spleen mononuclear cells. Spleen
mononuclear cells isolated from rats fed a diet containing 40ppb AFB1
(equivalent to 40g/kg) for 90 days showed a significant decrease in IL-2
secretion and a significant increase in IL-4, but there was no change in IL-10
compared to controls. However, when spleen mononuclear cells were exposed
to 20mol/l AFB1 in vitro, there was no change in IL-2, IL-4 or IL-10
secretion levels compared to controls (Theumer et al., 2003).
Bovine neutrophils, when exposed to very low doses of AFB1 (0.01-0.5
ng/ml), exhibited decrease myeloperoxidase activity, superoxide radical (O2-)
production, and phagocytic capacity. Although the function of the cells was
affected, AFB1 exposure did not affect the viability or percentage of apoptotic
cells in comparison to untreated neutrophils (Mehrzad et al., 2011).
Hematopoietic progenitor cells are also targets of aflatoxin toxicity. Roda
and colleagues (2010) investigated the effect of AFB1 on erythroid and
myeloid progenitors. They found a dose dependent decrease in the formation

of erythroid colony forming units (CFU-E) and granulocyte monocyte colony
forming units (CFU-GM) from human or murine progenitor cells incubated
with AFB1. Of note, there was a species-specific effect. The IC50 values for
human granulocyte-monocyte progenitors was four times lower than that of
mice, indicating increased sensitivity (CFU-GM IC50: 2.45 1.08 in human
cells, versus 11.08 2.92 in murine cells) (Roda et al., 2010).
There are several investigations into the effect of aflatoxin exposure on
immunological parameters in humans. In these studies, aflatoxin exposure is
estimated by measuring the concentration of aflatoxin-albumin (AF-albumin)
adducts in the serum of study participants. Jiang and colleagues (2008)
investigated the effect of high or low AF-albumin levels in HIV+ an HIV
individuals. HIV+ individuals with high AF-albumin levels had significantly
lower percentages of T regulatory cells (Tregs), nave CD4+ cells, and B cells
compared to HIV+ individuals with low AF-albumin levels. In addition, high
AF-albumin levels accentuated HIV-related changes in T and B cells in HIV+
individuals (Jiang et al., 2008). In an earlier study, Jiang and colleagues (2005)
investigated the effect of AF-albumin adduct levels on leukocyte
immunophenotypes and monocyte phagocytic function. This study found that
individuals with high AF-albumin levels had lower percentages of
CD3+CD69+ and CD19+CD69+ cells. In addition, individuals with high AF-
albumin levels showed a decrease in proportion of perforin-expressing CD8+
and granzyme A and perforin expressing CD8+ T cells compared to low AF-
albumin individuals. There was no difference in monocyte phagocytosis
between the two groups (Jiang et al., 2005). These data indicate that CD8+ T
cell function is impaired in individuals exposed to high levels of aflatoxin,
which will have consequences for cellular immunity to infectious diseases.
Further studies in human monocytes incubated with AFB1 (0.05 50
pg/ml) showed a decrease in secretion of IL-1, IL-6 and TNF-. The mRNA
levels of the three cytokines studied were also reduced (Rossano et al., 1999).
Exposure to AFB1 in vitro significantly inhibited the phagocytic and
microbicidal activity of human monocytes (Cusumano et al., 1996). Other
investigations have found that human polymorphonuclear cells showed
significantly decreased chemotaxis after AFB1 treatment (Ubagai et al., 2008).
These results indicate that the ability of these cells to respond infection may be
affected by exposure to aflatoxin.
The effect of AFB1 treatment on immune cells varies between species and
cell type. There are several plausible explanations for this variability.
Aflatoxins are metabolized by cytochrome P450 enzymes into an epoxide
(Figure 2). It is through this intermediate that AFB1 exerts its toxic and
carcinogenic effects (Guengerich, et al., 1998; Guengerich et al., 2001). The
P450 enzymes are differentially expressed between species, and also between
cell types within a species. In a recent investigation by Bahari et al. (2013),
relative gene expression of cytochrome P450 isoforms CYP1B1 and CYP3A4
was significantly increased in human monocytes treated with AFB1 compared
to human lymphocytes exposed to AFB1 (Bahari et al., 2013). Induction of
cytochrome P450 would increase the conversion of AFB1 into AFB-epoxide,
thereby increasing its toxic and carcinogenic effect. Therefore, it is plausible
that myeloid cells are more susceptible to aflatoxin toxicity due to the
difference in cytochrome P450 isoform expression compared to lymphoid
cells.
It has been well established that aflatoxin metabolites have the ability to
bind to DNA. Another mechanism through which aflatoxin may exert its
immunosuppressive effects can form an adduct with the guanine nucleotide in
DNA (Bedard and Massey, 2006). The AFB1-epoxide can also be hydrolysed
to a compound which can bind to lysine residues in proteins (Guengerich, et
al., 1998; Guengerich et al., 2001). Aflatoxin-protein conjugates may disrupt
cell signalling pathways, especially if the AFB1-epoxide interacts with
enzymes or signalling molecules. Prior research has demonstrated that AFB1
binds to key cellular proteins including serine proteases (Cuccioloni et al.,
2009), albumin (Wild et al., 2000), and histones (Chih et al., 1993).
EFFECT OF AFB1 ON HUMORAL IMMUNITY

While aflatoxin exposure primarily affects cell-mediated immunity,
studies have investigated its effect on response to vaccination regimens.
Meissonnier et al. (2008) examined the effect of a diet containing AFB1 on the
humoral response in pigs immunised with OVA. There was no significant
change in the total concentrations of IgA, IgM and IgG, or of anti-OVA IgG
between AFB1-treated and control animals (Meissonnier et al., 2008). Another
study in pigs examined the effect of AFB1-contaminated feed on response to
Mycoplasma agalacticae vaccination. Marin et al. (2002) found that while
specific antibody responses to M. agalacticae were reduced in animals fed a
diet containing AFB1 compared to controls, the decrease was not statistically
significant (Marin et al., 2002). Investigations by Turner et al. (2003) found
that aflatoxin exposure was significantly associated with decreased levels of
secretory IgA in humans. There was a weak association between AF-albumin
adduct levels and response to pneumococcal vaccine type 23. However, there
was no statistically significant difference in the antibody response to
pneumococcal serotype 1, 5 and 14 vaccinations, or rabies vaccination,
between children with detectable and undetectable levels of AF-albumin
adducts (Turner et al., 2003).
Since antibody production and immune response to vaccines are reduced
by AFB1, this undoubtedly impacts an individuals susceptibility to infection.
Williams et al. (2004) refers also to an increased pace of disease rate following
exposure to AFB1. HIV and AIDS have been studied in relation to AFB1
exposure, it is speculated that there is an acceleration of disease when the
individual is simultaneously exposed to AFs again stemming from reduced
immunity. Altered CD4+ T cell function and the reduction in IL-2 would lead
to increased progression of HIV according to Williams et al. (2004). In 2005,
Oswald et al. (2005) reported a reduction and alteration of CD4+ T cells and
the related interleukin IL-2, in pigs treated with AFB1.
CONCLUSION
The immunomodulatory effects of AFB1 have been investigated in a
number of species and cellular targets. While the results of these various
studies differ, these differences are most likely due to variations in the assays
used, the target cell type investigated, and the mode of exposure (i.e. in vitro
or in vivo). Other factors that could contribute to the variability include
differences in dosages and route of exposure. Regardless of this variability,
when the data are considered together, it is apparent that AFB1 has the ability
to modulate the cellular immune response, however, humoral immunity is
largely unaffected. In particular, AFB1 inhibits the ability of macrophages and
T cells to respond to an infection by decreasing pro-inflammatory cytokine
secretion. Other macrophage functions that are affected include decreased
phagocytosis and decreased release of reactive intermediates which help fight
infection. T cell function is also affected, including decreased T cell
populations, in particular reduced numbers of perforin and granzyme A cells,
which mediate lysis of infected cells.
The data available to date makes it clear that aflatoxin is able to exert an
immunosuppressive effect in a number of species, but at present the
mechanism by which this effect is mediated remains unknown. A number of
studies have shown that aflatoxin has the ability to bind to both DNA
(Guengerich et al., 1998) and protein (Chiih, et al., 1993; Wild et al., 2000;
Cuccioloni et al., 2009) via reactive intermediates. It is possible that the AFB1-
8,9-epoxide has the ability to bind to signalling molecules that initiate the
inflammatory response, thereby impairing the ability of the immune system to
react to pathogen challenge. This is an area that warrants further investigation.
Several investigators have suggested that aflatoxin-induced
immunosuppression could inhibit the host response to infection, reactivate
chronic infections or decrease the efficacy of vaccination regimens (Bondy
and Pestka, 2000; Jiang et al., 2005; Oswald et al., 2005: Meissonnier et al.,
2008). This is an important area of research when considered in the context
high incidence of infectious diseases in areas where aflatoxin exposure is
common. It has already been proven that there is a strong correlation between
aflatoxin exposure and the development of HCC, particularly in individuals
with concurrent HBV infection (Kew, 2003; Sell, 2003). The high correlation
may be due in part to the immunosuppressive activity of repeated aflatoxin
exposure which would prevent the host from properly responding to chronic
HBV infection. In addition, high AF-albumin levels have been shown to
accentuate HIV-related changes in T and B cell population from HIV-infected
individuals (Jiang et al., 2008). The combination of HIV infection and
repeated aflatoxin exposure may contribute to accelerated disease progression
in infected individuals. Further research in this area is warranted to assess the
immunological impact of aflatoxin exposure on human and animal health, in
particular the synergism between aflatoxin consumption and disease
progression.
ACKNOWLEDGMENT
The financial support of Enterprise Ireland and the Biomedical
Diagnostics Institute is gratefully acknowledged.
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leukocytes. Toxicol In Vitro 2008, 22, 1115-1120.
Wild, CP; Yin, F; Turner, PC; Chemin, I; Chapot, B; Mendy, M; Whittle, H;
Kirk, GD; Hall, AJ. Environmental and genetic determinants of aflatoxin-
albumin adducts in the Gambia. Int J Cancer 2000, 86, 1-7.
Williams, JH; Phillips, TD; Jolly, PE; Stiles, JK; Jolly, CM; Aggarwal, D.
Human aflatoxicosis in developing countries: a review of toxicology,
exposure, potential health consequences, and interventions. Am J Clin
Nutr 2004, 80, 1106-1122.
Chapter 5
AFLATOXINS HAZARDS AND REGULATIONS

IMPACTS ON BRAZIL NUTS TRADE
Otniel Freita-Silva1,2, Renata Galhardo Borguini1

and Armando Venncio2
1
EMBRAPA Food Technology, Rio de Janeiro, RJ, Brazil
2
IBB - Institute for Biotechnology and Bioengineering,
Center of Biological Engineering, Universidade do Minho,
Campus de Gualtar, Braga, Portugal
ABSTRACT
Brazil nut is an important non-timber forest product produced in
Amazon region. This nut is used as food with high value in the
international market, due to its high nutritional and flavor characteristic
and to their association with environmental conservation and alleviation
of poor people living from Amazonia. Annually, several hundred tons of
Brazil nuts are produced in Brazil. However, they are susceptible to
aflatoxins (AF) contamination. Because of the detection of unacceptable
level of AF in Brazil nuts consignments arriving in European Union
ports, in 2003, special conditions were imposed on Brazil nuts entering
the European Union, decreasing the acceptable levels of AF. In 2010, the
European Union revised AF regulation on nuts; these new limits are more
adequate when considering the complexity of Brazil nut chain and the
Phone: ++55 21 3622-9645; Fax: ++55 21 3622-9713; E-mail: otniel.freitas@embrapa.br.

108 O. Freita-Silva, R. G. Borguini and A. Venncio
low risk related to its low consumption. This chapter points data on the
occurrence of AF in Brazil nuts, as reported by the Rapid Alert System
for Food and Feed (RASFF), and evaluates the efforts made by all sectors
involved in the agribusiness of Brazil nuts, in Brazil, in order to
contribute to protection of both domestic and international consumers
from possible health hazard caused by AF.
INTRODUCTION
Brazil nut is the main commodity from the Amazon rainforest
extractivism. The nuts are destined to national or international trade. Gatherers
pick and store the fruits; they are responsible for the initial handling and
processing, which is still done in the forest [1]. The main stages in Brazil nut
production are: production and collection in the forest (cleaning paths between
trees, gathering the fruit, opening the fruit and transporting them to the camp),
and processing (cleaning, drying and soaking, peeling the nuts, drying the
peeled nuts) and commercialization in the packing house [2].
Brazil nuts are a typical non-timber forest product (NTFPs) and, for its
characteristic flavor, high nutritional value and their association with
environmental conservation, have been increasingly valued in the market [3].
It is the only seed internationally traded that is gathered in the forest [4].
Its origin comes from the Amazon region, mostly in the north of Brazil
and neighbor countries (Peru, Guyana, Venezuela, Suriname and Bolivia), but
only Bolivia, Brazil and Peru export this nut [5].
According The International Nut and Dried Fruit Council Foundation
(INC) report [6] the world production of Brazil nuts in 2012 was estimated at
46,155 metric tons, a 94 percent increase from the previous year (32,130
metric tons). Bolivia accounts for 70% of total production with 32,130 metric
tons, followed by Brazil with 22 percent (10,200) and Peru with 8 percent
(3,825 metric tons) (Figure 1). Brazil nut is the most economically important
plant product that is harvested sustainably from Amazon rain forest. This
report also reinforces that close 70 percent of world supply comes from Pando
region, an area representing around 3percent of total Amazonian rain forest.
In 2008, Bolivia was responsible for 53% of in-shell Brazil nut world
production, compared with 39.35% and 0.37% from Brazil and Peru,
respectively [5, 6]. In recent years, producers countries have taken a series of
actions by their respective governments, research institutes, and Non-
Governmental Organizations among others, for controlling the Brazil nut
Aflatoxins Hazards and Regulations Impacts ... 109
contamination by aflatoxins (AF) with the aim to attend to the international

sanitary standards and consolidate the export markets.
Contamination by AF is a major problem for tree nuts, as well as for other
stored grains, milk and dry fruits, especially because the causal fungi,
Aspergillus flavus group, occurs as a natural contamination [7]. Industries and
producers have endeavored considerable efforts over the last 20 years to
minimize fungal growth and AF production in tree nuts, particularly in the
case of Brazil nuts, due to the hot and humid climatic conditions found in the
Amazon environment, with an average temperature of 26 C and relative
humidity of 8095%, which favors the production of these toxins. In addition,
the extractivism characteristics (temperature and relative humidity during
gathering and handling) are hard to be controlled, having a direct or indirect
effect on toxigenic fungi and on the production of AF. Since contamination is
usually associated with shelled nuts, proven processing/treatments that reduce
AF levels in Brazil nuts include shelling or sorting by size, specific gravity,
color or damage [8, 9, 10].
Figure 1. World production of Brazil nuts in 2012.
Background
Over the last 15 years in Brazil, Brazil nuts have experienced a significant
decrease in exports. According to a comparative assessment, one of the
explanations for the crisis caused by such decrease in the exports in the
Brazilian Amazon was the entry of Bolivia into the international market in
1996 [11], as well as a high incidence of AF in the nut recorded in Brazil.
The crash and crisis can still be justified by non-tariff barriers imposed by
the European Community since 1998, EC regulation 1525/98 and EC decision
493/2003 [12, 13]. Because of those decisions, Brazilian exports of Brazil nuts
in shell to Europe fell by almost 90% between 2000 and 2004. The first
regulation reduced the acceptable limit of total aflatoxins (AFT) in Brazil nuts
to 4 g/kg and 2 g/kg for aflatoxin B1 (AFB1) and rejected contaminated
consignments from Brazil [14]. While the second one imposed special
conditions on the import of Brazil nuts in shell originating in or consigned
from Brazil. Moreover, the domestic market absorbs only 10% of the Brazil
nut production, because what is left in Brazil is the lower quality product and
no marketing strategy for Brazilians consumers was established yet.
Although Brazil nuts are exported since 1800, only had a place in the
exports agenda of NTFPs after the beginning of the twentieth century [15].
After the decline of rubber production (Hevea brasiliensis), Brazil nuts
became the primary extraction product for export purposes in the Northern
Region of Brazil, and its exploitation has a key role in the socio-economic
organization of large areas of natural forest [5].
The Brazil nut industry comports with the principal objectives of
European policy on development co-operation (poverty reduction linked with
environmental protection) and forest conservation (maintaining forest cover).
However, European Regulation 1525-98 EC, which decreases acceptable
levels of AF in Brazil nuts to 4 g/kg, caused a crash in the Brazil nut trade.
Thus, European policies on food quality, development co-operation and forest
conservation are likely to operate cross-purposes. Brazil nut producer
countries had questioned the legal basis of the Regulation in terms of scientific
justification for the stricter limits on AF content and lack of conformity with
international standards set by Codex Alimentarius. The EU has countered by
invoking the precautionary principle [16].
An EU mission carried out in Brazil from 25 January to 9 February 2003,
from the Commission's Food and Veterinary Office (FVO), presented their
conclusion [13] imposing special conditions on the import of Brazil nuts in
shell originating in or consigned from Brazil, pointing out that:
1. Brazil nuts in shell originating in or consigned from Brazil have been

found, in many cases, to be contaminated with excessive levels of
AFB1 and AFT.
2. The Scientific Committee for Food has noted that AFB1, even at
extremely low levels, can cause cancer of the liver and is also
genotoxic.
3. Commission Regulation (EC) No 466/2001 of 8 March 2001, as last
amended by Regulation (EC) No 563/2002, sets maximum levels for
certain contaminants and in particular AF in foodstuffs. Those limits
have been frequently and largely exceeded in samples of Brazil nuts.
4. Such contamination constitutes a serious threat to public health within
the Community and it is therefore appropriate to adopt protective
measures at Community level.
5. To assess the control systems in place to prevent AF contamination
levels in Brazil nuts intended for export to the Community. The
mission revealed that: the national legislation provides an inadequate
sampling procedure; no adequate traceability system is in place,
neither during the process chain, nor in relation to the export
procedure and certification; control over the sample during the
dispatch to the laboratory is inadequate, some laboratories entitled to
perform analysis for the purposes of export certification do not
produce accurate or dependable results; on some AF certificates,
issued by private laboratories, lot identification is often inadequate to
enable dependable guarantees on the relationship between sample, lot
and certificate; the official controls on returned lots is inadequate. It is
therefore appropriate to subject Brazil nuts in shell originating in or
consigned from Brazil to special, strict conditions to provide a high
level of protection to public health.
6. It is necessary that Brazil nuts be collected, sorted, handled,
processed, packaged and transported following good hygiene
practices. It is also necessary to establish the levels of AFB1 and AFT
in samples taken from consignment immediately prior to their
dispatch from Brazil. The sampling and the analysis should be
performed in accordance with Commission Directive 98/53/EC of 16
July 1998 laying down the sampling methods and the methods of
analysis for the official control of the levels for certain contaminants
in foodstuffs, as amended by Directive 2002/27/EC of 13 March
2002.
7. Brazil should provide documentary evidence to accompany each
consignment of Brazil nuts, relating to the conditions of collection,
sorting, handling, processing, packaging and transport, as well as the
results of laboratory analysis of the samples taken from consignment

for levels of AFB1 and AFT.
8. From the findings of the FVO's mission, it may be concluded that
Brazil cannot ensure currently dependable analytical results or
guarantee lot integrity in respect of certification of consignments of
Brazil nuts. Therefore, any certificate issued for Brazil nuts from
Brazil raises serious doubts with regard to its reliability. Furthermore,
it may also be concluded that current official controls on returned lots
are inadequate. It is therefore appropriate to impose strict conditions
on the return of nonconforming lots. In the event that those strict
conditions are not complied with, subsequent non-conforming lots
should be destroyed.
9. It is therefore necessary in order to safeguard public health that all lots
of Brazil nuts imported into the Community, are subjected to
sampling and analysis for their AF level by the competent authority of
the importing Member State prior to release onto the market.
10. In the interests of public health, Member States should provide the
Commission with periodical reports of all analytical results of official
controls carried out in respect of consignments of Brazil nuts. Such
reports should be in addition to the notification obligations under the
Rapid Alert System for Food and Feed established under Regulation
(EC) No 178/2002.
11. The measures provided for in this Decision are in accordance with the
opinion of the Standing Committee on the Food Chain and Animal
Health.
Therefore the Commission adopted these decisions: (i) restrictions on

imports of Brazil nuts in shell originating in or consigned from Brazil, (ii)
sampling and analysis of Brazil nuts by the competent authority of Brazil, (iii)
coding at points of entry into the EU for consignments of Brazil nuts,
improving traceability (iv) obligations on member States as regards imports of
Brazil nuts from Brazil, (v) if a consignment is split, a copy of the analytical
report shall accompany each part of consignment and (vi) consignments of
Brazil nuts not complying with the maximum levels for AFB1 and AFT may be
returned to the original country [13].
Some years later, the Scientific Panel on Contaminants in the Food Chain
(CONTAM Panel) of the European Food Safety Authority (EFSA) adopted an
opinion related to the potential increase of consumer health risk by a possible
increase of the existing maximum levels for AF in almonds, hazelnuts and
pistachios and derived products in January 2007 (Question No EFSA-Q-2009-

00675).
This risk assessment was requested by the European Commission
following discussions at the Codex Committee for Food Additives and
Contaminants (CCFAC) where the setting of higher levels than the 4 g/kg,
the current EU maximum level for AF in almonds, hazelnuts and pistachios,
had been proposed.
The CONTAM Panel concluded in its assessment that changing the
maximum levels for AF from 4 to 8 or 10 g/kg in almonds, hazelnuts and
pistachios would have a minor impact on the estimates of dietary exposure,
cancer risk and the calculated margin of exposures [17].
Based on the information which was available in 2007 the CONTAM
Panel concluded that public health would not be adversely affected by
increasing the levels for AF from 4 g/kg to 10 g/kg for all tree nuts. The
CONTAM Panel, however, reiterated its previous conclusion that exposure to
AF from all sources should be as low as reasonably achievable, because AF
are genotoxic and carcinogenic, and that priority should be given to reducing
the numbers of highly contaminated foods reaching the market, irrespective of
the commodity involved [17].
Although the limits imposed by the European Union are too restrictive,
there was an attempt in recent regulations [18] to abide by the maximum
permitted limits of the Codex Alimentarius concerning tree nuts. An AF limit
of 10 and 15 g/kg, for ready-to-eat (RTE) and destined for further processing
(DFP), respectively, was established for Brazil nuts. Under these regulations,
this relaxation in the legislation does not result in increased consumer
exposure to AF [5, 17].
Data Analysis of Imports of Brazil Nuts from 2002-2010
An analysis on the Brazil nut, the most valued NTFP from Amazon
biome, exported to Europe, is focused on in this chapter. The period of this
studied was from 2002 to 2010.
This chapter discusses the EU import restrictions for Brazil nut from
Brazil, with focus on data of Brazil nuts according the Rapid Alert System for
Food and Feed (RASFF) also it was pointed the efforts made by the sectors
involved in the agribusiness of Brazil nuts in Brazil in order to contribute to
protection of both domestic and international consumers from possible health
hazard caused by AF.
The Rapid Alert System for Food and Feed (RASFF)

and Notifications in Brazil Nuts
The Rapid Alert System for Food and Feed (RASFF) was put in place to
provide food and feed control authorities with an effective tool to exchange
information about measures taken responding to serious risks detected in
relation to food or feed. This exchange of information helps Member States of
EU to act more rapidly and in a coordinated manner in response to a health
threat caused by food or feed.
The occurrence data in the RASFF notifications concerning mycotoxins
provide basis for the analysis of the situation and evaluation of tendencies in
AF Brazil nuts levels. This Chapter analyses the data collected by RASFF in
2003, 2004, 2005, 2006, 2007, 2008 and 2009 [19, 20, 21, 22, 23, 24, 25, 26]
related to AF and nuts contamination based on the data of the rapid alert
system of the EU. Once AF is the major mycotoxin, especially in-shell Brazil
nuts, and is found at concerning levels, imported products are 100% AF
regulated by specific Commission Decision [27].
According to the European Commission Directorate General for Health
and Consumer Affairs, through the Rapid Alert System for Food and Feed
(RASFF), the number of notifications based on AF in 2009 (638 notifications)
has significantly decreased compared to 2008 (902). AF findings in nuts, nut
products and seeds generated 518 notifications (Table 1). Brazil received 16
notifications, 7 of which concerned Brazil nuts (4 notifications on in-shell nuts
and 3 notifications on Brazil nut kernels from Bolivia) [26]. The Table 1
compares the border notification on general AF contamination in all products,
in nuts and seeds, and in Brazil nuts. Freitas-Silva and Venncio [5] pointed
that these numbers reflected a little progress on the production chain of Brazil
nuts in Brazil, on the steps represented by an implementation of good
manufacturing practices, appropriate sampling plans, favorable analysis
conditions and certification of the final product. On the other hand, the
numbers of the AF notifications in Brazil nut may suggest the option to export
shelled Brazil nuts instead of in-shell ones since the EU legislation only
requires 100% screening on import in-shell Brazil nuts from Brazil.
According to RASFF reports, since the AF control in Brazil nuts by
regulation was implemented [13], 101 lots of Brazil nuts were rejected (Figure
2). This regulation led to a greater control of the final product, with a high
reduction in the incidence of AF since 2005. An exception occurred in 2009,
which showed alarming levels of AF (Figure 2). These findings are the results
of a joint effort of governmental and nongovernmental associations to better

organization of the complex chain of Brazil nut.
Table 1. Border rejection notification on lots with aflatoxins in general

seed and nuts, and in Brazil nuts per yeara
Aflatoxins 2002 2003 2004 2005 2006 2007 2008 2009

In all 288 763 844 947 800 705 902 638
commodities
Nuts and seeds na 695 675 827 684 568 710 517
Brazil nuts 48 16 2 5 2 2 3 7b
a
Data adapted from RASFF Annual Report (2002-2009).
b
4 samples from Brazil and 3 from Bolivia.
na: not available.
This adjustment in regulation brought the greater control as it can be

observed in Figure 3. However, if the subsequent EU legislation of 2010
would have felt implemented since the beginning of the intervention about 10
to 15% of the lots disposed of, could have been sold (Figure 3). According to
Table 2, 11 samples would have been accepted if 2010 regulation was applied
before, to the RTE situation. This percentage is statistically significant at
p<0.001 (z=-3.423). The Table 2 also shows that 12 samples would have been
accepted if 2010 regulation was applied before, to the DFP situation. This
percentage is statistically significant at p<0.001 (z=-3.575). These values
represent an economic and social impact, considering the complex chain that
Brazil nut has.
Figure 2. Brazil nuts lots contaminated with AF (g/kg) rejected by the EU between
2001 and 2011. Number of rejected lots= 98. Data from RASFF Annual Report (2002
to 2011).
Table 2. Possibility of frequency and acceptance rate of Brazil nut

samples, before and after 2010 regulation, for RTE and DFP samples
(N=98)
Frequency Percentage Proportion Test

RTE samples
Accepted Samples with
0 0 -3.423a
regulation <2010
11 11.24%
regulation >2010
DFP samples
0 0 -3.575a
regulation <2010
12 12.20%
regulation >2010
a
significance at 0.001.
Data from RASFF Annual Report (2002-2009).
Iran pistachios were also regulated by EU and according to Cheraghali and

Yazdanpanah [28] the multi-approach intervention to prevent and control AF
contamination in pistachio nuts was fruitful, being observed a significantly
reduction of AF pistachios levels. Those authors also reported that it was very
difficult to convince both national authorities and private sectors to spend
more resources on research in this area, but the costs incurred because of
rejection of consignments would create a logic base for any development in
this area, since the importance of pistachio nuts for Iran economy.
This question has been addressed by Wu [29] which states that the
complex effects of regulatory limits for mycotoxins on price, trade, public
health, selling and purchasing decisions of nations affects the producing
countries that face economic losses. These losses are enhanced by other
indirect losses by far more dramatic for local populations. Since the highest
quality crop is exported to the developed countries, the lower quality one is
consumed internally, yielding to chronic to acute intoxication cases. This
author still points out its combination with the wide spread of malnutrition and
the lack of health care.
Number of lots
Figure 3. Estimated percentage of lots that could be commercialized in the EU from

samples discarded. Data adapted from RASFF Annual Report (2002-2009).
Another question is also addressed to a direct impact on the economy of

developing countries: due to a lack of monitoring at the export points, or if
monitoring is present a lack of confidence in the existing test management,
exported goods get rejected at the importing points of developed countries
leading to pricing pressure [30]. The value of RASFF system is unquestionable
and it fulfills its intended function, since that the system is a significant source
of valuable information. However, for risk assessment purposes, another

additional information is needed [31]. Szeits-Szab and Szab [31] suggested
RASFF to provide data on the ratio of all/tested/positive lots and to provide
not only the positive results, but also the exact mycotoxins level of every
analyzed sample. In this way it would be more interesting for risk assessment.
Brazilian Actions for Guarantee Brazil Nuts Food Safety
The incidence and frequency of Brazil nut contamination by AF has been

monitored by the Ministry of Agriculture of Brazil since 1998. The data
concerning the occurrence of AF in Brazil nut samples obtained from export
batches and batches rejected by importing countries between 2005 and 2006,
analyzing only the edible portion (kernels), demonstrated that about 85% of
294 samples showed no detectable levels of AFB1. In AF-positive samples, the
lower AFT limit ranged from 0.4 to 2.42 g/kg and only 13 samples (4.4%)
showed levels above 20 g/kg [1]. These results are favorable, in comparison
with previous years. In 2003, Brazil was audited by the EU with regard to the
production and processing of Brazil nuts. Exports of the product were totally
restricted since the publication of Decision 2003/493/EC [13]. This publication
established criteria and conditions for Brazil in order to export in-shell Brazil
nuts to the EU. This included the implementation of good manufacturing
practices, appropriate sampling plans, favorable analysis conditions and
certification of the final product [14]. This measure represented the suspension
of Brazilian exports, which caused an enormous socioeconomic impact on the
entire production chain and led to a stoppage of Brazil nut exports to the EU
[5].
In 2004, the Programa de Alimentos Seguros (PAS) Campo (Food
Safety Program - Field), a joint national action by SENAI / SEBRAE /
EMBRAPA, was structured as a program from farm to fork, aiming at
producing a document that would instruct producers on how to make a better
control and monitoring throughout the Brazil nut chain. The program
integrates activities of monitoring, control, inspection, and tracking of
contaminants, including mycotoxins. It should be implemented throughout the
production chain, promoting and establishing Good Agricultural Practices
(GAP) and Hazard Analysis and Critical Control Points (HACCP) principles
in order to certify their conformity with a food safe product [32].
Corrective and continuous actions to improve product quality are still
necessary for reduction AFT levels in this product [33].
To prevent and reduce contamination of the Brazil nuts by AF, the

Ministry of Agriculture, Livestock and Supply of Brazil through Instruo
Normativa (Normative Instruction) n 11, of march 2010 [32] established
under the National Security and Quality of Plant Products and the National
Plan for Control of Residues and Contaminants in Plant Products, the criteria
and procedures to control hygiene and sanitary conditions of the Brazil nut and
its by-products intended for human consumption in the domestic market,
import and export along the production chain [34].
Nowadays, AFT limit for Brazil nuts in Brazil varies from 10 to 20 g/kg,
to RTE and DFP, respectively. This new limit was set by the Ministry of
Health of Brazil through its regulatory agency (ANVISA) which has recently
reviewed and updated the national mycotoxins regulation [35]. The current
limit for AF in Brazil nuts is in agreement with EU levels, in order to meet
food safety standards, since the consumption of contaminated Brazil nuts can
pose a risk to the consumers health, both nationally and internationally, in
addition to causing serious losses to the economy and agribusinesses due to a
rejection of contaminated consignments by the market.
CONCLUSION
There is no easy or rapid solution to completely eliminate mycotoxins in
nuts or to put the commodities within the regulation limits. The impact of
regulations on Agro-food trade is not only a hindrance for exporters in
developing countries, because they cannot reach the limits or because their
products were not safe. It is mainly due the lack in infrastructure to assist all
the chain of monitoring, testing and certification. Without this infrastructure it
will be impossible to demonstrate compliance of products with the regulation
of the importing country.
With regard to AF, the management of the entire Brazil nut chain is still a
challenge. This wild commodity needs to be safely offered to consumers,
especially for the ones who expect to eat not only a nut but also a functional
food, because of its health-related appeal, and mostly because of its oil
characteristics and high selenium content. Prevention of contamination by
Aspergillus spp. through good handling practices is still the best measure to
avoid AF in Brazil nuts and to ensure the quality and safety of this product [5]
and needs to continuous investment to keep quality of the product [36, 37].
It still needs initiative to help to achieve food safety through of assisting
national governments and through working closely with regional economic
organizations to develop continuous local program for food safety which

optimize regional conditions for attaining food safety in trade policy area.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
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[31] Szeitz-Szab, M. & Szab, E. (2007). Presence of mycotoxins in food:
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risk assessment? Acta Aliment., 36, 127-138.
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(2004). Projeto PAS Campo (Qualidade e Segurana dos Alimentos).
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/SEDE. 61p.
[33] Lima, A. M.; Gonalves, E. C.; Andrade, S. S.; Barbosa, M. S.; Barroso,
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Chapter 6
POLYMORPHISMS OF DNA REPAIR

GENES AND TOXICOLOGICAL EFFECTS
OF AFLATOXIN B1 EXPOSURE
Xi-Dai Long1, 2,*, Jin-Guang Yao2*, Qian Yang3,

Cen-Han Huang4, Pinhu Liao4, Le-Gen Nong4,
Yu-Jin Tang4, Xiao-Ying Huang2, Chao Wang4,
Xue-Ming Wu3, Bing-Chen Huang3, Fu-Zhi Ban5,
Li-Xia Zeng6, Yun Ma7, Bo Zhai1, Jian-Jun Zhang1,
Feng Xue1, Cai-Xia Lu8 and Qiang Xia1
1
Department of Liver Surgery, Ren Ji Hospital, Shanghai Jiao Tong
University School of Medicine (RJHSJTM), P.R. China
2
Department of Pathology, the Affiliated Hospital of Youjiang Medical
College for Nationalities (AHYMCN), P.R. China
3
Department of Cell Biology and Anatomy, Louisiana State University
Health Sciences Center, Baton Rouge, LA, US
4
Department of Medicine, AHYMCN, P.R. China
*
Address all correspondence to: Xi-Dai Long, Department of Liver Surgery, RJHSJTM, No.
1630, Dongfang Road, Shanghai 200127, P.R. China; or Department of Pathology,
AHYMCN, No. 98, Chengxiang Rd., Baise City, Guangxi Zhuang Autonomous Region
533000, P.R.China. Email: sjtulongxd@263.net. Qiang Xia, Department of Liver Surgery,
RJHSJTM, No. 1630, Dongfang Road, Shanghai 200127, P.R. China. Email:
xiaqiang@medmail.com.cn or xiaqiang@sjtu.edu.cn. Tel.: +86 21 68383775. Fax.: + 86 21
58737232
These authors contributed equally to this work.

126 Xi-Dai Long, Jin-Guang Yao, Qian Yang et al.
5
Department of Test, the Affiliated Southwestern Hospital of Youjiang
Medical College for Nationalities, P.R. China
6
Department of Pathology, the Affiliated Tumor Hospital, Guangxi
Medical University (GXMU), P.R.China
7
Department of Pathology, the First Affiliated Hospital, GXMU,
P.R.China
8
Department of Medicine, Min Hang Hospital of Shanghai, P.R. China
ABSTRACT
Aflatoxin B1 (AFB1) is an important genic toxin produced by the
moulds Aspergillus parasiticus and Aspergillus flavus. AFB 1 is
metabolized by cytochrome P450 enzymes to its reactive form, AFB1-
8,9-epoxide (AFB1-epoxide), which covalently binds to DNA and induces
DNA damage. DNA damage induced by AFB1, if not repaired, may cause
such genic tox toxicological Effects as DNA adducts formation, gene
mutations and hepatocellular carcinoma (HCC). During the repair process
of DNA damage produced by AFB1, DNA repair genes play a central
role, because their function determines DNA repair capacity. In this
study, we investigated the association between seven polymorphisms
(including rs25487, rs861539, rs7003908, rs28383151, rs3734091,
rs13181, and rs2228001) in DNA repair genes XPC, XRCC4, XRCC1,
XRCC4, XPD, XRCC7, and XRCC3, and toxicological effects of AFB 1
using a hospital-based case-control study. Toxicological effects of AFB1
were analyzed by means of the levels of AFB 1-DNA adducts, the mutant
frequency of TP53 gene, and the risk of AFB1-related HCC. We found
that the mutants of XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and
XRCC3 had higher AFB1-DNA adducts levels, compared with the wilds
of these genes (3.276 vs 3.640 mol/mol DNA for rs25487, 2.990 vs
3.897 mol/mol DNA for rs861539, 2.879 vs 3.550 mol/mol DNA for
rs7003908, 3.308 vs 3.721 mol/mol DNA for rs28383151, 3.229 vs
3.654 mol/mol DNA for rs3734091, 2.926 vs 4.062 mol/mol DNA for
rs13181, and 3.083 vs 3.666 mol/mol DNA for rs2228001,
respectively). Furthermore, increasing risk of TP53 gene mutation and
HCC was also observed in these with the mutants of DNA repair genes.
These results suggested that polymorphisms of DNA repair genes might
modify the toxicological effects of AFB.
Keywords: Aflatoxin B1, DNA repair gene, toxicological effect,

polymorphism, hepatocellular carcinoma
Polymorphisms of DNA Repair Genes and Toxicological Effects 127
ABBREVIATIONS
AFB1, aflatoxin B1;
HCC, hepatocellular carcinoma;
DSB, double strand break;
NHEJ, the non-homologous end joining;
OR, odds ratio;
PCR, polymerase chain reaction;
TP53M, the hot-spot mutation at codon 249 of TP53 gene;
XRCC1, x-ray repair cross-complementing group 1;
rs25487-CC, the homozygotes of XRCC1 rs25487 C alleles;
rs25487-CT, the heterozygotes of XRCC1 rs25487 C and T allele;
rs25487-TT, the homozygotes of XRCC1 rs25487 T alleles;
rs861539-GG, the homozygotes of XRCC3 rs861539 G alleles;
rs861539-GA, the heterozygotes of XRCC3 rs861539 G and A allele;
rs861539-AA, the homozygotes of XRCC3 rs861539 A alleles;
rs7003908-AA, the homozygotes of XRCC7 rs7003908 A alleles;
rs7003908-AC, the heterozygotes of XRCC7 rs7003908 A and C allele;
rs7003908-CC, the homozygote of XRCC7 rs7003908 C alleles;
rs28383151-GA, the heterozygotes of XRCC4 rs7003908 G and A allele;
rs28383151-AA, the homozygote of XRCC4 rs28383151 A alleles;
rs3734091-GT, the heterozygotes of XRCC4 rs7003908 G and T allele;
rs3734091-TT, the homozygote of XRCC4 rs3734091 T alleles;
XPD, the xeroderma pigmentosum complementation group D;
rs13181-GG, the homozygotes of XPD rs13181 G alleles;
rs13181-GA, the heterozygotes of XPD rs7003908 G and A allele;
rs13181-AA, the homozygote of XPD rs13181 A alleles;
XPC, the xeroderma pigmentosum complementation group C;
rs2228001-GG, the homozygotes of XPC rs2228001 G alleles;
rs2228001-GA, the heterozygotes of XPC rs7003908 G and A allele;
rs2228001-AA, the homozygote of XPC rs2228001 A alleles.
1. INTRODUCTION
Aflatoxin B1 (AFB1) was am important member of aflatoxin family highly
substituted coumarins containing a fused dihydrofurofuran moiety [1-3]. This
aflatoxin is mainly produced by some strains of the moulds Aspergillus
parasiticus and Aspergillus flavus, and is structurally characterized by fusion
of a cyclopentanone ring to the lactone ring of the coumarin moiety [2, 3].
AFB1 was discovered as a contaminant of human and animal food, especially
peanuts (ground nuts), core, soya sauce, and fermented soy beans in tropical
areas such as the Southeastern China as a result of fungal contamination
during growth and after harvest which under hot and humid conditions. This
type of toxin has three toxicological effects: a. genotoxicity, mainly inducing
the formation of AFB1-DNA adducts and the hot-spot mutation of p53 gene; b.
the attraction of specific organs, especially liver; and c. carcinogenicity,
primarily causing hepatocellular carcinoma (HCC) [1-9]. Increasing evidences
have shown that DNA damage by AFB1 plays the central role of
carcinogenesis of HCC-related to this toxin in the toxic studies [2, 3, 10].
Today, AFB1 has been classified as a known human carcinogen by the
International Agency for Research on Cancer [1-3]. However, more and more
epidemiological evidence has exhibited that although many people are
exposed to the same levels of AFB1, only a relatively small proportion of
exposure person feature the toxicological effects of AFB1 such as showing
gene mutations and developing HCC [2, 3]. This indicates individual DNA
repair capacity related to AFB1-induced DNA damage might be associated
with the toxicological effects of AFB1 exposure. Here, we investigated the
effects of genetic polymorphisms in DNA repair genes XRCC1, XRCC3,
XRCC4, XRCC7, XPD, and XPC (including rs25487, rs861539, rs7003908,
rs28383151 and rs3734091, rs13181, and rs2228001) on the toxicological
effects of AFB1 exposure through the analysis of AFB1-DNA adducts amount,
TP53 gene mutation frequency, and HCC risk.
2. MATERIALS AND METHODS

2.1. Study Design
Because the toxicological effects of AFB1 exposure can be elucidated by

the analysis of AFB1-DNA adducts, TP53M, and HCC risk, a hospital-based
case-control study was designed and conducted (Figure 1). In this study, we
firstly analyzed the amount of AFB1-DNA adducts and the frequency of
TP53M in the liver cancer tissue samples, next evaluated HCC risk using
individual matching case-control design.
Figure 1. Study design. This study, including 1486 hepatocellular carcinoma (HCC)
patients and 1996 controls (matching to HCC cases based to age, sex, race, HBV
status, and HCV status), was conducted in the high AFB 1 exposure areas. The
corresponding samples, including peripheral blood samples for the analysis of
genotypes in the polymorphic loci of DNA repair genes and HCC risk, and cancerous
tissues samples for the analysis of AFB 1-DNA adducts and TP53 mutation, were
collected and tested.
2.2. Study Subject
Cases
A total of 1486 HCC cases treated at the affiliated hospitals of Guangxi
Medical University and Youjiang Medical College for Nationalities during the
period from January 2004 through December 2012 were recruited for
participation in the study. All cases were the residents of Guangxi Zhuang
Autonomous Region, a high AFB1 exposure area. The cases included in this
study, representing a significant proportion (>90%) of HCC patients in the
Guangxi population, were identified by histopathological diagnosis in 100% of
the HCC cases. All patients gave informed consent for participation and were
interviewed uniformly before surgery by a well-trained interviewer. The
questionnaire used in the interview sought detailed information on current and
past living habits, occupational history, family disease history, dietary history
and general demographic data. Clinical pathological data (including cirrhosis,
tumor size, PVT, and tumor stage), -fetoprotein (AFP), hepatitis virus B
(HBV) and hepatitis virus C (HCV) infection information, and therapeutic data
were collected from medical records in the hospitals by a Youjiang Cancer
Institution staff member.
At the same time of interview, 4 mL of peripheral blood was obtained for
the extraction of genomic DNA for each case. Additionally, surgically
removed tumor samples of all cases were collected for analyzing AFB1-DNA
adducts amount and TP53M frequency. Tumor tissue samples were surgically
dissected into small pieces, frozen immediately in liquid nitrogen and stored at
80 C. In this study, those hepatitis B surface antigen (HBsAg) positive and
anti-HCV positive in their peripheral serum were defined as groups infected
with HBV and HCV. Liver cirrhosis was diagnosed by pathological
examination, and stages of tumor were confirmed according to the tumor-
nodes-metastasis (TNM) staging system.
Controls
To investigate the oncogenicity toxicological effects of AFB1 exposure,
we designed and established case-control study. During the same period of
HCC investigation, controls without any evidence of liver disease were
randomly selected from a pool of healthy volunteers who visited the general
health check-up centers of the same hospitals for their routinely scheduled
physical examinations supported by local governments. In the present study, a
total of 2032 controls were enrolled and interviewed. Because of 36 volunteers
dropped out, 1996 controls were included in final analysis. To control the
effects of confounders which were associated with the distribution of
genotypes or the exposure of AFB1, controls were individually matched (1:1 or
2:1) to cases based on ethnicity (Han, Minority), sex, age ( 5 years), and
hepatic B virus (HBV) and hepatic C virus (HCV) infection. After giving
written consent, demographic information (including age, sex, race, medical
history, family disease history, dietary history, and living history), and HBV
and HCV infection information were collected in the hospitals using a
standard interviewer-administered questionnaire. At the same time, 4 mL of

peripheral blood was obtained for the extraction of genomic DNA.
Ethical Protocol
The study protocol was been carried out in accordance with "Ethical
Principles for Medical Research Involving Human Subjects" (World Medical
Association Declaration Of Helsinki, 2004) and approved by Institutional
review boards from Guangxi Cancer Institute, and the Medical Research
Council from the corresponding hospitals.
2.3. DNA Extraction
DNA was extracted from HCC cancerous tissues from all cancer patients
in a 1.5 mL microcentrifuge tube for deparaffinization and proteinase K
digestion, as described by standard procedures (Protocol #BS474, Bio Basic,
Inc., Ontario, Canada). DNA was stored at 48 oC until additional analysis. For
peripheral blood samples from all cases and controls, DNA was isolated using
classical phenol-chloroform extraction.
2.4. AFB1-DNA Adducts Assay
DNA Samples Preparation

In this study, the amount of AFB1-DNA adducts in cancerous tissues
samples were evaluated competitive enzyme-linked immunosorbent assay
(ELISA) [11-14]. To convert any N-7 adduct to AFB1-FAPy adducts, DNA
was treated with 15 mM Na2CO3 and 30 mM NaHCO3 (pH 9.6) for 2 hours,
precipitated with 2.5 volumes of 95% ethanol, and then redissolved in 10 mM
Tris-HCl (pH 7.0). The DNA samples were reprecipitated, dissolved in 1
PBS, and denatured by boiling for 5 min. After that, AFB1-FAPy adducts were
quantitated by ELISA using monoclonal antibody 6A10 (Novus Biologicals
LLC, catalog # NB600- 443).
ELISA
ELISA was accomplished as described previously [11-14]. Briefly,
Immulon 2 plates (Dynatech Laboratories, Chantilly, VA) were first coated
with 5 ng of imidazole ring-opened AFB1-DNA in 1 PBS by drying
overnight at 37 C. The test solutions contained unbound AFB1-DNA and
6A10 antibody. Goat anti-mouse IgG alkaline phosphatase (1:1500) and then
p-nitrophenyl phosphate (1 mg/mL in 1 M diethanolamine, pH 8.6) was added
to the DNA. After 90 min incubation at 37 C, absorbance at 405 nm was read
on a Bio-Tek Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT).
The standard curve for different imidazole ring-opened AFB1-DNA
concentration was drawn and adducts amount in samples were calculated
according to the corresponding standard curve. For the standard curve, highly
modified imidazole ring-opened AFB1-DNA was serially diluted with non-
modified denatured calf thymus DNA (R&D Systems, Inc., catalog # 9600-5-
D) such that 50 L contained from 0 to 1000 fmol adduct and 50 g DNA.
These samples were mixed with an equal volume of diluted 6A10 antibody (50
L, diluted 1:1.25106), added to the wells, and measured by competitive
ELISA [12-14].
For the test samples, 25 g heat-denatured DNA from cancerous tissues
samples in 50 L hydration solution was mixed with 50 L diluted antibody
before being added to the wells. The amount of AFB1-DNA in the test samples
was quantitated relative to a standard curve based on known concentrations of
AFB1-DNA. Each sample was measured in triplicate on the three different
assay dates. Adduct levels were ascertained according to the average of three
measures. The quality control for adduct assays was administered by blank and
positive controls.
2.5. TP53 Gene Mutation Analysis
Laboratory personnel were blinded to subject status. The hot-spot

mutation of TP53 gene (at codon 249, TP53M) was analyzed using the
TaqMan-PCR on iCycler iQ real-time PCR detection system (iQ5, Bio-Rad
Laboratories Inc., Hercules, CA). Primers for TP53M analysis were 5-TTG
GCT CTG ACT GTA CCA CCA T-3 and 5-TGG AGT CTT CCA GTG
TGA TGA TG-3; whereas probes were 5-FAM-ACC GGA GTC CCA TC-
MGB-3 and 5-VIC-AAC CGG AGG CCC AT-MGB-3 [15, 16]. Primers
and probes were synthesized by Introgen Bio., Ltd. (Shanghai, China) and
Applied Biosystems. PCR was carried out in a total volume of 25 L
consisting of 1 Premix Ex TaqTM (catalog # DRR039A, TaKaRa
Biotechnology (Dalian) Co., Ltd., Dalian, China), 0.2 M of each probe, 0.2
M of each primer, and 50 100 ng of genomic DNA. The PCR program had
an initial denaturation step of 2 min at 95 0C followed by 50 cycles of 10 sec at
95 0C and 1 min at 60 0C. For quality control, controls were included in each
run, and repeated genotyping and sequencing of a random 10% subset yielded
100% identical results. To analysis, the information of TP53M was divided
into two groups: TP53M negative status (spot mutation at codon 249 of TP53
gene not detected), and positive status (spot mutation at codon 249 of TP53
gene detected).
2.6. Polymorphisms Selection of DNA Repair Genes

and Genotyping
In this study, we only selected these single nucleotide polymorphisms

(SNPs) in the DNA repair genes that might modify AFB1-related HCC risk.
According to our previous results, a total of 7 SNPs , including rs25487 (in the
XRCC1), rs861539 (in the XRCC3), rs7003908 (in the XRCC7), rs28383151
and rs3734091 (in the XRCC4), rs13181 (in the XPD), and rs2228001 (in the
XPC), were finally analyzed in the present study (Table 1).
The genotypes of DNA repair genes were genotyped using the previous
TaqMan-PCR methods on iCycler iQ real-time PCR detection system (iQ5,
Bio-Rad Laboratories Inc.). Primer and probe sets and annealing temperatures
used for TaqMan-PCR assay are shown in Table 2. For quality control,
controls were included in each run, and repeated genotyping and sequencing of
a random 5% subset yielded 100% identical genotypes.
2.7. Statistical Analysis
Genotype data were analyzed as trichotomous variables, including wild

homozygotes (wild genotype), heterozygotes (heterozygotes genotype), and
mutant homozygotes (mutant genotype). For the association analysis between
polymorphisms in the DNA repair genes and the amount of AFB1-DNA
adducts, the different distribution of groups was tested using the Students t
test. In this analysis, each genotype was evaluated as a categorical variable
with three levels (homozygous low activity, heterozygous, and homozygous
high activity). The mean value (with SD and SE) of AFB1-DNA adducts levels
for each high activity group was calculated and compared with low activity
group (wild genotypes).
Table 1. The characteristics of polymorphisms in the DNA repair genes
Polymorph- Alleles Genotypes Codon Amino

ism Gene Chr:bp Wild Mutant Wild Heterozygotes Mutant No. acid Geno-typing
rs25487 XRCC1 19:44055726 C T CC CT TT 399 Arg/Gln TaqMan-PCR
rs861539 XRCC3 14:104165753 G A GG GA AA 241 Thr/Met TaqMan-PCR
rs7003908 XRCC7 8:48770702 A C AA AC CC / / TaqMan-PCR
rs28383151 XRCC4 5:82406873 G A GG GA AA 56 Ala/Thr TaqMan-PCR
rs3734091 XRCC4 5:82500734 G T GG GT TT 247 Ala/Ser TaqMan-PCR
rs13181 XPD 19:45854919 T G TT TG GG 751 Lys/Gln TaqMan-PCR
rs2228001 XPC 3:14187449 T G TT TG GG 939 Lys/Gln TaqMan-PCR
Table 2. Technical details of TaqMan-PCR analysis
Polymorphism Gene Primers Probes

rs25487 XRCC1 5'-GTGGGTGCTGGACTGTC-3' 5'-FAM-CCTCCCGGAGGTAA-MGB-3'
5'-GCAGGGTTGGCGTGTGA-3' 5'-VIC-CCCTCCCAGAGGTAA-MGB-3'
rs861539 XRCC3 5'-CCAGGGCCAGGCATCTG-3' 5'-FAM-CAGCATGGCCCCCA-MGB-3'

5'-CAGCACAGGGCTCTGGA-3' 5'-VIC-CAGCGTGGCCCCCA-MGB-3'
rs7003908 XRCC7 5'-CCTACCTCACGAACTCAGCAATT-3' 5'-FAM-CTAAGAGTCCGCTGTTT-MGB-3'

5'-GCTGCCAACGTTCTTTCCTTATAGT-3' 5'-Hex-CCTAAGAGTCAGCTGTTT-MGB-3'
rs28383151 XRCC4 C__58444701_10a C__58444701_10

rs3734091 XRCC4 5'-TGAGGAAAGTGAAAACCAAACTGATCT-3' 5'-FAM-CCTGAAGACAACCC-MGB-3'
5'-GCCCAAATAAGATATTCAACAGAGGAGAT-3' 5'-HEX-CCTGAAGCCAACCC-MGB-3'
rs13181 XPD 5'-AGTCACCAGGAACCGTTTATGG-3' 5'-HEX-CTCTATCCTCTGCAGCG-MGB-3'

5'-TCTGTTCTCTGCAGGAGG ATC-3' 5'-FAM-TATCCTCTTCAGCGTCT-MGB-3'
rs2228001 XPC 5-AGCAGCTTCCCACCTGTTC-3 5-FAM-CACAGCTGCTCAAAT-MGB-3

5-GTGGGTGCCCCTCTAGTG-5 5-Hex-CTCACAGCTTCTCAAAT-MGB-3
a
From the Applied Biosystems.
Frequency tables of independent variables (including TP53M variable and

HCC risk variables) and genotype data were evaluated for statistical
significance by Pearson's 2. To analyze the risk for gene mutation and HCC
associated with each genotype while adjusting for confounders, multivariable
logistic regression was done and odds ratios (OR) along with 95% confidence
intervals (95% CI) generated. In this type of the additive model, we treated
genotype as an ordinal variable (wild type coded as 0, heterozygote as 1, and
homozygotes variant as 2). For TP53M risk, unconditional logistic regression
model (enter method consisting of age, sex, race, AFB1 exposure, and HBV
and HCV infection status) was used for risk values. For HCC risk, based on
individually matched design of case-control study, we did conditional logistic
regression (with multivariate factors, including known causes of HCC among
the Guangxi population) to estimate ORs for risk of HCC and their 95% CIs.
In the present study, a P-value of < 0.05 was considered statistically
significant. All statistical analyses were done using SPSS version 18 (SPSS,
Inc., Chicago, IL).
3. RESULTS
3.1. The Demographic Characteristics of HCC Cases
The 1486 HCC cases were from high AFB1 exposure areas and included
in the final analysis and the demographic data of these patients is shown in
Table 3. The HBV and HCV infective rates were 72.95% (1084 of 1486) and
18.57% (276 of 1486), respectively. They were characterized by age, 49.32
11.43 years, and more commonly male. Among them, 72.68% (1080 of 1486)
cases featured liver cirrhosis and about 70% patients were in the second stage
of TNM system.
3.2. DNA Repair Genes Polymorphisms and AFB1-DNA Adducts

Levels
Cancerous tissue samples from the 1486 HCC patients were examined for
AFB1-DNA adducts. A mean SD of 3.296 1.710 mol/mol DNA was
observed in the tumor tissues samples (Table 4). To analyzed the represents of
AFB1-DNA adducts levels in cancerous tissue samples for AFB1 exposure, we
also investigated the levels of AFB1-DNA adducts in peripheral blood

leukocytes and AFB1-albumin adducts in peripheral blood serum, other two
important biomarkers for AFB1 exposure evaluation, according to previously
published methods (Table 4) [14, 16]. Results showed that AFB1-DNA
adducts levels in cancerous tissue samples were lineally related to these two
biomarkers, and higher DNA adducts levels were found in the tumor tissues
samples. Because the corresponding DNA adducts in cancerous tissues were
higher and more stable compared with peripheral blood, this adducts were
used for the following the toxicological effects of AFB1 exposure analysis.
Table 3. The characteristics of HCC cases
Characteristics
Total, n (%) 1486 (100.00)
Age (years)
Mean S.D. 49.32 11.43
Sex
Male, n (%) 1120 (75.37)
Female, n (%) 366 (24.63)
Race
Han, n (%) 700 (47.11)
Minority, n (%) 786 (52.89)
HBV
HBsAg(-), n (%) 402 (27.05)
HBsAg(+), n (%) 1084 (72.95)
HCV
anti-HCV(-), n (%) 1210 (81.43)
anti-HCV(+), n (%) 276 (18.57)
AFP
Negative, n (%) 747 (50.27)
Positive, n (%) 739 (49.73)
Liver Cirrhosis
No, n (%) 406 (27.32)
Yes, n (%) 1080 (72.68)
TNM stage
I, n (%) 112 (7.54)
II, n (%) 1010 (67.97)
III, n (%) 326 (21.94)
IV, n (%) 38 (2.56)
Table 4. The levels of AFB1 adducts in cancerous tissues and

peripheral blood
Adducts Mean SE SD
ADATa 3.296 0.044 1.710
ADABb 1.983 0.029 1.126
AAAc 28.390 1.162 44.798
ln(AAA) d 2.980 0.021 0.796
a
ADAT, AFB1-DNA adducts in HCC cancerous tissues (mol/mol DNA).
b
ADAB, AFB1-DNA adducts in peripheral blood leukocytes (mol/mol DNA).
c
AAA, AFB1-albumin adducts in peripheral blood (fmol/mg).
d
ln(AAA), logarithmical transformation of AAA (ln fmol/mg).
Table 5. Polymorphisms in DNA repair genes and AFB1-DNA

adduct levels
Adduct levela
Gene Polymorphism Genotype Mean SD Pb
XRCC1 rs25487 CC 3.276 0.063
CT 3.264 0.069 0.899
TT 3.640 0.149 0.026
XRCC3 rs861539 GG 2.990 0.078
GA 3.216 0.065 0.025
AA 3.897 0.089 4.96210-14
XRCC7 rs7003908 AA 2.879 0.084
AC 3.347 0.065 1.66310-5
CC 3.550 0.082 1.75110-8
XRCC4 rs28383151 GG 3.308 0.054
GA 3.405 0.094 0.069
AA 3.721 0.128 2.86710-4
XRCC4 rs3734091 GG 3.229 0.051
GT 3.439 0.113 0.095
TT 3.654 0.141 0.005
XPD rs13181 TT 2.926 0.066
TG 3.253 0.070 0.011
GG 4.062 0.097 4.26510-6
XPC rs2228001 TT 3.083 0.071
TG 3.332 0.067 0.001
GG 3.666 0.032 3.40410-22
a
AFB1-DNA adducts levels in the HCC cancerous tissues (mol/mol DNA).
b
Calculated by genotypes with mutant alleles compared with wild homozygote.
In this study, the genotypes of DNA repair genes XRCC1, XRCC3,

XRCC4, XRCC7, XPD, and XPC were tested using TaqMan-PCR techniques.
We found the mutants of these DNA repair genes had higher levels of AFB1-
DNA adducts, compared with their wilds (Table 5). For example, these with
XRCC7 rs7003908-CC genotype, compared to those with XRCC7 rs7003908-
AA genotype (2.879 0.084 mol/mol DNA), faced an increasing amount of
AFB1-DNA adducts (3.550 0.082 mol/mol DNA, P < 0.01).
3.3. DNA Repair Genes Polymorphisms and TP53M
The status of hotspot mutation in codon 249 of the p53 gene (TP53M) in
cancerous tissue samples was evaluated by TaqMan-PCR among 1486 HCC
cases. One thousand one hundred-sixteen (75.10%) of the 1486 HCCs showed
TP53M. The frequency of hotspot mutation was not related with HBV
infection and HCV infection (P > 0.05, data not shown). However, individuals
with the heterozygotes of XRCC1 rs25487 (namely: rs25487-CT) or the
variant homozygotes of XRCC1 rs25487 (namely: rs25487-TT) were more
probably to have higher frequency of TP53M than those with the wild-type
homozygote of XRCC1 rs25487 (namely: rs25487-CC). Its adjusted ORs
(95% CIs) were 2.419 (1.863-3.141) for rs25487-CT and 5.028 (2.490-10.153)
for rs25487-TT, respectively (Table 6). Similar risk role was also found in the
association analysis between other 6 polymorphisms (including rs861539 (in
the XRCC3], rs7003908 (in the XRCC7], rs28383151 and rs3734091 (in the
XRCC4], rs13181 (in the XPD], and rs2228001 (in the XPC]) and TP53M
(Table 6).
3.4. DNA Repair Genes Polymorphisms and HCC Risk
To explore the correlation between these 7 polymorphisms and another

biomarker of the toxicological effect of AFB1 exposure (also called HCC risk),
we conducted a hospital-based case-control study according to our previously
published methods [14-16]. A total of 1486 HCC cases and 1996 individually-
matched (based on age, sex, race, and HBV and HCV infection status) controls
were included in the present risk analysis (Table 7). There were no significant
differences between cases and controls in terms of distribution of age, sex,
race, and HBV and HCV status as a result of individual matching (P > 0.05).
More detailed information has been reported in our previous studies. These
results suggest that HCC patient data were comparable to control data.
Higher frequency of mutants of DNA repair genes was observed in the
HCC patients than in the controls. Conditional logistic regression analysis
exhibited that mutant alleles increased about 2 to 6 fold of HCC risk value.
This risk was more noticeable under the conditions of mutant homozygotes
(Table 7). For example, HCC risk for the genotype with XRCC4 rs3734091-
GT was 1.460 (1.195-1.783); whereas risk value was 2.244 (1.676-3.003) for
XRCC4 rs3734091-TT genotype. These results suggested the risk of HCC was
associated with the number of mutant alleles of DNA repair genes XRCC1,
XRCC3, XRCC7, XRCC4, XPD, and XPC.
4. DISCUSSION
4.1. The Evaluation of Toxicological Effects of AFB1
A main toxicological effect of AFB1 is to induce DNA damage, consisting

of AFB1-DNA adducts and the hot-spot mutation of tumor suppressor gene
p53 at codon 249 (TP53M) [5, 17-19]. Thus, the toxicological effects of AFB1
exposure might be elucidated using the analysis of AFB1-DNA-adducts levels
and TP53M frequency in the liver tissues or other tissues [2]. AFB1 can induce
several DNA adducts formation, including AFB1-N7-Gua adduct, AFB1-FAPy
adduct, and so on. Among these adducts, AFB1-FAPy adduct is the imidazole
ring-opened product of AFB1-N7-Gua adduct, also the stable form of the later
adduct, and may play an important role in the development of HCC.
Moreover, the accumulation of this adduct is time-dependent and non-
enzymatic, and may have potential biological importance because of its
apparent persistence in DNA. Thus, many researchers in the relative fields
regard AFB1-FAPy adduct as a validated biomarker of AFB1 exposure [1, 2, 4,
10]. Increasing evidences have exhibited that AFB1-FAPy-adducts levels in
the liver or placenta tissues are lineally correlated with AFB1 exposure levels
and HCC risk [14, 15], suggesting this adduct should be regarded as a
toxicological elucidation biomarker of AFB1. Our previous studies have shown
that peripheral blood leukocytes' adduct levels were positively and linearly
related to AFB1-DNA adduct levels of the HCC cancerous tissue. These data
suggested that the levels of peripheral blood leukocytes' DNA adducts were
representative of the tissues' DNA-adduct levels and might be regard as a
biomarker for AFB1 exposure [14, 15].
Table 6. Polymorphisms in DNA repair genes and TP53M risk
TP53M (-) TP53M (+)

Gene Polymorphism Genotype n % n % ORa 95% CI P
XRCC1 rs25487 CC 258 69.73 519 46.51 Reference
CT 103 27.84 505 45.25 2.419 1.863-3.141 3.37110-11
TT 9 2.43 92 8.24 5.028 2.490-10.153 6.65110-6
XRCC3 rs861539 GG 150 40.54 359 32.17 Reference
GA 148 40.00 486 43.55 1.380 1.057-1.802 0.018
AA 72 19.46 271 24.28 1.524 1.102-2.108 0.011
XRCC7 rs7003908 AA 126 34.05 237 21.24 Reference
AC 149 40.27 514 46.06 1.883 1.416-2.505 1.37210-5
CC 95 25.68 365 32.71 2.089 1.526-2.861 4.36810-6
GA 58 15.68 242 21.68 1.688 1.229-2.318 0.001
AA 13 3.51 126 11.29 3.829 2.129-6.888 7.38710-6
GT 29 7.84 196 17.56 2.799 1.856-4.222 9.19110-7
TT 9 2.43 113 10.13 5.104 2.556-10.190 3.82610-6
Table 6. (Continued)
TP53M (-) TP53M (+)

XPD rs13181 TT 165 44.59 384 34.41 Reference
TG 140 37.84 467 41.85 1.458 1.120-1.898 0.005
GG 65 17.57 265 23.75 1.744 1.256-2.422 0.001
XPC rs2228001 TT 166 44.86 380 34.05 Reference
TG 157 42.43 539 48.30 1.500 1.162-1.936 0.002
GG 47 12.70 197 17.65 1.818 1.258-2.625 0.001
a
Adjusted by age, sex, race, HBV status, and HCV status.
Table 7. Polymorphisms in DNA repair genes and HCC risk
Controls HCCs
XRCC1 rs25487 CC 1437 71.99 777 52.29 Reference
CT 520 26.05 608 40.92 2.155 1.861-2.495 9.91810-25
TT 39 1.95 101 6.80 4.774 3.264-6.981 7.61410-16
GA 539 27.00 634 42.66 3.321 2.848-3.872 5.67110-53
Controls HCCs
AA 27 1.35 343 23.08 5.846 3.907-13.747 3.39910-67
XRCC7 rs7003908 AA 1141 57.16 363 24.43 Reference
AC 608 30.46 663 44.62 3.434 2.921-4.037 1.66410-50
CC 247 12.37 460 30.96 5.867 4.828-7.129 7.47810-71
GA 217 10.87 300 20.19 2.248 1.857-2.722 1.03410-16
AA 62 3.11 139 9.35 3.690 2.708-5.029 1.34110-16
GT 226 11.32 225 15.14 1.460 1.195-1.783 2.06710-4
TT 81 4.06 122 8.21 2.244 1.676-3.003 5.55510-8
XPD rs13181 TT 1214 60.82 549 36.94 Reference
TG 611 30.61 607 40.85 2.193 1.884-2.551 3.21610-24
GG 171 8.57 330 22.21 4.270 3.458-5.273 1.84110-41
XPC rs2228001 TT 988 49.50 546 36.74 Reference
TG 804 40.28 696 46.84 1.570 1.357-1.817 1.38810-9
GG 204 10.22 244 16.42 2.185 1.764-2.706 8.23110-13
a
Adjusted by age, sex, race, HBV status, and HCV status.
Our present study found more amount of AFB1-DNA adducts in cancerous

tissues than in the peripheral blood. Thus, AFB1-DNA adducts in tumor tissues
were furthermore analyzed in the following study.
As regard of the mutations of p53 gene, AFB1 mainly induces the
transversion of G T in the third position at codon 249 of TP53M. The
frequent value of TP53M is more persistent biomarker and more directly
represents genic toxic effects compared with AFB1-DNA adducts [6, 18, 20,
21]. Our study also showed more than 75% of HCC cases had TP53M in the
cancerous tissues. Additionally, HCC is the most common malignant tumors
caused by AFB1 exposure. More and more epidemiological studies have
shown HCC risk is related to different the toxicological capacity of AFB1,
suggesting that tumor risk value might be regard as a selective elucidative
marker for AFB1 toxic effects [2, 15].
Because of the aforementioned reasons, the toxic effects of AFB1
exposure were evaluated through the following three biomarkers: (1) AFB1-
DNA adducts amount in HCC cancerous tissues, (2) the frequency of TP53M,
and (3) HCC risk, in this study. Our results also exhibited these biomarkers
reflected AFB1 exposure information and represented the toxicological
capacity of AFB1.
4.2. XRCC1 Polymorphism and Toxicological Effects of AFB1
XRCC1 gene also calls RCC and is one of three submits of DNA repair
complex in the SSBR pathway (Gene dbase from PubMed). This gene spans
about 32 kb on chromosome 19q13.2 and contains 17 exons and 16 introns.
Its encoding protein (633 amino acids), consists of three functional domains:
N-terminal domain (NTD), central breast cancer susceptibility protein-1
homology C-terminal (BRCT I), and C-terminal breast cancer susceptibility
protein-1 homology C-terminal (BRCT II) [22-24]. This protein is directly
associated with Pol , DNA ligase III, and PARP, via their three functional
domains and is implicated in the core processes in single-strand break repair
(SSBR) and base excision repair (BER) pathway [22-24]. More than 50 SNPs
in the coding region of XRCC1 gene that lead to amino acid substitution have
been described (SNP database). Among these polymorphisms, rs25487
polymorphism (also called codon Arg399Gln polymorphism) is of special
concern, because this polymorphism resides in functionally significant regions
(BRCT II) and may be related to decreasing DNA repair activity [22, 25-27].
In this study, to investigate the genic toxin effects of AFB1, we designed and
conducted a hospital-based HCC case study in the high AFB1 exposure areas.
Results showed that the HCC patients with XRCC1 genotypes with rs25487 T
alleles (namely: rs25487-CT or rs25487-TT) faced a significantly increasing
risk of TP53M than those with the wild-type homozygote of XRCC1 (namely,
rs25487-CC, OR = 2.155, 95% CI = 1.863-3.141 for rs25487-CT; OR = 5.028,
95% CI = 4.44-42.08 for rs25487-TT, respectively). Additionally, we also
found that rs25487 polymorphism was significantly associated with other two
toxic biomarkers: AFB1-DNA adducts and HCC risk. Supporting our results,
several studies from high AFB1 areas showed the XRCC1 rs25487 T alleles
were significantly correlated with higher levels of AFB1-DNA adducts and
higher risk of TP53M [19, 28]. As regards of risk biomarker for the
toxicological effects of AFB1 (namely AFB1-related HCC risk), sixteen studies
about XRCC1 rs25487 polymorphism were reported with the results being
contradictory in the several decades years [25-27]. Our results showed this
polymorphism could modulate HCC risk. Previous several meta-analysis
based on different AFB1 exposure levels supported our conclusion [20, 26, 27,
29]. These results suggest that the decreasing capacity of DNA repair resulting
from XRCC1 rs25487 polymorphism is contributed to the toxicological effects
of AFB1.
The protein encoded by XRCC3 gene is one of identified paralogs of the

strand-exchange protein RAD51 in human beings. This protein associates
directly with DNA breaks and facilitates of the formation of the RAD51
nucleoprotein filament, which is crucial both for homologous recombination
and HRR [30, 31]. Previous studies have shown that a common polymorphism
(rs861539) at codon 241 of XRCC3 gene (Thr to Met) modifies the function of
this gene [32-41]. Two reports from high AFB1-exposure areas all of world
supported above-mentioned conclusions [42, 43]. In the first frequent case-
control study in Guangxiese, we observed that the higher-frequency of
genotypes with XRCC3 codon 241 Met alleles (namely Thr/Met and Met/Met)
was observed in controls (33.01%) than HCC cases (61.48%, P < 0.001).
Regression analysis showed that Met alleles increases about 2- to 10-fold risk
of HCC and this running-up risk is modulated by the number of Met alleles
(adjusted OR 2.48 and 10.06 for one and two this alleles) [43]. The followed
relative size analysis and the present study not only found similar risk value of
AFB1-related HCC [42], but also found this polymorphism increased risk of
other two toxic biomarkers high AFB1-DNA adduct and TP53 gene mutation
[44]. Our present study supported aforementioned results. These data exhibits
that the polymorphism at codon 241 of XRCC3 gene is a genetic determinant
in the detoxication of AFB1.
DNA repair gene XRCC7, also called DNAPK, DNPK1, HYRC, HYRC1,
or p350) (Genbank ID. 5591), spans about 197 kb on chromosome 8q11 and
contains 85 exons and 86 introns (Gene dBase in PubMed). The protein
encoded by XRCC7 acts as DNA-dependent protein kinase catalytic subunit
(DNA-PKcs) that constitutes the large catalytic subunit of the DNA-PK
complex [45]. When DNA-PKcs is recruited to the site of DSBs by the
Ku70/Ku80 heterodimer, DNA-PK complex changes into its active form and
subsequently initiates the non-homologous end joining (NHEJ) repair, an
important DSBR pathway [46]. Murine mutants defective in the XRCC7 have
non-detectable DNA-PK activity, suggesting that XRCC7 is required for
NHEJ pathway protein [47, 48]. More than 100 polymorphisms have been
reported in the XRCC7 gene, some of which are correlated with malignant
tumors such as bladder cancer (dbSNP in NCBI Database). Of these genetic
polymorphisms in XRCC7 gene, we only investigated the relation between
rs7003908 polymorphism and the effects on AFB1 toxicological effects, and
found this polymorphism might be an important modifying factor for AFB1
toxic role. Supporting our findings, a previous study was also found that these
individuals with XRCC7 rs7003908 G alleles increased HCC risk compared
the homozygote of XRCC7 rs7003908 T alleles (XRCC7-TT), with OR value
3.45 (2.404.94) for XRCC7-TG and 5.04 (3.287.76) for XRCC7-GG,
respectively. Furthermore, this genetic mutation was correlated with higher the
levels of AFB1-DNA adducts (r = 0.142, P < 0.001) [49]. Taken together,
these results explored that genetic polymorphism of XRCC7 rs7003908 might
decrease AFB1-related DSBR capacity and result in an increasing
toxicological capacity of AFB1, inquiring more studies to support this
conclusion.
4.5. XRCC4 Polymorphisms and Toxicological Effects of AFB1
XRCC4, located on chromosome 5q14.2, is an important the

nonhomologous end-joining (NHEJ) gene [50, 51]. The encoded protein of
this gene consists of 336 amino acid residues (DDBJ/EMBL/Genbank
accession no. AAD47298) and interacts directly with Ku70/Ku80 in the NHEJ
pathway [50, 51]. It is hypothesized that XRCC4 serves as a flexible join
between Ku70/Ku80 and its associated protein, Ligase IV [50, 51]. XRCC4 is
required for precise end-joining of blunt DNA DSBs in mammalian
fibroblasts, and the mutant, XRCC4, results in more-deficient NHEJ capacity.
A gene-targeted mutation study has also shown that differentiating neurons
and lymphocytes strictly require XRCC4 end-joining proteins. The targeted
inactivation of this gene leads to late embryonic lethality accompanied by
defective neurogenesis and defective lymphogenesis. These results
demonstrate that XRCC4 is essential for the DNA repair capacity of NHEJ
[52-54]. More than 100 polymorphisms have been reported in the XRCC4
gene (SNP database), some of which are correlated with DNA adducts, gene
mutation, and malignant tumors (such as oral, gastric, liver, and bladder
cancers) [15, 16, 55-60]. In this study, we only analyzed 2 known SNPs
(rs28383151 and rs3734091) in the coding region of this gene because these
polymorphisms localize at conserved sites of this gene. They change the coded
amino acids and may be associated with a decreased DNA repair capacity and
an increased cancer risk [15, 16]. Our results exhibited that these two
polymorphisms increased AFB1-DNA adducts levels, TP53M risk, and AFB1-
related HCC risk, suggesting they should be important modified factors of
AFB1 toxicological effects.
4.6. XPD Polymorphism and Toxicological Effects of AFB1
XPD gene, also called excision repair cross-complementing rodent repair

deficiency complementation group 2 (ERCC2), COFS2, EM9, or TTD
(Genbank ID. 2068), spans about 20 kb on chromosome 19q13.3 and contains
23 exons and 22 introns [2, 61]. Its encoded- protein is one of seven central
proteins in the NER pathway and act as a DNA-dependent ATPase/helicase.
This protein is associated with the TFIIH transcription-factor complex, and
plays a role in NER pathway. During NER, XPD participates in the opening of
the DNA helix to allow the excision of the DNA fragment containing the
damaged base [61]. There are four described polymorphisms that induce
amino acid changes in the protein: at codons 199 (Ile to Met), at codon 201
(His to Tyr), at codon 312 (Asp to Asn) and at codon 751 (Lys to Gln) [62].
Among these polymorphisms, we only analyzed codon 751 polymorphism
(rs13181) in this study, mainly because our previous studies [14] found the
variant XPD codon 751 genotypes (namely Lys/Gln and Gln/Gln) detected by
TaqMan-MGB PCR was significantly different between HCC cases (35.9%
and 20.1% for Lys/Gln and Gln/Gln, respectively) and controls (26.3% for
Lys/Gln and 8.6% for Gln/Gln, P < 0.001). Individuals having variant alleles
had about 1.5- to 2.5-fold risk of developing the cancer (adjusted OR 1.75 and
95% CI 1.30-2.37 for Lys/Gln; adjusted OR 2.47 and 95% CI 1.62-3.76 for
Gln/Gln). Our present study (based on relative large sample size) suggested
that the genetic polymorphisms at conserved sequence of XPD gene such as at
codon 751 may have potential effect on AFB1-related HCC susceptibility. This
supports different AFB1-toxin capacity might be modified by genetic
polymorphisms at codon 751 in DNA repair gene XPD.
4.7. XPC Polymorphism and Toxicological Effects of AFB1
XPC gene (Genbank accession NO. AC090645) spans 33kb on

chromosome 3p25, and consists of 16 exons and 15 introns. This gene encodes
a 940-amino acid protein, an important DNA damage recognition molecule
which plays an important role in NER pathway [63-65]. XPC protein binds
tightly with another important NER protein HR23B to form a stable XPC-
HR23B complex, the first protein component that recognizes and binds to the
DNA damage sites. XPC-HR23B complex can recognize a variety of DNA
adducts formed by exogenous carcinogens such as AFB1 and binds to the
DNA damage sites [63-65]. Therefore, it may play a role in the decreasing
toxic effects of AFB1. Some studies have shown that low DNA repair capacity
resulting from the genetic mutation of XPC rs2228001 can progress the toxic
effects of AFB1 [63-65]. In the past several decade years, a total of three
studies reported XPC rs2228001 polymorphism was involved in AFB1
detoxication [66-68]. The first study is from Shunde area, Guangdong
Province which is characterized by high AFB1 exposure and high incidence
rate of HCC. In this study, researchers explored the correlation between this
polymorphism and risk of HCC via an 1-1 case-control study (including 78
HCC patients and 78 age- and sex-matching controls) method, and found the
mutation of XPC modified HCC risk (adjusted odds ratios [ORs] were 6.78
with 95% CI 2.03-22.69) [68]. Although they did not directly evaluated the
effects of XPC rs2228001 polymorphism on the toxic role of AFB1, study

population in their study is from high AFB1 exposure areas and high risk of
HCC for XPC mutant. The other two studies was conducted by our teams and
showed XPC codon 939 Gln alleles increased about 2-times risk of HCC [66,
67]. In our present study, we not only observed this mutation increased AFB1-
related HCC risk, but found more direct evidence of XPC polymorphism
modified the toxicological effects of AFB1 exposure through the analysis of
AFB1-DNA adducts the TP53M induced by AFB1 in the HCC cancerous
tissues. As a result, these data suggest that genetic polymorphism at codon 939
of XPC gene is a genetic determinant in the DNA repair process of DNA
damage induced by AFB1 exposure, and its low activity might result in
increasing strength of AFB1 toxin act.
4.8. Limitation
This study had several limitations. First, because the present study is a the
hospital-based study, potential selection bias might have occurred. Second, the
increased risk with AFB1 exposure status noted in this study was probably
underestimated, because the liver disease itself may affect the metabolism of
AFB1 and modify the levels of AFB1-DNA adducts. Third, in spite of the
status of TP53M was investigated in cases of HCC, other AFB1-related
mutations of the TP53 gene were not evaluated. Finally, we only analyzed 7
genetic mutations in DNA repair genes. Therefore, more genes deserve further
elucidation based on a large sample and the combination of genes and AFB1
exposure.
CONCLUSION
In conclusion, to the best of our knowledge, this is the first report to
investigate association between polymorphisms in DNA repair genes XRCC1,
XRCC3, XRCC4, XRCC7, XPC, and XPD and the toxicological effects of
AFB1 among Guangxi population from an high AFB1-exposure area. We find
that the genetic mutations in the DNA repair genes XRCC1, XRCC3, XRCC4,
XRCC7, XPC, and XPD might increase the amount of AFB1-DNA adducts,
the frequency of TP53M, and the risk of HCC, and the low DNA repair
capacity from genetic mutations of these genes should contribute to the
toxicological effects of AFB1. Given that AFB1 is am important genic agent
and a kind of I type carcinogen, our findings might have prevention

implications through identifying population with low DNA repair capacity,
once these findings are replicated by other studies based on a larger scale or
prospective studies.
CONFLICTS OF INTEREST AND SOURCE OF FUNDING

The authors declare no competing financial interests. This study was
supported in part by the National Natural Science Foundation of China (No.
81160255 and No. 81372639), the Innovation Program of Guangxi Municipal
Education Department (No. 201204LX674), Innovation Program of Shanghai
Municipal Education Commission (No.13YZ035), the Natural Science
Foundation of Guangxi (No. 2013GXNSFAA019251), and the Science
Foundation of Youjiang Medical College for Nationalities (No. 2005 and
2008).
ACKNOWLEDGMENT
We thank Dr. Qiu-Xiang Liang, Dr. Yun Yi, and Dr. Yuan-Feng Zhou for
sample collection and management; Dr. Hua Huang for molecular biochemical
technique. We also thank all members of Department of Medical Test and
Infective Control, Affiliated Hospital of Youjiang Medical College for
Nationalities for their help.
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Chapter 7
INCIDENCE OF ASPERGILLUS SECTION FLAVI

AND INTERRELATED MYCOFLORA IN
PEANUT AGROECOSYSTEMS IN ARGENTINA
Mara Alejandra Passone, Andrea Nesci,

Anala Montemarani and Miriam Etcheverry
Laboratorio de Ecologa Microbiana,
Departamento de Microbiologa e Inmunologa,
Facultad de Ciencias Exactas Fsico Qumicas y Naturales
Universidad Nacional de Ro Cuarto,
Ro Cuarto, Crdoba, Argentina
Members of the Research Career, Consejo Nacional de Investigaciones
Cientficas y Tcnicas (CONICET), Argentina
1. ABSTRACT
Studies in typical and new Argentinean peanut areas showed that
toxigenic Aspergillus section Flavi strains are widely distributed in soils
and seeds, with high probability of being transferred to the storage
ecosystem. Mycological analyses of soil showed that Aspergillus section
Flavi population were present in the two areas at similar counts (3.2x10 2
cfu g-1). Within this section, two fungal species were frequently isolated
with isolation percentages of 73 and 90% for A. flavus and of 27 and 9%
for A. parasiticus in soil samples from traditional and new areas,
respectively. The percentages of the different A. flavus phenotypes from
158 M. A. Passone, A. Nesci, A. Montemarani et al.
both peanut-growing areas showed that L strains were recovered in the

highest percentage and represented 59 and 88% of the isolates with
variable ability to produce aflatoxins (AFs). Peanut kernels collected at
harvest time from different localities of Crdoba and Formosa provinces
showed A. flavus and A. parasiticus contamination. The 42.8 and 70%
were classified as type L and the percentages of aflatoxigenic A. flavus
strains were 68.6 and 80.0% in samples from traditional and recent
peanut-growing areas, respectively. Highly toxigenic A. flavus S strains
were isolated with major frequency from soil and kernel samples coming
from traditional peanut-growing area. Aflatoxin contamination was
detected in peanut kernels from typical peanut growing area. Harvested
peanut were stored during 5 months in three storage systems (big bags,
wagons of conditioning and drying and stockpiled warehouse) and
mycological population succession was analyzed. Fungal isolation was
greater from pod (95%) than from kernel tissues. The most common fungi
identified included Penicillium, Aspergillus, Eurotium and Fusarium spp.
Within Aspergillus genus, the section Flavi had the greatest mean counts
of 1.4x104, 9.4x102, 5.2x102 cfu g-1 for big bags, wagon and warehouse,
respectively. A. flavus and A. parasiticus strains with variable ability to
produce AFs were isolated from peanut kernels stored in the three
systems at all sampling periods in the order of 1.5x102, 2.3x102 and 4.5
cfu g-1, respectively. .A. flavus S and L strains contributed to silo
community toxigenicity during all storage period. Total AF levels
ranging from 1.1 to 200.4 ng g-1 were registered in peanuts conditioned at
the higher aW values (0.940.84 aW) and stored in big bags. Despite the
water stress conditions registered in the stockpiled warehouse throughout
the storage period, AFB1 levels ranging between 2.9 and 69.1 ng g-1 were
registered from the third sampling.
Therefore, the interaction between biological and abiotic factors and
substrate may promote the Aspergillus contamination and the subsequent
AF accumulation in peanut from sowing to storage, highlighting the need
to promote good practices in order to avoid the risk of these metabolites
contamination in peanut food chain.
Incidence of Aspergillus Section Flavi and Interrelated Mycoflora ... 159
2. INTRODUCTION
Peanut is an economically important crop in Argentina, its annual
production in 2013 reached 0.9 million tons. Such importance lies in its
participation in international market. Peanut exportations fluctuate between
0.44 and 0.68 million tons since 2011, ranking the first position since 2012
(SIIA, 2013). The important role of Argentinean peanuts in the world market
has strictly two reasons; the internal consumption reduced (270 g annual per
capita) and the quality that allows it access to markets, such as EU the worlds
largest consumer market that is closed to other countries (Atayde et al., 2012;
Ding et al., 2012; Mutegi et al., 2013; SIIA, 2013). This nut is attractive
worldwide for their nutritional, sensory and health promoting attributes.
Peanuts are rich in energy and contain health beneficial nutrients, minerals,
antioxidants and vitamins giving it an exceptional nutrient profile that are
essential for optimum health (Jubeen et al., 2012).
Peanut is a dicotyledonous plant and the only species cultivated is Arachis
hypogaea L. Peanuts are annual, herbaceous, pubescent, erect or low-growing
plants. Their peculiarities are the aerial flowers and subterraneous fruits
(Ramantha Rao and Murty, 1994). In commercial plantations, once the plants
are uprooted, the pods are placed to dry in the sun in a windrow. This is still a
slow process; requiring 6-10 days under good weather conditions to reduce the
moisture content of peanut kernel from 40-50% to 20-25% (Schilling and
Misari, 1992). This is one of the most important stages of production since
poor drying can provoke a significant increase in fungal contamination
(Fonseca, 2010). The increase in the level of fungal contamination does not
only occur in the field, but also during the process of kernel formation,
harvesting, drying, transport and storage (Rossetto et al., 2005), as well as
during handling (Santos et al., 2001). The economic impact of fungal invasion
includes: reduction of seed germination rate and, more importantly,
compromise of product quality such as mold growth, discoloration, unpleasant
odor, loss of dry fabric, heating, cooking, chemical and nutritional alterations,
and mycotoxin production, particularly aflatoxins (AFs) that are strictly
regulated; all of which may make peanut products unsuitable for consumption
(Christensen, 1982; Paster and Bullerman, 1988). Aflatoxins in general and
specially aflatoxin B1 (AFB1) are a genotoxic, immunotoxic and
hepatocarcinogenic secondary metabolites (group 1) (IARC, 2002). Therefore,
the final regulations proposed by the European Union for maximum levels of
total AFs and AFB1 in peanuts was 4 and 2 ng g-1, respectively (Commission
Regulation, 2010). The objective of this chapter is to review the bibliography
concerning to the analysis of the total mycobiota population and aflatoxigenic

contamination in soils and seeds from two peanut growing areas of Argentina
(traditional and new) and in stored kernels in three storage systems (big bags,
wagons and stockpiled warehouse). This information will help to know the
distribution of potentially toxigenic Aspergillus section Flavi strains and the
risk of AF contamination in peanut kernels from sowing to storage.
3. CHARACTERIZATION OF SOIL ASPERGILLUS

SECTION FLAVI POPULATION FROM TWO PEANUT
GROWING AREAS OF ARGENTINA
3.1. Traditional Peanut-growing Area
The population under study was isolated from field soils within the peanut
growing region (General Deheza, Ro Cuarto, Charras) of Crdoba Province,
Argentina during the planting and harvest periods. The three regions evaluated
showed no significant differences in the incidence of filamentous fungi and
Aspergillus species from section Flavi (Table 1). The filamentous fungi
present in the soil samples as estimated from dilution plating ranged from
8.2x103 to 2.2x104 cfu g-1 of soil (mean 1.7x104 cfu g-1). Within each region,
filamentous fungi showed similar cfu g-1 at planting and harvest, except in
Charras. The same differences were observed in the isolation frequency of
Aspergillus section Flavi strains with counts ranged from 2.9x101 to 6.7x102
cfu g-1 (mean 3.2x102 cfu g-1) (Barros et al., 2003). Such differences between
planting and harvest could be explained by the environmental conditions
(temperature and rainfall) and soil temperature as demonstrated in previous
studies (Hill et al., 1983; Horn et al., 1994). Out of 506 Aspergillus section
Flavi isolates, 369 were A. flavus (73%) and 137 were A. parasiticus (27%).
The differences between the percentages of the different A. flavus phenotypes
isolated are shown in Table 2. The L phenotype (diameter of sclerotia > 400
m) was recovered in the highest percentage and represents 59% of the
isolates. In contrast, the recoveries of S (diameter of sclerotia < 400 m) and
non-sclerotial strains were 22 and 19%, respectively. Statistical analyses
showed significant differences in AF and cyclopiazonic acid (CPA) production
among L, S and NS producer strains (p<0.05). The S strains produced the
highest AF levels (3315.8 ng ml-1).
Table 1. Comparison of soil and peanut Aspergillus section Flavi populations
of two peanut-growing regions in Argentina
Traditional region New region

Filamentous section A. A. AFs Filamentous section A. A. AFs
fungi (cfu g-1) Flavi flavus parasiticus (g g-1) fungi (cfu g-1) Flavi flavus parasiticus (g g-1)
Samples Periods (cfu g-1) (%) (%) (cfu g-1) (%) (%)
4
Soil Planting 1.6x10 1.8x102a/ 76/90 19/9
2.6x102b
4
Harvest 1.9x10 1.4x103/ 87/88 13/12 3.6x104c 3.2x102 90 6
2.3x102
Peanut N.D. N.D. 60d 20 124.8+20.1a N.D. N.D. N.D. N.D. N.D.
N.D. not determined.
a
Barros et al., 2003; bAlaniz Zanon et al., 2013; cOrtiz et al., 2013; dVaamonde et al., 1995.
Table 2. Production of aflatoxin B and G groups and CPA by L, S and NS

strains of A. flavus isolated from two peanut-growing regions of Argentina
A. flavus strains (%) Mycotoxin production

Traditional region New region
Samples L S NS L S NS AFB AFG CPA
Soil 76.6 78.2 77.9 71* + n.d. +
11.1 8.0 9.9 10 n.d. n.d. +
- 10.0 - - + + +
11.3 3.8 9.1 - + n.d. n.d.
1.0 - 3.1 29 n.d. n.d. n.d.
Peanut 51.4* 89.3 33.3 44.0 + n.d. +
31.4 10.7 - 56.0 n.d. n.d. +
17.2 - 66.7 - + + +
n.d.: not detected; *Determined as the percentage of the total A. flavus strains.
Soil samples; Traditional region: L strain (n=218), S strain (n=81); NS strain (n=70)
(Barros et al., 2005); New region: L strain (n=340), S strain (n=10); NS strain
(n=37) (Ortiz et al., 2013).
Peanut samples; Traditional region: L strain (n=15), S strain (n=9); NS strain (n=11)
(Vaamonde et al., 1995); New region: L strain (n=28), S strain (n=3); NS strain
(n=9) (Pildain et al., 2004).
Similarly to the results for AFs, the S strains produced higher CPA levels
(65.3 g ml-1) than L and NS strains (mean levels = 54.7 and 39.6 g ml-1).
Only 4 out of 369 A. flavus isolates did not produce AFs or CPA, whereas
80% of the isolates produced both mycotoxins. Among the S strains, 10% of
the isolates showed an unusual pattern of mycotoxin production (AF group B
and G simultaneously with CPA) (Barros et al., 2005).
3.2. New Peanut-growing Area
To analyze the biodiversity of Aspergillus section Flavi soil population

from new peanut growing areas, soil samples were collected from Formosa, La
Pampa and south of Crdoba provinces. The mean values of total mycobiota
and Aspergillus section Flavi were 3.6x104 and 319 cfu g-1, respectively
(Table 1). Out of 430 strains isolated within the Aspergillus section Flavi 90%
were identified as A. flavus, 6% as A. parasiticus and 4% as A. caelatus.
Among the A. flavus isolated 88% were L strains, 3% were S strains and 10%
were not able to produce sclerotia (Table 2). Seventy one percent of A. flavus
were AF producers and 81% were CPA producers. A relatively large
Incidence of Aspergillus Section Flavi and Interrelated Mycoflora 163
proportion of A. flavus strains (n=111; 29%) isolates were not able to produce
AF. Molecular analysis of omt-A, ver-1, nor-1 and afl-R genes of 34 strains of
non aflatoxigenic A. flavus showed that 19 strains had absence of 1, 2 or 3 of
the genes analyzed. Only 1 strain showed absence of all 4 genes studied. There
were no significant differences in the mean level of AFB1 production among
strains isolated from the different new areas of peanut cultivation (Ortiz et al.,
2011; 2013).
Therefore, the data of the works conducted by Barros et al. (2003) and
Ortiz et al. (2011, 2013) showed that the total mycoflora count was higher and
the incidence of Aspergillus section Flavi was lower in samples collected from
the new peanut cultivation areas in comparison with the levels found in soil
samples collected in the main peanut growing area. Data on the incidence of
the soil native population from Las Acequias, located within the peanut
growing region of Crdoba, Argentina were recently reported by Alaniz Zanon
et al. (2013) and are consistent with those found by Barros et al. (2003) (Table
1). The inoculum level and the incidence of toxigenic isolates of native A.
flavus and A. parasiticus in soil at planting and pod maturation times were
similar among plots and A. flavus was the dominant specie from section Flavi,
showing an isolation percentage nearly to 90%. The remaining species
identified were A. parasiticus (9%) and the non aflatoxigenic specie A.
caelatus (1%). In relation to the incidence of toxigenic native isolates at
planting and harvest times, a total of 75 and 80% of A. flavus isolates were
toxigenic, whereas 98 and 100% of A. parasiticus isolates were toxigenic.
Similarly, Atayde et al. (2012) reported that A. flavus was the Aspergillus
species most frequently isolated (13.4%) from peanut soil after plant
emergence and 2 weeks prior to uprooting in four regions of So Paulo, Brazil,
with the higher frequency in samples from Jaboticabal (70.3%). The mean
frequency of A. flavus in soil varied according to sampling period and was
72% lower in the second sampling. In contrast, A. parasiticus was only
detected in 4 of the 40 samples analyzed. Although classified as a storage
fungus, the presence of A. flavus in soil samples indicates this substrate as the
primary reservoir of this fungus, a fact previously reported by Horn (2005) and
Gonalez et al. (2008).
The importance of determine type S, L or NS strains can be explained by
these resistance structures exhibit sporogenic germination. Thereby an
eventual preharvest control of A. flavus infection in that crops where AF is a
problem, agronomic practices designed to reduce the importance of sclerotia
as primary inoculum source are required (Wicklow, 1983). The results of these
studies provide new data on the communities of A. flavus in peanut soils in
Argentina, by associating of morphological and toxigenic characteristics of

strains isolated with the possible agroecological differences of the diverse
geographical areas from which strains were isolated. Therefore, the levels of
toxin produced by A. flavus strains isolated from new peanut growing area
were lower than those produced by strains isolated from areas with long term
history of peanut cultivation. Principal component analysis showed that fields
with recent history of peanut cultivation are closely related with the isolates
belonging to A. flavus L phenotype, producers of low AF levels, while typical
groundnut regions are closely related with high percentages of A. parasiticus
and A. flavus S phenotype, producers of higher AF levels. Such differences
may be due to: 1) the areas analyzed present different macro and microclimatic
conditions, 2) in the Crdoba province, the peanut is grown for several years
and over a larger area than in the other regions where this nut was developed a
few years ago and seeding in lesser extent, 3) agroecological regions analyzed
are at different latitudes and give another possible cause of variability (Cotty,
1997), 4) history of soil, type of crop, insect levels, rainfall, temperature and
cultural practices are also different (De Fina, 1992; Orum et al., 1997).
Therefore, the high presence of A. flavus in soil samples should have
contaminated the peanut pods. As the soil is a primary reservoir for A. flavus
and A. parasiticus and peanuts are an underground fruit, their pods should be
directly exposed to the two soil species (Horn et al., 1994).
4. INCIDENCE OF AFLATOXINS AND CHARACTERIZATION

OF PEANUT ASPERGILLUS SECTION FLAVI POPULATION
FROM TWO GROWING AREAS OF ARGENTINA
4.1. Traditional Peanut-growing Area
Natural Aspergillus section Flavi infection of peanut from 21 localities

(Baigorria, Holmberg, Alcira Gigena, Las Perdices, Corralito, Ro Cuarto,
Hernando, Matorrales, Oliva, Charras, Villa del Rosario, General Cabrera,
Carnerillo, Alejandro, Manfredi, Reduccin, Pampayasta, Villa Ascasubi, La
Carlota, Ticino and Colazo) situated in the traditional production areas of
Crdoba, Argentina was evaluated. From 21 locations and of the 32 samples
analyzed, a total of 44 strains were isolates (Table 1). A. flavus was isolated
from 60% of the samples, while A. parasiticus was present in 20% of them.
Strains of A. flavus and / or A. parasiticus were found in peanut kernels from
all locations, except two, Corralito and Hernando. The percentage of

aflatoxigenic A. flavus strains was very high (68.6%) as well as the percentage
of strains producing CPA, representing all the strains (100%) (Table 2). The
68.6% of the strains tested (n = 24) produced sclerotia under the following
culture conditions: Czapek agar, 30 C, 30 days in dark. The 25.7% were
classified as type S, of which most of them produced simultaneously AFB and
AFG (Vaamonde et al., 1995).
The AF analyses of peanut kernels from three regions showed a higher
frequency of contaminated samples from the Charras region (mean level of
total AFs = 158 g g-1/ 8 positive samples) in comparison with the General
Deheza and Ro Cuarto regions that showed mean levels of total AF
contamination of 7.4 and 209 g g-1 with 3 and 2 positive samples,
respectively (Table 1) (Barros et al., 2003).
4.2. New Peanut-growing Area
Table 2 shows characteristics of A. flavus strains (n = 40) isolated from

peanut kernels from Formosa province. Seventy eight percent of the isolates
were sclerotia producers under culture conditions and on peanut kernels. Three
of these sclerotia producers were classified as S strains. Eighty-nine percent of
the L strain isolates produced AFB and CPA, and no isolates that produced
only AF were observed (Pildain et al., 2004).
Recently, Alaniz Zanon et al. (2013) reported the density of A. flavus, the
only specie from section Flavi isolated from harvested peanut kernels in the
typical groundnut region. Peanut was subjected to two environmental
conditions of growth; with and without drought stress; with infection
percentage in the order of 6.5 and 57%, respectively. Incidence of Aspergillus
section Flavi in peanut kernels at harvest in different growing-areas of other
countries was also reported. Studies investigating the mycobiota and
mycotoxins in Brazilian peanut kernels from sowing to harvest reported that A.
flavus was detected in the pod filling (43.9%), full pod maturity (33.6%) and
dried pod (7.6%) stages. Moreover, A. parasiticus was found more frequently
than A. flavus in dry pod stage, 10.3 and 7.6% respectively, in spite of low aW
(0.71) (Gonalez et al., 2008). Similarly, Zorzete et al. (2011) reported that the
presence of A. flavus seems to vary according to the growth phase of the
peanut plant. For pods, all dried grain samples were positive for this fungus,
with an average contamination of 32.5%, whereas no contamination was
observed during the other phases. In kernels, increased presence of A. flavus
was observed during the stages of ripe grains (13%) and dried kernels (4%). A.
parasiticus was isolated from pod samples in low percentage (1%).
This indicate that A. flavus is the more aggressive specie and the
responsible for most of the AF contamination of peanuts.
Natural occurrence of AF and CPA contamination in peanuts coming from
the nucleus of the Argentinean peanut-growing area was investigated. Co-
occurrence of CPA and AF was detected in two of 50 samples analyzed. The
levels of these toxins found in positive samples were 4300 and 493 g kg-1 for
CPA, 625 and 435 g kg-1 for AFB1, and 625 and 83 g kg-1 for AFG1,
respectively (Fernandez Pinto and Patriarca, 2001). In the Charras region the
high AF-producing potential of Aspergillus species from section Flavi at
harvest time, together with predisposing conditions for peanut infection, could
explain the higher number of samples contaminated with AFs (Barros et al.,
2003). On the one hand, Alaniz Zanon et al. (2013) demonstrated that
temperatures close or above to 30 C during several days summed to drought
stress were conducive conditions for AF accumulation in peanut. A household
survey was carried out in Busia and Homabay districts in western Kenya,
which were chosen, based on their significance in terms of peanut production
and because they offered a contrasting environment under which peanuts are
cultivated. The levels of AFs ranged from 0 to 2687.6 g kg-1 and from 0 to
7525.0 g kg-1 in samples from Busia and Homabay districts, respectively.
There was a highly significant association between agroecological zones and
AF levels. While 10.70% of samples from Busia district had AF levels >20 g
kg-1, only 4.09% of samples from Homabay were in this category (Mutegi et
al., 2013). On the other hand, Gonalez et al. (2008) analyzed AF content of
peanut kernels during the different crop growth stages and showed that AF
concentrations increased when the kernels aW decreased (0.71), which
occurred at dried pod stage. Dorner et al. (1989) reported that immature peanut
pods are more resistant to fungi and AF contamination because they produce
more total phytoalexins than mature peanuts, at high aW. Attack by moles also
was found to be significantly associated with AF levels. Damage by moles
predisposes pods to colonization by AF-producing fungi, and similar damage
by terrestrial arthropods has been reported (Dicko et al., 1999). Pod damage
also exposes the kernels to colonization by AF-producing and other
saprophytic fungi (Chapin et al., 2004).
Therefore, peanut kernels harvested from different peanut-growing areas
of Argentina contain mycelia and spores of aflatoxigenic fungi, which can
result in a significant decrease in grain quality when they are stored.
Table 3. Effect of storage time on total mycoflora (cfu g-1) from peanut
pods and kernels stored in different storage systems
Log cfu g-1 + S.D.a

Time Big bagc Wagond Warehousee
b
(months) (250 kg) (4,000 kg) (26,000 ton)
Pods Kernels Kernels Kernels
First 3.5 + 2.8 b < b 5.2 + 4.9 b 3.6 + 0.4 b
Second 4.9 + 4.6 a 3.6 + 2.9 a 6.2 + 5.9 a 3.7 + 0.5 a
Third 4.4 + 3.4 a 3.4 + 2.5 a 5.9 + 5.4 a 4.6 + 1.1 a
Fourth 4.4 + 3.7 a 3.5 + 3.0 a 5.4 + 4.9 a 4.7 + 0.9 a
Fifth 4.8 + 3.9 a 3.3 + 2.8 a 5.7 + 5.3 a 4.5 + 1.2 a
a
Mean + S.D. based on 120 independent pod and seed samples.
b
Storage period: July-November for big bag (0.76+0.02 aW); July-November for
wagons; May-October for warehouse.
c
Passone et al. (2009a).
d
Doprado (2008).
e
Nesci et al. (2011).
Data not sharing a common letter in the same group are significantly different
according to Tukey Test (P < 0.05).
5. INCIDENCE OF FUNGAL POPULATION, AFLATOXINS

AND CHARACTERIZATION OF ASPERGILLUS SECTION
FLAVI IN PEANUT FROM DIFFERENT STORAGE SYSTEMS
5.1. Incidence of Total Mycoflora in Stored Peanut
5.1.1. Analyses of Peanut Stored in Big Bags Conditioned at Different aW

Levels
Mycoflora analyses were conducted on peanuts artificially dried up to
0.94 + 0.01, 0.88 + 0.01, 0.84 + 0.01 and 0.76 + 0.02 aW levels by using a
continuous dryer that insufflated air at 35 C and stored in big bags with a
capacity for 250 kg in-pod peanuts. Ten samples were monthly collected from
each big bag during a 5-month period.
Fungal populations from 200 peanut kernel samples harvested for human
consumption and stored in four big bags (250 kg) in Storage Company in the
south of Crdoba province were analyzed. Total fungal counts were higher in
peanuts conditioned at high aW levels (big bags 1 and 2). Total mean counts of
6.9x107 and 3.6x107 cfu g-1 were recorded in big bags with 0.94 and 0.88
initial aW, respectively. Meanwhile, mean fungal counts in big bags 3 and 4
were estimated at 6.7x106 and 2.1x104 cfu g-1, respectively. Analyses of fungal
populations from 50 peanut kernel and pod samples did not demonstrated
significant differences between the incidences in each sampling period.
Isolation from pod tissue yielded more fungi than from kernel, 85% of the
fungi isolated were from pod tissue. The mean fungal counts during the six
sampling periods for pods and kernels were 4.0x104 and 2.3x103 cfu g-1,
respectively. Most of the fungi were isolated in the last sampling period. The
total counts in the first sampling were 3.1x103 and < 103 cfu g-1, while after a
5-month storage period, the count increased to 6.1x104 and 2.0x103 cfu g-1 in
pods and kernels, respectively (Table 3) (Passone et al., 2009a).
During monitoring, Aspergillus, Penicillium and Eurotium were the
genera commonly isolated from peanut kernels during all sampling periods;
Fusarium spp. were only detected in the first storage period (1-3 months). The
two higher initial water stress conditions assayed (0.84 and 0.76 aW) mainly
affected the development of genera such as Fusarium and filamentous fungi
group. On the contrary, Eurotium spp. counts increased within the second
storage period and the highest inoculum of this genus (mean = 7.8x105 cfu g-1)
was found in peanut kernels conditioned at the middle aW levels (0.88 and 0.84
aW) (Table 4) (Passone et al., 2009b).
A deeper identification, at section levels were done for samples from big
bag 4 and the most frequently occurring fungi are presented in Table 5.
Ninety-eight percent of the fungal isolates were Deuteromycetes and
Ascomycetes, and the remaining were Zygomycetes. A mycological survey of
90 peanut pod and kernel samples showed the presence of three principal
genera of filamentous fungi (Penicillium, Aspergillus and Fusarium spp.). The
fungal genera that showed a relatively low frequency of isolation and that were
not important mycotoxin producers (Eurotium, Monascus, Alternaria,
Cladosporium, Byssochlamys, Rhizopus, Mucor, Absidia spp. and sterile
mycelium) were all included in the filamentous fungi group.
Penicillium spp. had the greatest mean frequency levels in pod and kernel
tissues during the research period with a count of around 1.8x104 and 1.5x103
cfu g-1, respectively. Penicillium species sorted in three sections
Divaricatum, Furcatum and Simplicia were isolated from both tissue type
during the storage period. The highest frequency of isolation corresponded to
the Simplicia section (44.7%), followed by the Furcatum (32.7%) and
Divaricatum sections. Aspergillus spp., a common peanut contaminant, was
isolated from both pod and kernel tissues at the six sampling periods.
Table 4. Incidence of total mycoflora in kernels from in-pod peanuts
conditioned at different aW levels and stored in big bags
aW cfu g-1a
1-3 months 4-5 months
Aspergillusb Penicillium Fusarium Eurotium Filamentous Aspergillus Penicillium Eurotium Filamentous
fungic fungi
Big bag 1 1.3x106 4.8x107 2.3x104 3.3x102 2.4x105 1.5x105 1.9x107 5.0x103 1.1x104
(0.94+0.01)
Big bag 2 4.6x106 1.9x107 1.0x103 < 2.1x105 9.7x105 1.0x107 1.1x106 3.3x102
(0.88+0.01)
(0.84+0.01)
(0.76+0.02)
a
Mean values based on ten replicate data in DRBC and in DG18 medium.
b
Includes the sections: Nigri, Circumdati and Fumigati.
c
Includes the genera: Absidia, Alternaria, Cladosporium, Monascus, Paecilomyces, Rhizopus, Trichoderma, dematiaceous and non-
sporulating fungi.
Passone et al. (2009b).
Table 5. Incidence of mycobiota in peanut pod and kernel tissues stored in

different storage systems
Mycobiotab Log cfu g-1 + S.D.a

Big bagc Wagond Warehousee
(250 kg) (4,000 kg) (26,000 ton)
Pods Kernels Kernels Kernels
Sterile mycelium 4.76 + 5.06 3.91 + 4.09 3.03 + 2.99 1.40 + 1.64
Zygomycetes
Absidia 3.54 + 3.81 2.26 + 2.48 < <
Mucor 4.08 + 4.23 2.30 + 2.32 < <
Rhizopus 2.22 + 2.61 < < <
Ascomycetes
Byssochlamys < < 3.86 + 3.86 <
Eurotium 2.92 + 3.31 < 3.37 + 3.37 0.10 + 0.24
Monascus 4.69 + 4.59 3.09 + 3.25 2.48 + 2.48 <
Talaromyces < < 1.83 + 1.69 <
Deuteromycetes
Alternaria 3.87 + 4.25 2.22 + 2.14 < 0.34 + 0.78
Aspergillus
Section Flavi 3.56 + 0.30 2.29 + 2.19 3.46 + 3.16 1.72 + 1.64
Fumigati < < 1.23 + 1.23 0.18 + 0.72
Nigri 3.54 + 1.48 2.32 + 2.55 2.86 + 2.55 0.28 + 0.80
Terrei < < 1.23 + 1.23 <
Cladosporium < < < 1.08 + 1.10
Fusarium 3.33 + 3.71 2.30 + 2.80 3.23 + 2.88 0.94 + 1.24
Paecilomyces < < 2.81 + 2.81 <
Penicillium 2.72 + 1.82
Section < < 2.18 + 2.18 <
Aspergilloides
Exilicaulis < < 1.92 + 1.81 <
Divaricatum 2.16 + 2.59 1.57 + 1.79 3.75 + 3.48 <
Furcatum 3.36 + 3.62 2.02 + 2.16 3.55 + 3.42 <
Simplicia 4.20 + 4.30 3.15 + 2.99 4.98 + 4.52 <
Trichoderma 2.22 + 2.61 1.23 + 1.61 < <
Total filamentous 6.69 5.79 5.08 4.22
fungi
a
Mean + S.D. based on 120 independent pod and seed samples.
b
Distribution of genera and section into group according to Pitt (2000), Klich (2002),
Samson et al. (2002).
c
Passone et al. (2008), Big bag (0.76+0.02 aW).
d
Doprado (2008).
e
The cfu counts were 7.1x103 and 4.0x102 cfu g-1 in pod and kernel
samples, respectively. Two sections of Aspergillus genus were identified from
pod and kernel tissues. The Aspergillus section Flavi had the greatest mean
frequency (49.9%). Isolation frequency of Fusarium spp. was sporadic
throughout the study. The mean counts for this genus were 2.1x103 and
2.0x102 cfu g-1 in pod and kernel tissues, respectively. Monascus spp. was
consistently isolated from pods and kernels during the last 3 months of the
assay. It is notable that Monascus spp. was isolated at numerically greater
levels from pods (97.6%) than from kernels during the study. Sterile
mycelium, Absidia, Mucor, Rhizopus, Alternaria, Eurotium, Paecilomyces and
Trichoderma spp. were all isolated in low frequency (mean: 4.5x104 cfu g-1)
during the 5-month of storage. The Zygomycetes such as Absidia, Mucor and
Rhizopus spp. were consistently isolated from pod and kernel tissues at the
first two sampling periods, whereas the incidence of Monascus spp. and
Eurotium spp. increased at the end of the storage time. Alternaria and
Trichoderma spp. were isolated at low levels during the assay (Passone et al.,
2008).
The determination of physical properties of the samples revealed
considerable differences in aW and temperature between the first and the fifth
sampling. Water availability levels in peanut conditioned at four aW decreased
at mean level of 0.63 + 0.04 aW. Temperature values of peanut from the four
big bags increased from 12.6 + 0.6 C to 29.3 + 0.9 C between the first and
fifth sampling period and pH values were maintained relatively stable (mean:
6.7) (Passone et al., 2009b).
5.1.2. Analyses of Peanut Stored in Wagons of Conditioning and Drying

Mycoflora analyses were conducted on harvested peanut kernels with 0.87
+ 0.04 aW levels and stored in wagons of conditioning and drying with a
capacity for 4000 kg in-pod peanuts. Ten in-pod peanut samples (500 g) were
taken random at time zero, after filling the silo, and every 30 days over a
period of 5 months (July-November). Total fungal of all samples obtained
from DRBC agar, and counts of xerophilic fungi obtained from DG18 agar
were over 1x105 cfu g-1, showing a high degree of contamination (Table 3).
Eight genera of filamentous fungi were isolated from peanut kernels, with
prevalence of Penicillium, Aspergillus, Fusarium and Eurotium which showed
a relative density (RD) of isolation of 86.0, 3.4, 2.0 and 1.4%, respectively.
While Monascus, Byssochlamys, Talaromyces and Paecilomyces spp., that
make up the filamentous fungi group were isolated in lower RD (0.25, 6.0,
0.05, 0.54%) over the 5 months of storage (Table 5).
Of all isolates found in the Penicillium genus, species of the following

sections were recovered: Simplicia, Aspergilloides, Exilicaulis, Furcatum,
Divaricatum and Penicillium, with a prevalence of 88.0, 0.2, 0.1, 7.7 and
0.3%, respectively. In the Aspergillus genus, species from sections Flavi,
Fumigati, Nigri, Restricti, Circumdati, Nidulantes and Terrei were isolated
with a prevalence of 54.2, 29.7, 13.8, 1.7%, respectively and 0.2% for the
three last sections.
Considering the environmental variability, storage temperatures increased
gradually during the 5 months of storage of 15 to 28 C. In relation to records
of aW, the level medium was 0.72 aW, a reduction was observed during the first
4 months of storage from 0.87 to 0.63 (Doprado, 2008).
5.1.3. Analyses on Peanut Stored in Stockpiled Warehouse

Analyses of fungal populations in 95 peanut kernel samples did not
demonstrated a significant sampling period effect (p=0.578) on the incidence
of the total fungi isolated in peanut kernel during May, June, August,
September and October. The values were in a range of 3.6-4.7 (log cfu) (Table
3). Penicillium, Fusarium, and Aspergillus section Flavi were the principal
filamentous fungi isolated between the first and fifth sampling periods. Other
fungal communities present as minor components of the mycoflora included
Cladosporium, Aspergillus section Nigri, Alternaria, Eurotium, Aspergillus
spp. and sterile mycelia (Table 5). The great fungal diversity was observed
during the second and fourth sampling periods, Aspergillus section Flavi,
Penicillium, Aspergillus section Nigri and sterile mycelia were isolated during
all incubation period. The lowest incidence of Aspergillus section Flavi was
detected in the first sampling period, with an increase in the fourth sampling
period.
The temperature varied between 9 and 19 C during the sampling period.
The lowest temperature was detected in June and the highest in October.
Water activity of samples ranged from 0.43 to 0.57 during 180 days of storage.
Water activity values were higher in the first and fourth months than in the
fifth month of the experiment (Nesci et al., 2011).
Also fungi were detected in all peanut samples analyzed, a low incidence
of fungal colonization was observed during five sampling period in peanut
kernels stored in big bag (0.76 + 0.02) and warehouse. Fungal counts in big
bag were higher from pods (95%) than from kernels. The low incidence of
fungal colonization in kernels supports earlier research showing the
importance of injury for invasion by microorganisms and the role of the seed
coat as a barrier for invasion (Carter, 1973). However, the level of fungal
populations from peanut kernels stored in the wagon exceeded 1x105 cfu g-1
and was maintained throughout the storage period despite the environmental
factor variations. These results suggested a high fungal activity and count
levels exceeded the recommended limits to ensure peanut hygienic quality
(Atlanta Poland, 2013). These differences were not due to the storage system
employed if not, the conditioning of the grain before storing, because peanut
entered more wetter (initial aW = 0.87) in the wagon. Therefore total fungal
count in the wagon was similar to peanut stored in big bag 3. In a study
conducted in Indonesia by Bulaong and Dharmaputra (2002) shelled peanuts
of Gajah var. with initial moisture content of 7% were stored at 11 kg/bag in
four bag types namely: jute bag, polypropylene bag, jute bag doubled with thin
polyethylene (PE), and jute bag doubled with thick PE. Storage was done for 6
months under warehouse conditions with monitoring of relative humidity and
temperature. Statistical analyses showed that moisture content and fungal
population were significantly higher in jute (JB) (8.2%; 4.3x104 cfu g-1) and
polypropylene (PB) bags (8.3%; 3.3x104 cfu g-1) than in PE-doubled jute bags
(7.6%; 1.1x103 and 7.5%; 2.4x103 cfu g-1).
Our studies revealed some distinct trends in the relative density of fungal
populations in peanut kernels. The mycological population succession
observed in three storage systems showed that Penicillium and Aspergillus
were the most prevalent genera throughout the storage time and that Eurotium
spp. counts increased after the third month. Similarly, Bhattacharya and Raha
(2002) observed that during harvest field fungi such as Fusarium, Alternaria,
Curvularia and Rhizopus spp. mostly induced the infection of peanut seeds,
but their numbers decreased gradually during storage probably because they
were replaced by storage fungi, mainly by different species of Aspergillus as
found by other researchers (Adebesin et al., 2001, Magnoli et al., 2006). In our
studies, disappearance of field fungi after the first month was evident due to
reduction of aW in kernels, as most of the field fungi were unable to continue
growing at seed moisture of less than 90% RH as pointed out by Lacey (1989).
Bulaong and Dharmaputra (2002) also reported differences in the peanut
fungal population stored in different bag types throughout the storage period.
For all bag types, the significant increase in fungal count was attributed to 2
fungal species, i.e. Penicillium funiculosum and Aspergillus penicillioides. In
JB, the dominant fungus was P. funiculosum from month 0 to month 4. At
month 5 and 6, the dominant species was A. penicillioides. For PB, the
dominant species from month 0 to month 4 was P. funiculosum, while A.
penicillioides became dominant in PB from month 4 to month 6. Eurotium
amstelodami was the second dominant species from month 5 to 6 in JB and
PB. In JB+1 P. funiculosum was dominant from month 0 until month 6.

Eurotium chevalieri was the second dominant species at 1.70 and 17.6% of
population for months 5 and 6, respectively. In JB+2, P. funiculosum was
dominant from month 0 to month 6. Eurotium chevalieri contributed 2.08 and
0.75% to the total population at months 5 and 6 respectively. Therefore, in the
complex system where several fungal species exist together, the dominance of
a species may not be solely due to its ability to tolerate existing moisture
conditions. Several types of interactions exist among fungi. In addition to
competition for space and nutrients, there could be inhibitory interactions due
to release of metabolites of derailing biochemical path ways (Choudhary,
1992).
The most important environmental determinants on fungal growth are aW
and temperature (Pitt, 1975; Troller and Christian, 1978). In these studies,
temperature values were not a limiting factor for the fungal development since
from around the seventh week in all storage systems, the level was the
minimum enough (15 C) to allow the growth of mesophilic fungi. However,
aW values were low and it tended to reduce even more (mean level = 0.60) at
the end of the storage. This fact limited fungal development such as Fusarium
spp. and filamentous fungi group (Beuchat, 1983), favoring the growth of only
the xerophilic fungi that dominated in peanut samples from the three storage
systems.
5.2. Incidence of Aspergillus Section Flavi and Aflatoxins in

Stored Peanut
5.2.1. Analyses of Peanut Stored in Big Bags Conditioned at Different aw

Levels
Peanut kernels showed natural infections by members of Aspergillus from
section Flavi during the 5-month storage period, at the different aW initial
values (big bag 1=0.920.01, big bag 2=0.880.01, big bag 3=0.840.01 and
big bag 4=0.760.02) (Figure 1). Peanut samples taken at different storage
times to determine the density of Aspergillus section Flavi spp. by
conventional and molecular methods, showed correlation between the data
obtained (r=0.613; p<0.0001). However, the cfu values obtained by RT-PCR
were usually higher (0.51 log units) than those obtained by conventional
counts (CC). Both count methods showed that the highest cfu values were in
peanut kernels conditioned at the two highest aW and that this counts were
relatively stable during the first three months of storage (Figure 1). The mean
counts obtained between the first and fourth samplings were 4.9106 cfu g-1
(CC) and 2.6107 cfu g-1 (RT-PCR), and 1.0107 cfu g-1 (CC) and 2.6107 cfu
g-1 (RT-PCR), for big bags 1 and 2 respectively. A reduction of Aspergillus
section Flavi spp. counts [2.34.6 log units (CC) and 1.72 log units (RT-
PCR)] was observed at the end of the storage period. In peanut samples from
big bag 3, Aspergillus section Flavi spp. counts were relatively constant
(7.91057.1106 cfu g-1) during the whole storage period. Similar to the
results observed in big bags 1 and 2, Aspergillus section Flavi spp. counts
were reduced at about 51% at the end of the storage period. In samples taken
at the third and fourth months of storage from big bag 4, RT-PCR was able to
detect nor-1 copies estimated at 92 and 17 cfu g-1, respectively, while the
conventional method gave <100 cfu g-1.
8
Big bag 1
Big bag 2
Big bag 3
Big bag 4
6 Wagon
Warehouse
Log cfu g-1
0
First Second Thirth Fourth Fifth
Sampling Periods
Figure 1. Colony forming units per gram of peanut (log cfu g -1) of Aspergillus section
Flavi spp. present in peanut seed samples from big bags 1, 2, 3 and 4, wagon and
warehouse during a 5-month storage period (Doprado, 2008; Nesci et al., 2011;
Passone et al., 2010).
Figure 2 shows that two species of Aspergillus section Flavi, A. flavus and
A. parasiticus, were identified from peanut pod samples, with a predominance
of A. flavus (58.6%). A. flavus was predominant in all peanut samples showing
isolation percentages of 88.9, 96.4, 100 and 89.2% for big bags 1, 2, 3 and 4,
respectively. A. parasiticus was isolated in low percentage in big bags 1, 2 and
4 ranging from 3.7 to 13.0%. A. parvisclerotigenus were sporadically isolated
from big bags 1 and 2 in a mean level of 2.2%. Only two A. caelatus and A.
bombycis isolates from big bag 1 and at the third and fourth sampling were
identified.
4
A. flavus
A. parasiticus
A. caelatus
A. bombycis
3 A. minisclerotium
A. parvisclerotigenus
A. spp.
Log cfu g-1
0
Pods Seeds* Seeds Seeds
Big bags Wagons Warehouse
Figure 2. Propagules of Aspergillus section Flavi species per gram of peanut stored in
different storage systems. (*Mean of four big bags) (Doprado, 2008; Nesci et al., 2011;
Passone et al., 2010).
Two hundred and twenty seven (70%) out of 323 strains of Aspergillus
section Flavi, were AF producer in culture (Table 6). Toxin levels ranged from
1.3 to 76530.5 ng ml-1 of culture medium (mean level: 1003.9 ng ml-1). A.
parasiticus was the specie with the highest percentage of AF-producing
isolates (100%), their AF production levels ranging from 10.8 to 76530.5 ng
ml-1 (mean level: 4537.3 ng ml-1). Sixty eight percent of A. flavus were AF
producer with mean levels ranging from 19.9 to 3285.6 ng mL-1. Out of 296 A.
flavus isolates, 4, 45 and 51% were classified as S, L and non sclerotial (NS)
strains, respectively. One hundred percent of S strains were AF producers,
whereas L strains produced the highest AF levels. Among S strains, 69% of
the isolates showed an unusual pattern of mycotoxin production (AF group B

and G simultaneously).
Table 6. Aflatoxins production by species in the Aspergillus section Flavi

isolated from stored peanut seeds in different storage systems
Positive Range (ng ml-1) b Mean levels

Species
strains a B aflatoxins G aflatoxins (ng ml-1) + SE c
Big bagsd
A. flavus S strains 4/13 6.4 45.6 23.4 + 9.8
9/13 117.3 232.8 15.5 150.7 212.2 + 39.1
A. flavus L strains 28/132 <1
66/132 1.5 79.9 20.2 + 2.8
38/132 108.0 30612.5 3285.6 + 1193.3
A. flavus NS strains 66/151 <1
58/151 1.3 95.7 19.9 + 3.1
27/151 100.6 1218.8 388.1 + 70.6
A. parasiticus 3/20 9.4 78.6 0.7 26.3 53.3 + 27.7
17/20 119.3 75468.0 16.2 5670.0 9021.2 + 5336.5
A. parvisclerotigenus 4/4 1.5 58.7 25.8 + 12.4
A. caelatus 2/2 <1
A. bombycis 1/1 1.3 + 0.0
Wagone
A. flavus S strains 3/4 <
1/4 2 40500
A. flavus L strains 51/93 <
41/93 2 40500
1/93 40600 82000
A. flavus NS strains 25/32 <
7/32 2 40500
Warehousef
A. flavus 52/79
A. parasiticus 2/2 <2 2500.0 2700 + 5700
SE: standard error. Detection limit: 1 ng ml-1 medium.
a
Ratio of AF-producing strains vs. total strains tested.
b
Range levels of AFB and AFG between 1-100 ng ml-1 and 101- maximum production
ng ml-1.
c
Mean levels of total AFs.
d
Passone et al. (2010).
e
Doprado (2008).
f
Table 7. Aflatoxin levels in peanut kernel samples analyzed over 5 months

of storage
Aflatoxins concentration ng g-1 (mean + SE)a

Samplingb Big bag 1c Big bag 2 Big bag 3 Warehoused
First 86.8 + 27.8 a 3.9 + 0.2 c 1.1 + 1.1 b <1
Second 158.2 + 38.1 a 50.7 + 3.8 bc 1.2 + 0.1 b <1
Third 139.6 + 28.7 a 82.6 + 21.8 ab 10.6 + 0.3 a 2.9 + 5.2
Fourth 153.4 + 47.5 a 83.6 + 41.2 ab 13.8 + 3.5 a 68.9 + 152.9
Fifth 200.4 + 69.1 a 140.9 + 14.9 a 12.5 + 3.0 a 69.1 + 152.1

a
Mean levels of total aflatoxins (AFB1 + AFB2 + AFG1 + AFG2) from peanut seed
samples.
SE: standard error, n = 5. Detection limit: 1 ng g-1.
b
Storage period: July-November for big bags; May-October for warehouse
c
Big bag 1 0.94 + 0.01 aW; big bag 2 0.88 + 0.01 aW; big bag 3 0.84 + 0.01 aW
(Passone et al., 2010).
d
Values in columns with not letters in common are significantly different (p < 0.05)
according to Fisher LSD test.
Aflatoxin B1, B2, G1 and G2 concentrations of the one-hundred peanut

kernel samples collected are shown in Table 7. Aflatoxin levels in samples
from big bags 1 and 2 increased with the time of storage, although with
different time rates. When samplings of the first and fourth months were
compared, toxin increases were 57 and 97% for big bags 1 and 2, respectively.
The mean value of total AFs obtained in samples from big bag 3 at the third
sampling was significantly higher (89%) than from those obtained in the first
and second samplings (p<0.05). Aflatoxins were not detected at any analyzed
peanut sample from big bag 4. Statistical analysis showed a very poor negative
correlation between cfu and AFs accumulation (r=0.514; p=0.05) in peanut
samples.
The determination of physical properties of the samples revealed marked
differences in aW and temperature among individual samples in the four big
bags throughout the storage period. Water activity levels in peanut samples
from big bags 1 and 2 decreased relatively faster during the first three months
of storage, from 0.920.01 aW to 0.760.00 aW and from 0.880.01 aW to
0.670.03 aW, respectively. Thereafter the aW decreased at a slower rate until

the end of storage period (mean=0.650.01 aW). However, aW levels in peanuts
from big bag 3 were lower during the five months of storage (from 0.840.01
aW to 0.600.04 aW). Peanut samples aW from big bag 4 significantly
decreased (p<0.05) during the second month of storage and after that remained
relatively constant until the end of storage period, reaching similar values
(0.660.01 aW) to those registered in big bags with the highest initial aW. Due
to the gradual increase of ambient temperature at Argentinean spring,
temperatures registered in peanuts from the four big bags showed a constant
increase during the four months of storage, from 12.70.45 C to 29.20.6 C.
These environmental variations significantly affected fungal density
(F=584.413; p<0.001) and total AF accumulation (F=32.475; p<0.001).
Statistical analysis carried out with data registered from big bags 1, 2 and 3
showed a negative correlation between aW and toxin accumulation (r=0.643;
p=0.031) and a positive correlation between temperature and total AFs
(r=0.658; p=0.0225). In both cases the correlation was significant (Passone et
al., 2010).
5.2.2. Analyses on Peanut Stored in Wagons of Conditioning and Drying

Aspergillus section Flavi species were isolated at low count levels,
between 1x101 and 4.6x102 cfu g-1, at all sampling carried out. Within this
section two species, A. flavus, with the highest prevalence (89.3%), and A.
parasiticus were identified (Figure 2). Throughout the five samplings carried
out between July and November, 144 isolated from Aspergillus section Flavi
were identified, of which 129 and 16 were A. flavus and A. parasiticus,
respectively. In relation to production of sclerotia, of the total A. flavus strains
isolated, 75.2% were producers and of these 95.9% were classified as L
strains, while only 4.1% as S strains (Table 7). When the toxigenic ability of
A. flavus isolates was examined, it was observed that only 1 isolate produced
AFG1 and the others produced type B AFs. L strains produced, on average,
higher AF levels than S morphotype strains. Of the 16 A. parasiticus isolates,
only 4 (25%) produced undetectable AF levels (<1 ppm). All toxigenic strains
produced AFB1, 2 of them also produced AFB2 and 4 produced AFB1 and
AFG1.
Of the 60 samples analyzed, no AFs were detected in any of them
(Doprado, 2008).
5.2.3. Analyses on Peanut Stockpiled Warehouse

The lowest incidence of Aspergillus section Flavi was detected in the first
sampling period, with an increase in the fourth sampling period (Figure 1).
Figure 2 shows the incidence of Aspergillus species that colonize the stored
grain during five sampling moments. Among the Aspergillus populations
recovered from peanut kernels, A. flavus was the most frequently isolated
(79%), followed by A. caelatus (7.6%), A. parasiticus (5.9%), A. spp. (5.1%)
and A. minisclerotium (1.7%).
Analyses of toxigenic potential of Aspergillus section Flavi strains
isolated from the peanut samples revealed the production of AFB1 by 52
strains out of 79 of A. flavus with mean levels ranging from <2 to 225000 ng
ml-1 and 2 strains of A. parasiticus with mean levels ranging from <2 to 25000
ng ml-1 (Table 6). The highest proportion of A. flavus toxigenic strains (87.5%)
was obtained from the second sampling period (June). However, the highest
number of A. flavus strains was isolated in September, as were the strains with
ability to produce the maximum AFB1 level (225000 ng ml-1). A. parasiticus
strains were isolated during the third sampling period (August) and produced
AFB1 levels between <2 and 25000 ng ml-1.
In samples corresponding to sampling periods of May and June, no AFB1
were detected. Aflatoxin B1 accumulation in peanut kernels during all
incubation periods was determined (Table 7). The greater levels of AFB1 were
detected in September and October with a mean of 68.86 and 69.12 g kg-1,
respectively, coinciding with the highest Aspergillus section Flavi count
(Nesci et al., 2011).
Stored peanuts represent a complex ecosystem in which seed spoilage by
fungi is determined by a range of factors which can be classified into four
groups; intrinsic nutritional factors, and extrinsic, processing and implicit
factors (Magan et al., 2004). Alone or in combination among them, these
factors affect the composition of the fungal population, inducing changes
throughout the storage period. In our studies, reductions of peanut aW and
increases of temperatures were registered in all big bags and wagon
throughout the storage period and these physical changes were reflected by a
reduction of Aspergillus section Flavi growth, independently of the
quantification methodology applied. When the CC method was used, count
reductions of these fungi were estimated in 35.2, 65.1, 33.6, 100 and 55.9% for
big bags 1, 2, 3, 4 and wagon, respectively. It is important to emphasize that
similar results were obtained with RT-PCR assays, whose cfu reductions were
around 36.0%. Similarly, Bulaong and Dharmaputra (2002) reported that A.
flavus, the only specie isolated from section Flavi, constituted a minor
percentage of the total population (from 0.001 to 1.45%) during month 4 to

month 6 in the four bag types, with a mean percentage of 15.6% at the
beginning of the assay. On the contrary, we observed an increase in the
frequency of Aspergillus section Flavi in peanut stored in warehouse,
estimated in 90.3% at the two last sampling periods. In their study,
Bhattacharya and Raha (2002) detected a high percentage of peanut seeds
contaminated by A. flavus (40%) at the beginning of the storage and the
incidence of this fungus increased to 78% after the tenth month. The results of
the work conducted by Nakai et al. (2008) on stored peanut in 25 kg bags
during 12-month period showed that the relative frequency of A. flavus
increased from 19.4 to 26.7% at the end of the storage. The presence of
Aspergillus and Penicillium in peanut kernels, is because the great adaptation
of these fungi to this substrate, especially during storage (Rossetto et al.,
2005).
These results indicate that Aspergillus species from section Flavi were
found at important counts from stored peanuts in the different storage system
in Argentina and demonstrated to be highly toxigenic. Among representatives
of the section Flavi, A. flavus was the prevalent specie in the three storage
system analyzed followed by A. parasiticus in big bags and wagon and by A.
caelatus in warehouse. It is noteworthy that, although the fungal composition
analyses from peanut pods were performed only in the big bag with the lowest
initial aW content (0.76+0.02), the mean counts of A. flavus and A. parasiticus
were 12.2 and 79.9% higher than the mean counts obtained from peanut
kernels of the four big bags. Tannis, waxes, amino compounds and structural
features in the peanut seed coat have been implicated in resistance to invasion
by A. flavus and A. parasiticus (Amaya-F et al., 1977; LaPrade et al., 1973;
Sanders and Mixon, 1978; Zambettakis and Bockelee-Morvan, 1976). A.
flavus have previously been reported as the prevalent specie from section Flavi
in peanut stored in different storage systems and markets. In addition to A.
flavus (21.2%), A. niger was isolated but at low frequency (0.6%) from stored
peanut kernels in Brazil (Nakai et al., 2008). Recently, Zorzete et al. (2013)
found species from sections Flavi and Nigri (mean fungal frequency= 10.1, 1.5
and 5.2% for A. flavus, A. parasiticus and Aspergillus section Nigri,
respectively) in peanut seed cultivars Runner IAC Caiap and 886 stored in 25
kg sacks, stacked on wooden pallets during April to September 2006 in Brazil.
Bulaong and Dharmaputra (2002) also reported that A. flavus (4.6%) and A.
niger (1.9%) were isolated during all storage period between other Aspergillus
species with high occurrence but only during the first (A. fumigatus 22.8%, A.
versicolor 0.9%, A. wentii 2.1%) or the last (A. penicillioides 47.6%) two
months of storage in Phillipines. In unshelled peanuts obtained from five

fresh-produce markets in Kenya the Aspergillus species isolates were A. flavus
(28%), A. parasiticus (32%), A. niger and A. ochraceous (41%) (Gachomo et
al., 2004). In a survey conducted in Nairobi, Nyanza and Western provinces in
Kenya between March and July 2009 with 1263 peanut products sampled out
of which 705 samples were microbiologically analyzed; six Aspergillus
species were detected in the samples and were in decreasing order of cfu g-1:
A. flavus (808), A. niger (156), A. tamari (27), A. alliaceus (21), A. parasiticus
(10), and A. caelatus (5). The combined incidence of A. flavus and A.
parasiticus was varied significantly (p<0.05) with peanut product: peanut flour
(69%), shelled raw peanuts (53%), spoilt peanuts (49%), boiled podded
peanuts (45%), podded peanuts (39%), peanut butter (31%), fried peanuts
(22%) and roasted peanuts (20%) (Wagacha et al., 2013). Main fungi present
in 20 samples of 50 g roasted and salted peanuts and 16 samples of 50 g unsalted
peanuts (pure) collected from Zanjan BAZAR (India) were A. flavus (39.1%),
Penicillium (9.2%), Rhizopus (7.2%), Mucor (2.5%), Alternaria (1.03%) and
Nigrospora (0.5%) (Rostami et al., 2009).
In our studies the low aW levels detected in peanut stored in big bag 4 and
wagon ensured the absence of AFs, however A. flavus S, L and NS strains and
A. parasiticus with different AF-producing potential were present between the
first and the fifth sampling periods. Therefore, if the aW of the grains remains
below 0.65, molds are unable to grow and the stored grain will be stable.
However, localized pockets of higher moisture may be present if temperature
and moisture gradients develop in the storage by the biological activity of
insects and rodents, opening the way for mold germination, growth and
consequently AF production (Hocking, 2003). A low percentage of peanut
samples from warehouse were contaminated with AFB1, but with values
exceeding the maximum limit of 20 g kg-1 allowed for peanut without
shelling, shelling, raw or toasted, by the Mercosur regulations (56/94) for the
sum of AFs (B1+B2+G1+G2) (FAO, 2004). The production of AFB1 was
detected in 67% of strains. Studies conducted in Brazil showed that 33.3% of
peanut samples were contaminated with AFB1 at mean levels ranging from 7.0
to 116 g kg-1 of which 24 samples exceeded the maximum limit established
by the aforementioned legislation and 93.8% of A. flavus strains isolated were
AF-producers (Nakai et al., 2008). Recently surveys analyzed the occurrence
of AF and the incidence of AF-producing fungi in peanuts and peanut products
traded in Kenyan markets (Wagacha et al., 2013). Seventy three percent of A.
flavus and A. parasiticus isolates produced at least one of the AF types, with
66% producing AFB1. The total AF level among peanut products ranged from
0 to 1629 mg g-1; and there was a positive correlation (r = 0.2711; p<0.05)

between the incidence of A. flavus and A. parasiticus, and total AF level.
Thirty seven percent of the samples exceeded the 10 mg kg-1 regulatory limit
for AF levels set by the Kenya Bureau of Standards (2007). Raw podded
peanuts had the lowest AF levels, 96% having levels less than 4 mg kg-1 and
only 4% having more than 10 mg kg-1. The most AF-contaminated products
were peanut butter and spoilt peanuts, with 69% and 75% respectively,
exceeding 10 mg kg-1. The AF concentrations in peanuts from big bags 1, 2
and 3 increased toward the end of storage period. The increases in the second
bimonthly ranging between 15.8 and 55.1%, which is about twice lower than
those registered in the first two months. These results appear related to the
reduction of the Aspergillus section Flavi population throughout the storage
period. It is possible that a balance occurs in the closed silos environment,
between the cumulative increase in AFs concentration and the lower marginal
efficiency in their production and/or release, due to the fungus population
decrease. Despite the fungal population reduction observed at the end of the
storage period, we suggested that the environmental stress could produce an
increase in AF levels. Gunterus et al. (2007) reported that no correlation was
observed between inoculum level and AF accumulation. Aflatoxin
concentration increased with decreasing inoculum size. The lowest inoculum
(102 conidiospores g-1 of peanuts) generated highest AF levels (1.000 g g-1 of
peanuts).
CONCLUSION
Based on these results, A. flavus and A. parasiticus strains are widely
distributed in soil and groundnut kernels coming from the traditional and new
peanut-growing areas and during the storage period in the three peanut storage
systems analyzed in Argentina. The ecophysiology of these fungi implies a
risk of AF contamination if environmental conditions became conducive,
highlighting the need to promote good practices at all stages of the peanut
value chain in order to minimize market access by non-complying products.
ACKNOWLEDGMENTS
This work was carried out through grants from the Agencia Nacional de
Promocin Cientfica y Tecnolgica and from the Secreterara de Ciencia y
Tcnica, Universidad Nacional de Ro Cuarto.
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Chapter 8
TOXICOLOGICAL EFFECTS,
RISK ASSESSMENT AND LEGISLATION
FOR AFLATOXINS
Marina Goumenou1, Dimosthenis Axiotis2*,

Marilena Trantallidi5, Dionysios Vynias2,
Ioannis Tsakiris3, Athanasios Alegakis2,
Josef Dumanov4 and Aristidis Tsatsakis2
1
General Chemical State Laboratory, D Division of Athens,
Athens, Greece
2
Laboratory of Toxicology, Department of Medicine,
University of Crete, Voutes, Heraklion, Greece
3
TEI of Western Macedonia, Florina Branch, Department of Agricultural
Products Management and Quality Control, Terma Kontopoulou,
Florina, Greece
4
Mycological Institute EU US
5
Dept. of Biomedical and Clinical Sciences, University of Milan,
Milan, Italy
*
Corresponding author: Dimosthenis Axiotis, Email: dimaxiot@gmail.com, Mob: +30 693 7
656182, +39 345 6694328.
192 Marina Goumenou, Dimosthenis Axiotis, Marilena Trantallidi et al.
SUMMARY
Aflatoxins are toxic metabolites produced by the fungus Aspergillus. The
main representatives are aflatoxins B1, B2, G1, G2. Their occurrence in food
like nuts, cereals and cereal-derived products is a result of fungal
contamination before harvest and during storage. Milk can also be
contaminated by aflatoxin M1 (main metabolite of B1) as a result of animals
exposure to feed contaminated by the aflatoxin B1.
Aflatoxins manifest acute and chronic toxicity. Evidence of acute
aflatoxicosis in humans involving a range of symptoms from vomiting to death
has been reported mainly in Third World Countries. In relation to chronic
toxicity aflatoxins are well known for their genotoxic and carcinogenic
properties while recent studies evident a series of other possible effects like
reprotoxicity, impaired growth in children, intestinal functions, chronic fatigue
syndrome, compromise immunity and interfere with protein metabolism and
multiple micronutrients that are critical to health.
The critical step for aflatoxins risk assessment is the estimation of the real
exposure. For this reason a number of surveys are conducted globally using
tools like biomarkers of exposure and modeling. In addition new parameters
like the climate change are now taken into consideration in order to predict
possible current and future changes of exposure to aflatoxins. As aflatoxins are
compounds of natural origin and their presence in food cannot be totally
eliminated the risk management is based on keeping the total exposure as low
as reasonably achievable taking into account the social-economic impact of
crop and livestock losses. Exposure reduction is achieved mainly by reducing
the number of highly contaminated foods reaching the market by regulatory
control but also applying detoxification strategies. According to the EU
regulatory framework minimization of the exposure to aflatoxins is based on
setting maximum levels of aflatoxins in different foodstuffs (4 10 g/kg total
aflatoxins) and feed (EC/1881/2006, Directive 2002/32/EC). Products
exceeding the maximum levels should not be placed on the EU market.
Methods of sampling and analysis for the official control of aflatoxins, are also
set (EC/401/2006) in order to ensure common sampling criteria to the same
products and that certain performance criteria are fulfilled. The United States
Food and Drug Administration (FDA) has established the action levels for
aflatoxin present in food to the 20 g/kg (0.5 g/kg for milk) and up to 300
g/kg for feed. Finally an action level of 10 g/kg total aflatoxins is also used
from Japan authorities.
Toxicological Effects, Risk Assessment and Legislation 193
1. INTRODUCTION
The acute effects from Aspergillus flavus have been well studied when its
primary toxins, the aflatoxins, were isolated from peanut meal in 1961 during
the investigation of an epizootic of "Turkey X" disease in England [1]. At that
time, these toxins were identified as secondary metabolites of some strains of
Aspergillus flavus and were considered as the etiologic agents of the disease in
turkeys following contamination of their feed by A. flavus. Since then,
numerous studies have identified Aflatoxins to be the causative agent of
adverse health effects with liver toxicity as the major end-point.
Initially, four major aflatoxin types were recognized, referred to,
collectively, as aflatoxins and designated B1, B2, G1, and G2 due to their
fluorescence and retention factor values on thin-layer chromatographic plates.
Upon ingestion of aflatoxin B1 by humans and animals, production of
aflatoxin M1, as a metabolite, was discovered in mother's milk in exposure
levels of ng. Furthermore, aflatoxin M2, another potent hepatotoxic and
hepatocarcinogenic aflatoxin has been identified in milk of cows fed on meal
contaminated with aflatoxin B2 [2]. In addition to the aforementioned
aflatoxins, the aflatoxin Q1, a major metabolite of B1, was found in in vitro
liver preparations of other higher vertebrates [3].
Currently, at least 14 different types of aflatoxin are known [4]. Aflatoxin
B1 is considered as the most toxic and the most highly monitored and
regulated mycotoxin in foodstuffs produced by both Aspergillus flavus and
Aspergillus parasiticus. Aflatoxin G1 and G2 are produced exclusively by A.
parasiticus. Although Aspergilli are often present in food and feed products,
their occurrence does not consist necessarily an indicator of harmful levels of
aflatoxin as they do not all produce aflatoxins.
It is well documented that aflatoxins manifest acute and chronic toxicity.
Apart from their strongly evident acute liver toxicity and carcinogenicity
potential, recent studies manifest a series of other possible effects, such as
reprotoxicity, impaired growth in children, intestinal malfunctions,
neurotoxicity and chronic fatigue syndrome, nephrotoxicity, compromised
immunity and interference with protein metabolism and multiple
micronutrients that are critical to health. Part of aflatoxinstoxic potential is
their very fast absorption by the small intestine, as indicated by studies in rats
[5].
Risk assessment of aflatoxins requires in addition to thorough knowledge
of health effects (hazard identification and characterization), exposure data.
About 4.5 billion people, mostly in developing countries, are at risk of chronic
exposure to aflatoxins from contaminated food crops [6]. The great majority of
studies for aflatoxins mainly focus on the contamination levels in certain food
commodities. However, since consumers may be exposed to aflatoxins
through a normal diet, dietary studies are crucial in order to accurately
estimate exposure and its variations between different groups (age,
geographical distribution, diet habits etc).
According to the classic risk assessment approach for non-genotoxic
substances, risk characterization is based on the comparison of a substances
derived no-effect level, based on the evaluation of toxicological data, with the
estimated exposure levels. Such an approach is not possible to be followed for
aflatoxins as a threshold is not considered to exist. Alternatively, the As-Low-
As-Reasonably-Achievable (ALARA) approach is used by setting maximum
acceptable amounts of aflatoxins in foodstuffs. The ALARA level, reflects the
concentration of a substance that cannot be eliminated from food without
involving the discard of that food altogether or without severely compromising
the availability of major food supplies [7].
The protection of public health, consumers and workers from aflatoxins is
addressed in different aspects of legislation. Aflatoxins are subject to
regulations at national, regional and international level. A well-structured legal
framework should guarantee safe products (foodstuffs and feedstuffs) intended
for human and animal consumption, including regulatory limits, measures for
monitoring compliance with limits, guidance to the food industry, cooperation
between agencies on food safety and enforcement action. Legislation in
Europe, USA and elsewhere contributes to the global effort of controlling
aflatoxins adverse health effects with respect to any social-economic
reasoning.
2. HEALTH EFFECTS
Health effects caused by aflatoxins are mainly divided into acute and
chronic toxic effects. Among chronic effects, carcinogenicity is by far the
most studied. However, a number of other chronic adverse health effects are
known nowadays, and include reproductive toxicity, impaired growth in
children, intestinal malfunctions and compromised immunity.
2.1. Mechanisms of Action
The numerous health effects caused by aflatoxins are the result of

common or different underlined mechanisms of action that can be or not
crossed in the complex net of biochemical reactions in the human body. At
molecular level, a key player in aflatoxin toxicity is the epoxide derivative of
aflatoxin B1. This epoxide has the ability to bind on DNA and to disrupt
transcription and translation activities, thus initiating carcinogenesis. Due to
the oxidative nature of this toxic derivative it can release free radicals causing
cell damage (Figure 1).
The evolution of molecular techniques like microarrays and PCR was
critical in understanding more precisely the mechanism of action of aflatoxins,
starting from molecular level, to genes, cells and organ level. Recent gene
expression studies have shown that aflatoxins can cause:
a) down regulation of carnitine palmitoyltransferase (CPT) system in

mitochondrion leading to decrease of body weight gain
b) down regulation of fatty acid metabolism pathway increasing liver
weight and causing fatty liver
c) up-regulation of cell proliferation pathway causing carcinoma, and
d) down regulation of B cell activation lowering immunity in birds fed
aflatoxin.
In addition, aflatoxins can impair protein biosynthesis by forming adducts

with DNA, RNA and protein, inhibit RNA synthesis, DNA-dependent RNA
polymerase activity, and cause degranulation of endoplasmic reticulum [9, 10].
Susceptibility to aflatoxin is higher in younger ages; it is sex-related
(testosterone-dependent) and with a great inter- and intra-species variability
depending on the biochemical detoxification abilities [11].
2.2. Acute Effects
The acute toxic effects of aflatoxins have been studied for many decades
[12]. Epidemiologic, experimental and clinical studies have shown that high
exposure to aflatoxins (above 6000 mg), through digestion, can cause severe
intoxication, which results in direct liver damage and subsequent illness or
death [13,14,15]. The mechanism of aflatoxin acute toxicity is related to
malfunction of the liver, which is the target organ of aflatoxin toxicity.
Aflatoxin metabolites, such as the aforementioned electrophilic B1, 8-9

epoxide [16] react with cell components causing serious disturbance in cell
lipid and carbohydrates metabolism. Lipids infiltration from the liver cells
causes hepatocytes death and liver cirrhosis and necrosis. The manifestation of
the cellular mechanism of aflatoxicosis at whole organism level regards
mainly the reduction of critical blood proteins, icterus, vomiting, edema and
abdominal pain [17].
No animals are immune to the acute toxic effects of aflatoxins. Animal
studies have found two orders of magnitude difference in the median lethal
dose for aflatoxin B1. Susceptible species such as rabbits and ducks have a
low (0.3 mg/kg) median lethal dose, whereas chickens (18 mg/kg) and rats
have greater tolerance. Adult humans usually have a high tolerance of
aflatoxin; in the acute poisonings reported, usually children are the ones who
die [11, 9].
Figure 1. Mechanism of cell damage in aflatoxins toxicity [8].
Outbreaks of aflatoxicosis affecting up to several hundred people at a time

have occurred sporadically, most recently in eastern Kenya in early 2004
[18,19]. The Kenya incidence was attributed to poor harvest of maize that had
been damaged and stored in warm and humid conditions making it susceptible
to mold by drought. From January to June 2004, 317 people sought hospital
treatment for symptoms of liver failure, and 125 died.
2.3. Genotoxicity and Carcinogenicity
Aflatoxins as toxins are among the most carcinogenic substances known

and the major cancerous hepatocellular carcinoma (HCC) risk factor. Cancer is
broadly defined as any disease in which normal cells are damaged and do not
undergo programmed cell death (apoptosis) as fast as they divide via mitosis.
Aflatoxin B1 is metabolized by cytochrome-P450 enzymes to the reactive
intermediate AFB1-8,9 epoxide (AFBO) which binds to liver cell DNA,
resulting in DNA adducts. DNA adducts interact with the guanine bases of
liver cell DNA6. This is thought to cause mutations at the codon 249 hotspot
in exon 7 of the p53 gene, an important gene in preventing cell cycle
progression, when there are DNA mutations, or signaling apoptosis [20].
These mutations seem to affect some base pair locations more than others
for example, the third base of codon 249 of the p53 gene appears to be more
susceptible to aflatoxin-mediated mutations than nearby bases [21]. As a
consequence of aflatoxin genotoxicity potential, even short-term exposure can
lead to a risk of developing liver cancer.
Although this mechanism of genotoxicity is well recognized and
understood, non-genotoxic effects also exist and resulting cancers in the form
of enzymatic necrosis from the hyphae of pathogenic Aspergilli with the
production the hyphal digestive enzymes: the primary are proteases (protein),
amylases (carbohydrate), and lipases (fats).
From neoplasia (tumor) to necrosis, cancers are recognized in over 200
differing pathologies referred to by cell, tissue or organs affected. Aflatoxins,
as mentioned above, can be classified as genotoxic (p53) or non-genotoxic. In
neoplastic disease, the neoplasms may be benign, pre-malignant as a
carcinoma in situ or a malignancy as a cancer. Immunohistological
Aspergilloma, resulting from invasive Aspergillus fumigatus is the part of
immunological defensive mechanism in the formation of encapsulating fibroid
tumors known as a mycetoma or fungus ball rarely developing into a cancer.
In any case, the carcinogenic potential seems to increase due to malnutrition,
especially in cases of pyridoxine deficiency [22].
2.4. Other Health Effects
2.4.1. Reproductive Toxicity

The intensive research for aflatoxin-induced reproductive toxicity started
long after the study of acute toxicity and carcinogenicity. However, there is up
to now a considerable number of studies that provide evidence for the
association of aflatoxin exposure with testicular toxicity, infertility,
developmental toxicity and possible mechanisms involved.
According to the study of Verma and Nair [23], aflatoxins can inhibit the
stereoidogenesis in mice by reduction in 3b- and 17b-hydroxysteroid
dehydrogenases probably due to alterations in mitochondria, inhibition of
protein synthesis/enzyme activity, alteration in plasma membrane of Leydig
cell due to lipid peroxidation and/or irregular protein biosynthesis. In the study
of Ibeh and Ogonor[24], a correlation between infertility in men and aflatoxin
concentration in semen was suggested. In addition, higher frequency in sperm
count, morphology and motility was observed in the higher exposed infertile
men. In the same study,similar results were observed by intentionally exposing
rats. Additional studies in animals have shown spermatotoxic activity of
aflatoxins supporting these conclusions [25, 26, 27, 28]. Possible mechanism
involve disruption on sex hormone synthesis, protein synthesis inhibition, fat
metabolism disruption and, possibly, direct lysis of sperm cell membrane.
There is a considerable number of studies related to the association of
exposure to aflatoxins and low birth weight [29]. However, the overall result is
inconclusive as there are both positive and negative associations and in many
cases additional possible causes were not considered. In any case, the
possibility of such an association remains of high concern as birth weight
lower than 2500 g is closely associated with fetal and neonatal morbidity and
mortality, inhibited growth, poor cognitive development, and chronic diseases
later in life [30].
2.4.2. Impaired Growth in Children

Chronic, subclinical exposure does not lead to symptoms as severe as
acute aflatoxicosis. Children, however, are particularly affected by aflatoxin
exposure, which leads to delayed development [31] and impaired growth [32].
Food borne aflatoxin exposure is common in many areas of Africa and Asia
where childhood stunting and underweight are also frequent, due to a variety
of possibly interacting factors such as enteric diseases, socioeconomic status,
and suboptimal nutrition [33]. Gong et al. performed in 2002 [34] a cross
sectional study in young children from Benin and Togo in relation to the
dietary aflatoxin exposure and impaired growth revealing a striking

association between exposure to aflatoxin in children and both stunting (a
reflection of chronic malnutrition) and being underweight (an indicator of
acute malnutrition). In this study, children still partially breast-fed had lower
exposure, almost certainly reflecting lower toxin levels in milk than in
weaning and family foods. Thus, growth faltering occurs at a time of change to
solid foods, when there is co-exposure to aflatoxin and a plethora of infectious
hazards, such as malaria, diarrhea and respiratory infections. Studies in Togo,
Gambia, Ghana, Iran, Kenya, and the United Arab Emirates have shown
similar findings, outlined in a review published by Wu and colleagues in 2011
[35, 33]. However, studies with cross sectional design cannot always confirm
if the association between aflatoxin exposure and impaired growth is a direct
result of aflatoxin toxicity or reflects consumption of fungus affected food of
poor nutritional quality. Apart from any association between children exposure
to aflatoxins and after birth growth impairment, aflatoxins can cause genetic
defects at fetal stage as they can cross placental barriers [36].
2.4.3. Intestinal Malfunctions

Intestinal cells are the first cells to be exposed to aflatoxins [37]. Rapidly
dividing and activated cells and tissues with a high protein turnover are
predominant in gut epithelium; consequently, the effect of aflatoxins as
disruptors of protein synthesis is of great importance. Intestinal homeostasis
depends on the balance between nutrition, immune system and gut microbiota
and this homeostasis is crucial for the health of the GIT, the absorption of
nutrients and the functions of the immune system. For these reasons, intestinal
investigations have gained significant interest over the last decade.
Evidence on the impairment of growth after exposure to aflatoxins was
questioned as related to malnutrition of exposed populations. However, there
is evidence that such malnutrition is not just the output of socio-economic
conditions, but probably also a consequence of aflatoxin exposure which can
compromise the absorption of nutrients. Several studies in animals support the
reduction of nutrient requirements, absorption, digestibility and utilization due
to aflatoxins [40, 38, 39, 40, 41]. In addition, disturbance on digestive
enzymes has been reported [41, 43, 42] without however to be clear if this is a
cause or the output of other aflatoxin-related effects. Studies on birds revealed
effects in the morphology of the GIT, as well, while the results for other type
of effects, such as nutrients utilization and microbiota sensitivity on these
studies are rather controversial [43].
2.4.4. Neurotoxicity
The effects of aflatoxins in the nervous system are related to the action of
their metabolite AFB-8, 9-epoxide and its ability to create DNA and protein
adducts, to inhibit protein, RNA and DNA synthesis, to promote mitochondrial
directed apoptosis of the nerve cells and to produce Oxygen Reactive Species
(ROS) [44, 45, 46, 47]. Myelin depletion and disturbance of brain chemistry
by aflatoxins is also reported [50, 48, 49]. Aflatoxins have been reported to
cause tumors in both the central and peripheral nervous system [54]. Several
neurotransmitters are additionally reported to be affected by aflatoxins in
animals, such as acetylcholinesterase enzymes that may affect the memory,
learning and cognitive functions[50], dopamine, serotonin as well as tyrosine
and tryptophan, leading to neurocognitive decline and alteration of sleep cycle,
dullness, restlessness, muscle tremor, convulsions, loss of memory, epilepsy,
idiocy, loss of muscle coordination, and abnormal sensations [51, 52, 53, 54].
AFB1 has also been reported to increase the central and peripheral nervous
system Na/K ATPase, -glucuronidase and -galactosidase while inhibiting
the Mg-ATPse in experimental animals; this is also important in the normal
functioning of the glutamate neurotransmitter and their NMDA receptors [55,
56].
Liver malfunction caused by aflatoxicosis has as secondary effect, i.e. the
gathering of non-detoxified ammonia in the brain. Ammonia can pass the brain
barrier causing encephalopathy. Toxic encephalopathy due to aflatoxin
involves multiple symptoms such as loss of balance, recent memory decline,
headaches, lightheadedness, spaciness/disorientation, insomnia, loss of
coordination; an association with a Reye-like Syndrome in Thailand, New
Zealand, Czech Republic, Slovakia, the United States, Malaysia, Venezuela
and Europe [57] has been reported.
2.4.5. Immunosuppression
Many studies prove that aflatoxin B1 is immunosuppressive in animals,
with particularly strong effects on cell-mediated immunity and increased
susceptibility to bacterial and parasitic infections [58]. According to these
studies, performed mainly to birds and rats, aflatoxins decrease complement
activity leading to impairment of phagocytosis and reduce chemotactic ability
of leucocytes. Immunosuppression caused by exposure to aflatoxins is the
output of their interference with normal function of B and T-cells [59],
reduction of the phagocytosis by macrophages and of the activity of vitamin K
[60]. In addition, the impairment of protein synthesis caused by dietary
aflatoxin could account for the lack of humoral immunity without the
necessity of B and T cell destruction [61].
Human monocytes treated with aflatoxin B1 resulted in impaired
phagocytic and microbicidal activity and decrease in specific cytokine
secretion [61]. Studies have linked human exposure to aflatoxins to increased
prevalence of infection [62]. According to the study of Turner et al. (2003)
[63] sIgA in saliva may be reduced because of dietary levels of aflatoxin
exposure in children in rural Gambia that are frequently exposed to high levels
of aflatoxin. Aflatoxins can also cross the human placenta [68]. This finding is
also supported by the study of Pier et al. [64] where the immunosuppressive
effects of aflatoxin were also shown to be transferred across the porcine
placenta and to affect the unborn fetus. Finally, it is important to mention that
various recent studies have focused on ameliorating the effects of aflatoxin by
supplementing or amending the diet [65].
The immunosuppressive potency of aflatoxins is also responsible for the
decrease of the vaccination value reducing the antibody response in animals
like poultry, rabbits and cattles [11]. Considering that aflatoxins exposure
occurrence is mainly reported in regions of Africa and Asia, where infectious
diseases are common and vaccination is of great importance, one can easily
understand the additional implication that could be created by this aspect of
aflatoxins adversity if relevant for humans. However, existing studies in
humans do not support clearly such relevance. According to the study of
Turner et al. (2003) [64] mentioned above, antibody response to one of four
pneumococcal serotypes, but not rabies vaccine, was weakly associated with
higher levels of serum aflatoxin-albumin (AF-alb) adducts (as biomarker of
long term exposure) in children exposed to aflatoxin in Gambia. Furthermore,
no association between cell-mediated immunity responses to test antigens and
AF-alb was reported. Allen et al. (1992) [66] also observed absence of
correlation between Af-alb concentration and the malaria specific antibody.
2.4.6. Miscellaneous
Other health effects mentioned in the open literature concern the
respiratory system [17], the renal system [70] and incidences of teratogenicity.
More information about aflatoxins health effects can be found in the literature
collection Additional Studies on Aflatoxin and Health published by the
PACA (Partnership for Aflatoxins Control in Africa) [67].
3. RISK ASSESSMENT
3.1. Exposure
Aflatoxins are mainly observed during and after post-harvest food and
feed contamination, mainly due to temperature and humidity conditions which
favor their production. Weather conditions and cultivation techniques may also
favor spoilage during field stage. The food chain is the main pathway through
which aflatoxins enter the human body. In a worldwide basis, it is difficult and
in some cases impossible to estimate the degree of exposure to aflatoxins
based on the consumption of specific food commodities and the levels of
contamination. Worldwide trade of food products and different dietary habits
may also increase the difficulty of exposure assessment to aflatoxins [68].
The most common food commodities in which aflatoxins were detected
are: a) cereals and small grains i.e. wheat and barley b) milk and dietary
products and c) nuts and dried fruits. The aforementioned foodstuffs are
consumed by all age groups and some of them constitute a crucial part of daily
diet i.e milk for children. There are several studies reporting the levels of
aflatoxins in food and, at the same time, estimating the exposure of population
through consumption. A number of them are full diet studies while the rest are
referred to a specific food commodity.
Jager et al. (2013) present an assessment of aflatoxin intake in Brazil [69].
In this study, the levels of aflatoxins were determined in peanut, corn, bean
and milk milk products collected directly from home of residents during
2011 and 2012. A total of 240 samples were analyzed. The results point out
that peanuts and derivatives were the most important source of exposure for
the population. The estimated daily intake of aflatoxins through peanut
consumption was 13.7 ng kg-1 b.w day-1. These results are much higher
compared to the intake levels of aflatoxins from other countries such as
France, Australia and United States (0.345 ng kg-1 b.w day-1, 0.15 ng kg-1 b.w
day-1 and 0.26 ng kg-1 b.w day-1, respectively). Milk represents a high portion
of the daily intake with a maximum value of 0.3 ng kg-1 b.w day-1 which, in
this case, was in accordance with values reported for French population.
In a study performed in Spain, a total of 603 samples were collected from
Catalonia supermarkets during 2008 and 2009 [70]. The samples represented
the most common food commodities consumed in Catalonia and included
peanuts, pistachios, dried figs, sweet corn, breakfast cereals, corn snacks, dried
red pepper and baby food. In this study, the levels of population exposure to
aflatoxins through diet were ranged from 0.036 up to 0.788 ng kg-1 b.w day-1.
In another study in France a total of 577 food samples (food products based on
cereals, milk, dairy products, solid food products, beverages and breakfast
cereals) were analyzed for mycotoxins during the period 2006-2007.
According to the results the levels of exposure to aflatoxins through
consumption of selected foods ranged from 0.001 ng kg-1 b.w day-1 up to 0.198
ng kg-1 b.w day-1 for children, while for adults from 0.001 ng kg-1 b.w day-1 up
to 0.188 ng kg-1 b.w day-1 [71]. In Japan from 2004 to 2006, a total of 884
food samples (chocolate, buckwheat, cocoa, almond, peanut butter, peanut, red
pepper, pistachio and white pepper) were collected and analysed for aflatoxin
residues [72]. The estimated daily intake based on 95th values ranged from
0.003 up to 0.014 ng kg-1 b.w day-1 according to the different scenarios based
on the age of the consumers.
In some cases, studies focus on more specific food commodities, which
represent local diet habits, such as spices in China. Zhao et al. (2013)
estimated the exposure of the Chinese population to aflatoxins through the
consumption of different spices (pepper, chili prickly ash, cinnamon, aniseed,
fennel, curry powder, cumin and ginger) [73]. A total of 480 samples were
collected from retailers and analyzed during 2009. In this study, several
scenarios were followed for the estimation of aflatoxins daily intake. In a
worst-case scenario, the estimated daily intake mean values based on
consumptions of spices in New Zealand, Europe, US, India and Thailand were
0.057, 0.115, 0.461, 1.097 and 1.672 ng kg-1 b.w day-1, respectively.
Literature provides extended data for the exposure to aflatoxins through
milk and dairy products consumption mostly based on the results from
monitoring studies. In a study that took place in Brazil a total of 125 different
milk types were collected from Sao Paulo during 2006 and analyzed for
aflatoxins [74]. The estimated daily intake of aflatoxins based on local milk
consumption was 1 ng kg-1 b.w day-1 for children and 0.188 ng kg-1 b.w day-1
for adults. In another study,176 milk samples were collected in Serbia during
2013 [75]. The highest estimated daily intake of aflatoxin through milk
consumption was 36.8 ng kg-1 b.w day-1.
As already mentioned, peanuts are considered as a potential source of
aflatoxins. In a study that took place in Brazil, 100 samples were collected
from the market and analyzed during 2006-2007 [76]. According to the results,
the estimated daily intake varied from 0.6 up to 10.4 ng kg-1 b.w day-1. In a
study conducted in China, a total of 1040 samples were collected from four
different cultivation areas during 2009 2010 and analyzed for aflatoxin
residues. According to the results and based on local consumption figures the
estimated daily intake for children was 0.218 -0.222 ng kg-1 b.w day-1 while
for adults 0.106-0.108 ng kg-1 b.w day-1, [77] .
Studies have shown that a widely eaten staple food in West African
countries such as Ghana; Kenkey typically contains large amounts of
aflatoxin-producing Aspergillus even after fermentation [78]. Pica (an eating
disorder among pregnant women which involves the ingestion of non-nutritive
substances such as raw maize, soil, gum, ash and other substances that may be
contaminated by Aspergillus moulds) is another source of increased aflatoxin
intake [79,80].
What is clear up to now is that the great majority of studies for aflatoxins
mainly focus on the contamination levels in certain food commodities, and on
new trends of analytical techniques. When it comes to exposure, and since it is
not clear which food commodity contributes more to exposure, there are many
variables, which may affect the estimation, such as dietary habits. Since
consumers may be exposed to aflatoxins through a normal diet, exposure
studies constitute a crucial parameter for estimating variations in exposure
between different groups (age, geographical distribution, diet habits etc). In
any case, the genotoxic potential of aflatoxins makes any level of exposure to
be a risk factor.
3.2. Risk Characterization
3.2.1. Introduction
Risk assessment is the scientific evaluation of the probability of
occurrence of known or potential adverse health effects resulting from human
exposure to (food-borne) hazards; it is the primary scientific basis for the
establishment of regulations [81]. The availability of toxicological data, as
well as the availability of data on the occurrence of contaminants in various
commodities, are the main factors required in order to perform an assessment
of risks. Usually, the methodology followed in risk assessment implies the
identification of a No-Observed-Adverse-Effect-Level (NOAEL) for the
critical effect from toxicological studies and the application of relevant
uncertainty (or assessment) factors for the derivation of limits of exposure
(e.g. tolerable intake levels). These limits of exposure are subsequently
compared with the outcome of the exposure assessment for the final step of the
risk assessment procedure, namely the risk characterization, in order to
conclude on the existence (or non-existence) of risk.
In the case of aflatoxins, however, this approach cannot be followed due

to the fact that the critical effect is carcinogenicity. No-effect concentration
limits cannot be established for genotoxic carcinogens; any small dose will
have a proportionally small probability of inducing an effect. Exposure to this
type of compounds should, in theory, be completely prevented; nevertheless,
exposure of the population to some level of aflatoxins has to be tolerated, as
these are naturally occurring food contaminants. It is, therefore, recommended
that the level of the contaminant in food should be reduced so as to be As Low
As Reasonably Achievable (ALARA). The ALARA level, which reflects the
irreducible level for a contaminant, is defined as the concentration of a
substance that cannot be eliminated from a food without involving the discard
of that food altogether or without severely compromising the availability of
major food supplies [81].
In the European Union, the opinion of the Scientific Committee (SC) of
the European Food Safety Authority (EFSA) addresses approaches beyond the
ALARA principle, allowing a level of potency assessment of specific
substances which are present in food and which are both genotoxic and
carcinogenic. Such an approach does not substitute for minimizing exposure to
all such substances. It ensures that, where resources are limited, the highest
priority is given first to those substances which present the greatest risk for
humans. The SC of EFSA recommends the application of the margin of
exposure (MOE) approach as a harmonized methodology for assessing the risk
of genotoxic and carcinogenic substances which may be found in food,
irrespective of their origin. The margin of exposure is defined as the reference
point on the dose-response curve (usually based on animal experiments in the
absence of human data) divided by the estimated intake by humans [82].
Evaluation of risks to public health arising from dietary exposure to
aflatoxins has been performed by various expert groups throughout the years.
In this section, the principal risk assessment approaches related to aflatoxin
exposure are presented.
3.2.2. The Joint FAO/WHO Expert Committee on Food Additives

(JECFA) Approach
A quantitative risk assessment was performed in 1997 by the Joint
Committee on Food Additives (JECFA) - a scientific advisory body of the
World Health Organization (WHO) and the Food and Agriculture
Organization (FAO), focusing on the increase in primary liver cancer [13].
The Committee reviewed the experimental evidence concerning the
carcinogenicity of the aflatoxins, evaluated their potencies, linked these
potencies to intake estimates, and discussed the impact of hypothetical

standards on sample populations and their overall risks. The conclusions of the
Committee regarding aflatoxin potency were contingent upon the dynamics of
hepatitis B infection (HBV) in a human population, since the said potency
appears to be significantly enhanced in carriers of hepatitis B virus - as
determined by the presence in serum of the hepatitis B surface antigen
(presence denoted HBsAg+ and absence denoted HbsAg-). Overall, based on
the weight of scientific evidence (epidemiological data, laboratory animal
studies, in vivo and in vitro metabolism studies) the Committee considered that
aflatoxins should be treated as carcinogenic food contaminants, the intake of
which should be reduced to levels as low as reasonably achievable.
Regarding aflatoxin B1 (AFB1) potencies, the Committee reviewed the
potency estimates from the epidemiological studies which showed a positive
association between aflatoxins and liver cancer and selected separate potency
estimates and ranges for HBsAg+ and for HBsAg- individuals. Potency values
of 0.3 cancers/year per 100 000 population per ng aflatoxin/kg of body weight
per day (uncertainty range: 0.05-0.5) in HBsAg+ individuals and of 0.01
cancers/year per 100 000 population per ng aflatoxin/kg of body weight per
day (uncertainty range: 0.002-0.03) in HBsAg- individuals were chosen.
Linking the potency estimates (risk per unit dose) to the estimates of
aflatoxin intake (dose per person), the fraction of the incidence of liver cancer
in a population attributable to aflatoxin intake could be derived (population
risk), based upon the prevalence of hepatitis B infection in various regions.
Relative estimates of mean dietary intake of aflatoxins for various regions
were provided using regional diets from the Global Environment Monitoring
System - Food Contamination Monitoring and Assessment Programme
(GEMS/Food) combined with data on levels of aflatoxin contamination.
Comparing two hypothetical standards (10 g/kg and 20 g/kg aflatoxin
B1 in food) and considering the limitations and assumptions inherent in this
approach, the Committee concluded that populations with a low prevalence of
HBsAg+ individuals and/or with a low mean intake (less than 1 ng/kg of body
weight per day) are unlikely to exhibit detectable differences in population
risks for standards in the range of the hypothetical cases; populations with a
high prevalence of HBsAg+ individuals and high mean intake of aflatoxins
would benefit from reductions in aflatoxin intake. Specifically, reducing the
hypothetical standard from 20 g/kg to 10 g/kg, would yield a drop in the
estimated population risk of approximately 2 cancers/year per 109 people in
case of a low prevalence of hepatitis B, and of 300 cancers/year per 109 people
in case of a high prevalence of hepatitis B.
Overall, it was concluded that when two alternative standards for aflatoxin
contamination in food are considered, the higher standard will yield essentially
the same risk as the lower standard if the fractions of samples excluded under
the two standards are similar. When a substantial fraction of the current food
supply is heavily contaminated with aflatoxins, reducing the levels of
contamination may result in detectable reductions in rates of liver cancer.
Conversely, when only a small fraction of the current food supply is heavily
contaminated, reducing the standard by an apparently substantial amount may
have little appreciable effect on health. FAO and WHO encourage
governments and the Codex Alimentarius Commission (CAC) to make use of
the aforementioned evaluation in deciding on the appropriate standards to
apply to aflatoxins. However, this requires a significant amount of information
at national level including monitoring data and information on dietary patterns
and the prevalence of hepatitis B in the population.
3.2.3. The European Food Safety Authority (EFSA) Approach

In 2007, the Scientific Panel on Contaminants in the Food chain
(CONTAM) of the European Food Safety Authority (EFSA) - an independent
body of the European Commission - was asked to provide a scientific opinion
on the potential increase of consumer health risk associated with a proposed
change of the EU maximum level of 4 g/kg for total aflatoxins (sum of
aflatoxins B1, B2, G1 and G2) in ready-to-eat almonds, hazelnuts, pistachios
and derived products to 8 or 10 g/kg, taking into account exposure to all
aflatoxins from other food sources, including aflatoxin M1 [83]. For this
evaluation, the following were considered: (i) occurrence data provided and
uncertainties related to the heterogeneous distribution of aflatoxins; (ii)
specific consumption patterns of the relevant food commodities in the different
Member States; (iii) specific (vulnerable) population groups, including
children, hepatitis carriers and high level consumers; (iv) relative proportion of
AFB1 to total aflatoxins. Additionally, EFSA was requested to estimate the
margin of exposure (MOE) for the presence of aflatoxins (total
aflatoxins/AFB1) in food, considering vulnerable population groups.
For the current opinion, occurrence data on aflatoxins in a range of food
products were mainly collected from random and targeted monitoring and
surveillance activities in Member States over a seven year period. For those
samples where aflatoxins were detectable, AFB1 was generally the major
contributor to total aflatoxins. Data relating to concentrations of aflatoxin M1
(the major metabolite of AFB1) in commercial milk samples were generally
below 0.05 g/kg and taking into account the lower carcinogenic potency of
M1, these were not further considered.
For the assessment of the impact of a possible change in the maximum
levels for almonds, hazelnuts and pistachios, the CONTAM Panel estimated
dietary exposure excluding occurrence data above 4, 8 and 10 g/kg,
respectively. The estimates indicated that increasing the maximum levels for
total aflatoxins in almonds, hazelnuts and pistachios from 4 to 8 or 10 g/kg
would result in an increase in the average total dietary exposure to aflatoxins
in the region of 1 %. In the case of consumers with the highest level of
consumption, the estimates indicated that increasing the maximum levels for
total aflatoxins from 4 to 8 or 10 g/kg could increase total dietary exposure to
aflatoxins by up to 20 %. If, as is expected, nuts exceeding the maximum
levels are occasionally consumed, the total long term average dietary
exposures might be higher, but the relative impact of raising the maximum
level from 4 to 8 or 10 g/kg in the three nuts would be less.
Considering liver carcinogenicity as the pivotal effect for the risk
assessment, the Panel considered dose-response modelling of experimental
data from both animal studies and epidemiological studies for the MOE
calculation, based on the risk assessment approach for genotoxic carcinogens
[82]. The available database for dose-response modelling was only sufficient
for AFB1.
Taking into account that AFB1 constituted a major proportion of total
aflatoxins in the samples analysed, for the purposes of this evaluation, the
Panel made the precautionary assumption that the potency of total aflatoxins is
equivalent to that of AFB1.
Overall, based on the information available in 2007, the CONTAM Panel
concluded that public health would not be adversely affected by changing the
maximum levels for total aflatoxins from 4 to 8 or 10 g/kg in almonds,
hazelnuts and pistachios; minor effects would be expected on the estimates of
dietary exposure, cancer risk and the calculated MOEs.
The CONTAM Panel, however, reiterated its previous conclusion that
exposure to aflatoxins from all sources should be as low as reasonably
achievable, because aflatoxins are genotoxic and carcinogenic, and that
priority should be given to reducing the numbers of highly contaminated foods
reaching the market, irrespective of the commodity involved.
4. LEGISLATION
4.1. Introduction
Aflatoxins are subject to regulations at national, regional and international

level. The principle objective behind this action is to protect public health,
consumers and workers as well as to ensure safety on food supply (farm to
fork approach). A well-structured legal framework should guarantee safe
products (foodstuffs and feedstuffs) intended for human and animal
consumption. Thus, an effective legal framework should typically include:
Regulatory limits;
Monitoring to ensure compliance with limits;
Guidance to the food industry;
Cooperation between agencies on food safety;
Enforcement action.
Limits for contaminants are set with the purpose to reduce contamination
in food and feed. The case of aflatoxins is particular due to the fact that these
toxins are genotoxic carcinogens. Actions on their exclusion from food and
feed would have to be imposed; however, aflatoxins are natural occurring
contaminants and exposure cannot be completely prevented. In this context a
range of regulatory limits are established by national, regional and
international authorities depending mainly on scientific and socioeconomic
grounds.
Since 1998 harmonized limits exist for aflatoxins in various foodstuffs and
feedstuffs in the EU; EU regulations regarding aflatoxins are of the most
extensive and detailed compared to other regions of the world. This is also true
for most of the developed market economies that have more stringent
standards than developing countries. The U.S. Food and Drug Administration
issued for the first time Action Levels for aflatoxins in 1969. Based on an
assessment of FAO which was carried out by the National Institute for Public
Health and the Environment (The Netherlands) 39 countries in Europe,
accounting for approximately 99% of the continents population, had specific
mycotoxin regulations in 2003 [81, 84].
Improvements in food regulations have taken place constantly and reflect,
in a great extent, the increase of exports and imports of food and feed
products. This increase is a result of the growing globalization of trade.
However, the ease of moving products raises the risk of the spread of food
hazards and the need for the countries to ensure confidence in the safety of
their products.
Due to the importance of food trade and safety, EU and U.S. have
established agreements and partnerships with international organizations such
as WTO (World Trade Organization), FAO (Food and Agriculture
Organization) and WHO (World Health Organization). The EU is committed
to multilateralism and has acknowledged the fundamental importance of WTO
in the international trade system. WTO works towards multilateral rule-
making, trade liberalisation and sustainable development. FAO and WHO
approved in 1963 the establishment of the Joint FAO/WHO Food Standards
Programme with the Codex Alimentarius Commission as its principal organ,
which develops harmonised international food standards, guidelines and codes
of practice to protect the health of the consumers and ensure fair practices in
the food trade. The Commission represents the EU in the Codex Alimentarius
Commission and its bodies, and as a member of the WTO should apply the
Codex Alimentarius standards and meet its obligations under the World Trade
Organisation Agreement on Sanitary and Phyto-sanitary Measures (SPS
Agreement). Under this agreement the EU and other members have the right to
apply stricter standards than the CODEX standards, as long as those are based
on science (e.g. risk assessment) [85].
The setting of aflatoxin regulations is a complex activity that involves
many factors and interested parties. Recently, EUs more stringent limits have
been debated and the European Commission requested the European Food
Safety Authority (EFSA) the EU risk assessment body for food and feed
safety - to give its opinion on possible adverse effects on humans after an
increase of limits for certain kind of nuts. The EFSAs conclusion permitted
the increase and limits were set higher. However, differences between
regulatory limits and in general regulations of developed and developing
countries still exist reflecting how socioeconomic criteria affect legislation.
4.2. EU - Legal Framework
Historically the European Union guided its food policy towards advanced
food safety as an integral part and contributor to food security. In the first
decades after the 2nd World War, EU dealt with food security issues, animal
diseases and new consumer habits. As a result, new Regulations and
Directives were born to tackle animal diseases; CAP Common Agriculture
Policy improved food security; RASFF Rapid Alert System for Food and
Feed was established as a back-up measure to health risks from food and feed;
and the European Foundation for the Improvement of Living and Working
conditions dealt, amongst other issues, with food and nutrition. Although EU
legislation covered numerous aspects in food and feed safety, there was not in
place an integrated legal framework which could effectively address and cover
all sectors of the food chain as a whole, from primary production to retail sale,
as well as a unique authority which could provide with rigorous scientific
advice the European Commission, and clear communication for the risks[86].
The aforementioned gap was first addressed in the Commissions Green
Paper [87] on Food Law (1997) which was followed by the White Paper on
Food Safety [88], one of the most important milestones in the history of food
policy in the EU, issued in 2000. The main scope of the White Paper was to
suggest and set out actions which will eventually lead to a new integrated
policy on food safety in the EU. The actions were based on the following
strategic priorities of the White Paper:
High level of consumer health protection;

Establishment of an independent European Food Authority;
Consistent implementation of a farm to table approach in food
legislation;
Clear attribution of primary responsibility of safe food production to
industry, producers and suppliers;
Application of Risk analysis (Risk Assessment, Risk Management -
precautionary principle where appropriate - and Risk Communication)
in legislation. The Precautionary principle may apply when adverse
health effects have been identified but scientific uncertainty persists;
Traceability of products through the whole food chain;
Greater transparency at all levels of Food Safety policy;
Official Controls at both national and EU level (all parts of the food
production chain must be subject to official controls);
Ability to take rapid, effective safeguard measures in response to
health emergencies throughout the food chain;
Well informed consumers and re-establishment of confidence.
As a result, in 2002, the General Food Law [89] was drawn up by the
European Commission laying down the general principles and requirements of
Food Law, establishing procedures in the context of food safety, and setting up
the European Food Safety Authority. It could be described as a solid base upon
which further important food safety rules can be provided.
On this basis, and taking into account of general principles and concepts
(provided in the General Food Law) such as the high level of human life and
health; the free movement of safe food and feed, and the measures which
guarantee that unsafe food is not placed on the market, a legal framework on
contaminants in food and feed has been put into effect, in line with the existing
Council Regulation (EEC) No 315/93[90] on Community procedures for
contaminants in food. The latter regulation lies down, among other, that food
containing a contaminant in an amount which is unacceptable from the public
health viewpoint, and in particular at a toxicological level, shall not be placed
on the market. In addition, contaminant levels shall be kept as low as
reasonably achievable by following good practices at all stages (production,
processing, treatment, storage, etc). Subsequent amendments (EC No
1882/2003, EC No 596/2009) enable the Commission, where necessary, to
establish maximum tolerances for specific contaminants. Prior to provisions
adoption which may have an effect upon public health, consultation of the
EFSAs Scientific Committee on Food is obligatory.
Maximum Levels
Due to the fact that aflatoxins occur naturally in food it is not possible to
impose total ban of them. Therefore, it was necessary to take restriction
actions on their maximum concentration, minimizing any risk to human health.
Replacing the former EC No 466/2001 regulation [91], the Commission
Regulation EC No 1881/2006 - setting maximum levels for certain
contaminants in foodstuffs [92] - entered into force.
This was subsequently amended by the EC No 165/2010 regulation [93]
increasing certain levels for certain foodstuffs respecting at the same time
EUs agreement with WTO. The latest amendment had previous been justified
according to EFSAs opinion on the potential increase of consumer health risk
by a possible increase of the existing maximum levels for aflatoxins in
almonds, hazelnuts and pistachios and derived products in 2007, and by its
statement related to the effects on public health of an increase of the levels for
aflatoxin total for tree nuts other than almonds, hazelnuts and pistachios in
2009. Previous and new levels of concentration for aflatoxin in certain
foodstuffs are reported in Table 1.
Table 1. Maximum levels for aflatoxins (B1 and total) in foodstuffs before
and after the amendment of EC No 1881/2006[95] regulation
EC No EC No EC No EC No
1881/ 165/ 1881/ 165/
2006 2010 2006 2010
Total* Total*
B1 (g/ B1 (g/
(g/ (g/
kg) kg)
kg) kg)
Treenuts Almonds, Ready to eat 2 8 4 10
Pistachios, For further 5 12 10 15
Apricot processing
kernels
Hazelnuts, Ready to eat 2 5 4 10
Brazil nuts For further 5 8 10 15
processing
Other tree Ready to eat 2 2 4 4
nuts For further 5 5 10 10
processing
Oilseeds Peanuts Ready to eat 2 2 4 4
(not for For further 8 8 15 15
crushing) processing
Other Ready to eat - 2 - 4
oilseeds For further - 8 - 15
processing
Cereals Corn Ready to eat 2 2 4 4
For further 5 5 10 10
processing
Rice Ready to eat 2 2 4 4
For further - 5 - 10
processing
Other Ready to eat 2 2 4 4
cereals For further - - - -
processing
* Total aflatoxins B1, B2, G1, G2
Limits have been developed considering that aflatoxins are genotoxic

carcinogens, and are set at a level which is as low as reasonably achievable
(ALARA). Limits exist for ready to eat and for further processing
foodstuffs, and apply to aflatoxin B1, which is the most potent for human
health, total aflatoxins (B1, B2, G1, G2) and aflatoxin M1.
Vulnerable groups, such as babies, infants and young children, are
protected under more stringent limits for certain food products. In other cases,
however, foodstuffs may be placed on the market when concentration levels of

aflatoxins exceed limits as soon as they are not intended for human
consumption, they do not exceed the maximum limits for these products which
are to be sorted before human consumption, and labelled clearly showing their
use.
Similar to food, feedstuffs for animals are subject to tolerance levels for
aflatoxin, as well. Commission Directive 2002/32/EC [94], amending
Commission Directive 1999/29/EC and all subsequent amendments of it, lays
down rules to ensure agricultural productivity and sustainability and to ensure
public and animal health, animal welfare and the environment.
Official Control
Maximum levels for aflatoxins set by the EU should be subject to official
control in order to ensure and enable effective enforcement. To this end,
competent authorities throughout Community should perform the same
sampling criteria and achieve the same analysis performance in accordance
with Commission Regulation EC No 401/2006 [96] amended by EC No
178/2010 regulation [97].
Official controls are necessary to ensure safe imported and exported
products. EU is a major importer and exporter of foodstuffs worldwide. To
protect public health and to ensure, amongst other, safe products at export and
import, EC has put into effect regulations and decisions which safeguard
contaminated products to aflatoxins. Commission Regulation (EC) No
1152/2009 [98] imposes special conditions governing the import of certain
foodstuffs from certain third countries due to contamination risk by aflatoxins
[99]. A Common Entry Document is used in order for the authorities to be
informed prior to the shipment of the consignment. Commission Regulation
(EC) No 669/2009 implementing regulation EC No 882/2004 dealing with the
increased level of official controls on imports of certain feed and food of non-
animal origin (also referred to as high risk legislation) provides for the use of
Document.
In other cases, in order to further facilitate decrease of controls at import
of products into the EC, pre-export controls on aflatoxin can be applied as
described under article 23 of Commission Regulation EC No 882/2004 [100].
After inspection of the Food and Veterinary Office on USAs control system
and related laboratories for aflatoxin levels in peanuts, approval of pre-export
was granted by Commission Decision to USA in 2007.
Monitoring and Reporting

Monitoring and reporting of aflatoxin levels after controls are essential
actions which assist to identify potential risks to human health. For that reason,
EC has put into effect the Rapid Alert System for Food and Feed. In addition,
an EU Reference Laboratory [101] has been established for mycotoxins, and
guidance on different aspects regarding contaminants in food and feed has
been issued assisting related authorities, namely the Guidance document for
competent authorities for the control of compliance with EU legislation on
aflatoxins [102].
EU legislation on aflatoxins in food and feed is regularly amended and
updated in light of new scientific evidence, EFSAs opinions and statements,
and in line with the international Codex Alimentarius. This is essential in order
to safeguard public health, place in the market safe and wholesome products
and safeguard the important role of trade.
4.3. USA - Legal Framework
In the early 1900s, USA enacted the Wiley act (named after Harvey W.
Wiley) with the official title Pure Food and Drug Act. Prior to the Act, a
number of products had been released in the market, and deemed legal without
the need of pre-market approval. In many cases such products provoked
adverse health effects to consumers. On the basis of the Wiley Act, interstate
commerce of adulterated and misbranded food and drugs was prohibited [103].
Certain deficiencies of the obsolete Act and a major outbreak due to an unsafe
drug gave pace to a substantial amendment. After about 30 years, in 1938, a
new act was drawn up mandating, among other, food standards, and improved
food packaging policy [104]. The Federal Food, Drug and Cosmetic Act
FD&C Act [103] increased substantially the level of government intervention
in the food, drug and agricultural markets and established the legal framework
within which the Food and Drug Administration - FDA operates. In this
context, significant power was given to FDA (former Bureau of Chemistry), as
part (since 1968) of the Public Health Service [105]. The federal agency is
charged with the FD&C Act enforcement ensuring that food is safe, pure,
wholesome and appropriately labelled. Although FDA is responsible for the
safety of quite all food, there are other federal agencies (about 15)
administering food safety related laws, and of the most importance is Food
Safety and Inspection Service FSIS (meat inspections) which is part of the
Department of Agriculture [106]. Together with the Centers for Disease
Control and Prevention CDC [107], which is the link between foodborne
illness and food safety systems (governmental and food producers), these three
agencies cooperate closely to enforce the US food safety Law.
The case of aflatoxins appears in the FD&C Act under section 402(a)(1),
where food and feed containing naturally occurring contaminants are
considered to be adulterated if they are deemed by FDA to be injurious to
human or animal health. In order for the FDA to evaluate whether adulteration
of food or feed (domestic or imported) has taken place, regulations and
guidance are developed. FDA regulations have a federal character and are
based on the laws set forth in the FD&C Act. FDA guidance is not legally
binding to the public or FDA and defines how the agency deals with a
regulatory issue. In addition to the above, FDA provides a convenient and
organized system for statements Compliance Policy Guides Manual- of its
compliance policy including those statements which contain regulatory action
guidance information [108].
Maximum Levels
By the 1960s, aflatoxins as food contaminants were identified and at that
time about half of the food supply was subject to standards in the U.S. [109].
With the scope to reduce natural occurred contamination in foodstuffs and
feedstuff, FDA issues policy guidance (or enforcement pronouncements) in
one of the following three forms:
Advisory Levels - Provide guidance to the industry concerning levels

of a substance present in food or feed but enforcement is not the
fundamental purpose of an advisory level.
Action Levels - Specify a precise level of contamination at which the
agency is prepared to take regulatory action. Action levels alert the
industry that FDA believes it has the scientific data to support
regulatory and/or court action if a toxin or contaminant is present at
levels exceeding the action level if the agency chooses to do so.
Regulatory Limits Are established after issuing valid regulations
under the public notice and comment rulemaking procedures.
For aflatoxins, the FDA has established Action Levels according to the
intended use of food (CPG sec. 555.400, 570.200, 570.500 and 570.375) and
feed (CPG sec. 683.100) [103]. Table 2 depicts such levels for a number of
products.
The Center for Food Safety and Applied Nutrition - CFSAN is the branch
of the FDA that is responsible for establishing standards of tolerance levels.
Table 2. FDA Action Levels for aflatoxins in human food,

animal feed and animal feed ingredients
Aflatoxins
Products Intended Use Level Notes
(ppb)
Milk Direct 0.5 Identity of aflatoxin
consumption M1 is confirmed by
the chemical
derivative test
Foods, peanuts and Direct 20 Not raw peanut
peanut products, consumption products*. Identity
brazil nuts, and of aflatoxin B1 is
pistachio nuts confirmed by
chemical derivative
Corn and peanut Finishing beef 300 I.e. feedlot
products cattle
Cottonseed meal Beef cattle, 300 Regardless of age or
swine, or poultry breeding status
Corn and peanut Finishing swine 200
products 100 pounds
Corn and peanut Breeding beef 100
products cattle, breeding
swine, or mature
poultry
Corn, peanut Immature 20 Excluding
products, and other animals cottonseed meal
animal feeds and intended for
feed ingredients immature animals
Peanut products, Dairy animals, 20
cottonseed meal, and animal not listed
other animal feeds above, or when
and feed ingredients the intended use
is unknown
* The USDA has a comprehensive program involving raw peanuts which can be
expected to result in proper processing or destruction of any high aflatoxin raw
peanuts.
Official Control and Inspections

FDA has issued the below compliance programs for mycotoxins,
including aflatoxins, which are updated and reissued periodically due to
changes in methodology, and number of products to be collected.
Mycotoxins in Domestic Foods (CFSAN)

Mycotoxins in Imported Foods (CFSAN)
Feed Contaminants Program (CVM)
The programs objectives are to control, monitor and report, among other
contaminants, aflatoxins. More specifically they aim to collect and analyse
domestic and import samples of various food products and to determine the
occurrence and levels of aflatoxins.
In addition to the above programs, prevention (pre-harvest post-harvest)
and decontamination strategies exist for grains [110].
Recent Developments in U.S. Food Safety Policy

The Federal Food, Drug and Cosmetic Act concerning Food Safety (food
contaminants) was subject to numerous amendments through the years. In
2011, the US congress enacted the FDA Food and Drug Modernization Act
FSMA [111], the most extensive amendment of the Food Safety Laws in the
last 7 decades. The amendment was deemed necessary due to incidents of
foodborne illness outbreaks and the need of a higher level of Food Safety in
general (e.g. protection against bioterrorism). In contrary to previous food
safety policy which was structured to respond in contamination of food, the
FSMA makes use of a systematic approach to food safety management and
aims to prevent contamination of food and feed.
The basic Law enhancements of SFMA are listed below:
Preventive controls: Food facilities are required to implement a

written preventive control plan; FDAs produce safety standards;
FDA regulations to prevent intentional contamination of food and
feed.
Inspection and Compliance: the FDA ensures compliance of
producers and processors to the preventive control standards by
establishing a mandated inspection frequency for food facilities based
on risk; accessing records of food safety plans; and assures that testing
laboratories are accredited meeting high-quality standards.
Imported Food Safety: the FDA ensures that imported products meet
U.S. standards by: (i) regulating importers responsibility to verify
that their foreign suppliers have adequate preventive controls in place;
(ii) qualifying third parties to certify foreign facilities which comply
with U.S. food standards; (iii) requiring third party certification of
compliance (or other assurance) of high-risk imported foods; (iv)
establishing a voluntary qualified importer program; (v) and having
the authority to deny entry of food from a foreign facility which
refuses access.
Response: the FDA uses tools in order to respond effectively to food
contamination when preventive controls fail to. Mandatory recall of
unsafe food, expanded administrative detention of suspect food,
suspension of registration of a facility, enhanced track and trace
system for both domestic and imported foods and feeds and additional
recordkeeping for high risk foods are such tools.
Enhanced Partnerships: the FDA initiates a formal system of
collaboration with other domestic and foreign government agencies in
order to achieve public health goals.
The above enhancements, among other objectives, aim to prevent

contamination of food and feed from aflatoxins, and improve food quality of
domestic and imported products. Depending also on the considerable funding
needed, the implementation of the new Act will take time until rule making
process, driven by the FDA, issues final rules and regulations with
transparency after public consultation [112].
4.4. Other Countries
Other countries such as Turkey, Bosnia and Herzegovina and Switzerland

seem to be influenced by the EU Regulation and tend to have comprehensive
legislation controlling the level of aflatoxins. As for international markets,
China followed by Brazil and Mexico have the most comprehensive legislation
on aflatoxins. Specific maximum limits are set for aflatoxin B1 and/or total
aflatoxin in several foodstuffs in these countries. Other countries such as
Canada, Australia and New Zealand, Gulf Cooperation Council (GCC) and
Nigeria lay down specific limits for total aflatoxins mainly in nuts as indicated
in Table 3 [113].
Table 3. Total aflatoxins limits in Australia/New Zealand, Canada,

Codex, GCC, Nigeria, India, USA and South Africa
Total
Country Foodstuffs aflatoxins
(g/kg)
Australia/ Peanuts 15
New Zealand Tree nuts
Canada Nut and nut products 15
Codex Peanuts, almonds, shelled Brazil nuts, hazelnuts 15
GCC(a) pistachios intended for further processing
Nigeria Almonds, hazelnuts, pistachios, shelled Brazil nuts, 10
ready-to-eat
India Wheat, maize, jawar (sorghum) and bajra, rice, whole 30
and split pulse (dal) masur (lentil), whole and split
pulse urd (mung bean), whole and split pulse moong
(green gram), whole and split pulse chana (gram),
split pulse arhar (red gram), and other food grains
Groundnut kernels (shelled) (peanuts); 30
USA Brazil nuts, peanuts and peanut products, pistachio 20
products
South Africa Peanuts 15
(a) Members of GCC are Saudi Arabia, United Arab Emirates (UAE), Kuwait,
Bahrain, Oman, Yemen and Qatar.
Countries such as India establish general maximum levels for total

aflatoxins for foods: 30 g/kg and 20 g/kg, respectively. In South Africa, a
general maximum limit for total aflatoxins is set at 10 g/kg and additionally a
general maximum limit for aflatoxin B1 is set at 5 g/kg for all foodstuffs.
Recently, Japan has established strict tolerance levels also for total
aflatoxins in all foodstuffs which must not exceed 10 g/kg.
It is evident that established tolerance levels among countries vary in a
great extent. This reflects on great differences in risk perception of each
country and/or region. Developing countries, mainly located in tropical areas,
encounter greater contamination in commodities from aflatoxins. A major part
of these countries export a significant amount of such commodities to
developed countries which tend to emanate more stringent standards. In that
context, developing countries are forced to balance their policy taking into
account trade interests, food security and food safety issues; a task which is
not trivial and leads to higher tolerance levels. On the contrary, developed
countries, having in place more advanced and efficient agricultural practices
and manufacturing processes are able to achieve lower levels of aflatoxins in

commodities. Thus, they focus more on public health issues and food safety
resulting in stringent limits.
Table 4. Aflatoxin M1 limits
Aflatoxin M1
Country Foodstuffs
(g/kg)
EU Raw milk, heat-treated milk and milk for the 0.050
Bosnia and manufacture of milk-based products
Herzegovina Infant formulae and follow-on formulae, 0.025
Turkey including infant milk and follow-on milk (products ready
to use)
Dietary foods for special medical purposes 0.025
intended specifically for infants (products ready
to use)
China Milk and milk products (for milk powder, 0.5
calculated on a fresh milk basis)
Formulated foods for infants (milk or milk 0.5
protein based) (calculated on
a dry powder
basis)
Formulated foods for older infants and 0.5
young children (milk or milk protein based) (calculated on
a dry powder
basis)
Formulated foods for special medical 0.5
purposes intended for infants (calculated on
a dry powder
basis)
Codex, GCC, Milk 0.5
India, Kenya,
USA
Argentina Milk, liquid including milk used in the 0.5 [1]
manufacture of milk and milk products and
reconstituted milk
Milk, powder 5.0
Milk formula ND
Mexico Pasteurised, ultrapasteurised, sterilised and 0.5 [1]
dehydrated milk, milk formula and combined
milk products
South Africa Milk 0.05
ND: Not Detectable, [1] Given in g/l.
Significant effort dealing with above aspects is made in an international

level. In order to achieve harmonization and avoid misuse of the
precautionary approach Codex Alimentarious sets standards to be respected
from countries under WTO international agreement. However, countries may
establish different limits upon scientifically based evidence maintaining
current differences of standards among countries.
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Chapter 9
FOOD SOURCES AND OCCURRENCE

OF AFLATOXINS:
THE EXPERIENCE IN GREECE
Ioannis N. Tsakiris,1 Elisavet Maria Renieri,2

Maria Vlachou,2 Eleftheria Theodoropoulou,2
Marina Goumenou2 and Aristides M. Tsatsakis,2
1
TEI of Western Macedonia, Florina Branch,
Department of Agricultural Products Marketing
and Quality Control, Terma Kontopoulou 53100 Florina, Greece
2
Laboratory of Toxicology, Department of Medicine, University of Crete,
Voutes 71003, Heraklion, Greece
ABSTRACT
This paper presents a review of the occurrence of aflatoxins in
different food commodities in Greece, based both on results represented
in literature as well as results derived from monitoring programs of the
Center of Toxicology Science & Research, Medical School, University of
Crete. Aflatoxins, can pose a severe threat to food safety, since they are
characterized carcinogenic to humans, IARC Group 1. They may be
formed or developed in any stage of the agricultural production (primary
Author for correspondence: Ioannis N. Tsakiris. Email: tsakiris@teikoz.gr, Mob: 0030

6977270004.
234 Ioannis N. Tsakiris, Elisavet Maria Renieri, Maria Vlachou et al.
production, processing and storage) as a result of transitional weather

conditions or of poor storage. Studies, monitoring programs and surveys,
which have been carried out in Greece, are mainly focused in milk and
dairy products. In this context, several studies have been conducted in
animal feeds as well, since there is notable evidence that they are
potential sources of aflatoxins in milk production. Additionally, both
black and green olives have been examined for possible contamination by
aflatoxins, due to the fact that they are damaged during harvest and
processing and thus providing a substrate for aflatoxin development.
Finally, a limited number of studies investigate the presence of aflatoxins
in different processed products like breakfast cereals. The above
foodstuffs have been studied on account of their high nutritional value
and the fact that they are consumed by different population groups.
Results indicate that residue levels of aflatoxins which are presented in
fresh as well as processed agricultural products, do not pose any
considerable risk for the Greek population groups. The most important
factors influencing the levels of aflatoxins in major agricultural products
appear to be the growing and cultivation techniques, as well as the food
safety parameters during harvesting, storage and processing. An
additional issue, which seems to raise concern internationally, is the fact
that climate change in combination with modifications in the cultivation
techniques may affect the frequency and severity of aflatoxin residues in
agricultural products.
LIST OF ABBREVIATIONS
AFB1 Aflatoxin B1
AFM1 Aflatoxin M1
ELISA Enzyme linked immunosorbent assays
GC Gas chromatography
GAP Good Agricultural Practice
HACCP Hazard Analysis and Critical Control Point
HPLC High performance liquid chromatography
IARC International Agency for Research on Cancer
LC-MS/MS liquid chromatographytandem mass spectrometry
NIRS Near infrared reflectance spectroscopy
TLC Thin-layer chromatography
Food Sources and Occurrence of Aflatoxins 235
1. INTRODUCTION
Aflatoxins are a group of toxic secondary metabolites produced by certain
strains of the fungi Aspergillus flavus, Aspergillus parasiticus and the rare
Aspergillus nomius (Rahimi et al., 2010, Cano-Sancho et al., 2010), which
contaminate agricultural products both primary and processed. Aflatoxins may
subsequently enter the food chain creating serious risks to animal and human
health. The production of these compounds is highly influenced by several
environmental factors such as soil composition, insect infestation, temperature
and water availability (aw) both pre- and post- harvest (Paterson and Lima,
2010). Aflatoxins may be present in a wide range of food commodities,
particularly cereals, oilseeds, spices and tree nuts (Pitt et al., 2013). Maize,
peanuts, pistachios, brazils, black pepper, dried fruit and figs are all known to
be high-risk foods for aflatoxin contamination, but the toxin has also been
detected in many other commodities. Milk, cheese and other dairy products are
also known to be at risk of contamination by aflatoxins (Tsakiris et al., 2013a;
Tsakiris et al., 2013b ). The highest levels are usually found in commodities
from warmer regions of the world where there is a great deal of climatic
variation.
Aflatoxins are the most studied mycotoxins and mainly include aflatoxin
B1, B2, G1 and G2 with aflatoxin B1 being the most toxic of these
metabolites. A.flavus produces exclusively B aflatoxins, while A. parasiticus
and A. nomius produce B and G types (Fallah, 2010; Sidhu et al., 2009).
Aflatoxins are extremely toxic and present teratogenic, mutagenic and
carcinogenic effects that have been demonstrated in several studies (Green et
al., 1982; Wangikar et al., 2005; Mckean et al., 2006; El-Sayed and Khalil,
2009). Aflatoxin B1 (AFB1) has been classified as a Group 1 human
carcinogen by the International Agency for Research on Cancer (IARC)
(IARC, 1993). The main monohydroxylated derivative of AFB1 is aflatoxin
M1 (AFM1) which is also of great interest since it is formed in liver and
excreted into milk. Even though AFM1 is less toxic than AFB1, it has been
classified as a Group 2B possible human carcinogen by the IARC. Figure 1
presents the chemical structure of AFB1 and AFM1.
In Greece, most studies and monitoring programs which deal with the
occurrence, risk assessment and control of aflatoxins in foodstuff are mainly
focused in milk and dairy products (Roussi et al., 2013; Malissiova et al.,
2013; Tsakiris et al., 2013a ). In this context, interest has also been laid on
animal feeds and the possible hazards they represent for the food chain
(Vlachou et al., 2004).
Figure 1. Chemical Structure of Aflatoxin B1(a) and M1(b).
Occurrence of aflatoxins has also been investigated for black and green
olives originating from Greece, since its a product of high nutritional value
and a major component of the Mediterranean diet (Ghitakou et al., 2006). A
more limited number of studies have been carried out on crops and processed
products such as breakfast cereals (Villa and Markaki, 2009). Other products,
less essential but considerably frequent components of the Greek diet such as
pistachio nuts and bee pollen, have been under investigation for possible
aflatoxin contamination.
Aspergillus lipases are involved in the mechanisms that follow fungal
invasion as A. flavus primarily destroys the lipid body formation. The fatty
acid contained in the lipid bodies of olive seeds is composed of palmitic, oleic
and linoleic acid which promote the Aspergillus spp. growth. Olives support
aflatoxin production and their biosynthesis is affected by lipid oxidation.
Therefore, it is possible for toxinogenesis to occur which may transfer into
olive oil (Leontopoulos et al., 2003; Ghitakou et al., 2006).
Cereal crops are exposed to fungal invasion both before harvest in the
field and post-harvest during storage and processing. Growing and cultivation
techniques seem to influence aflatoxin levels in cereal crops. Bee pollen which
is increasingly consumed due to its beneficial properties may be a substrate for
aflatogenic fungi and AFB1 production is likely even following a minor
contamination (Pitta and Markaki, 2010). High levels of AFB1 found in
pistachio nuts on the other hand, render it dominant among other types of
aflatoxins also detected in them. Pistachio nuts may be contaminated in every
stage from maturity till storage with maturity being the most critical stage for
aflatoxin contamination (Cheraghali et al., 2007; Georgiadou et al., 2012).
The aforementioned products are of great economic importance and most

of them are consumed on a daily basis by all age groups. It should be noted
that even if these products contain low levels of aflatoxins, they contribute to
the daily intake causing a cumulative effect over a period of years. Excessive
intake of products containing aflatoxins at high levels on a daily basis can
create considerable health problems. Therefore, it is necessary to establish a
critical control point system in order to monitor exposure and minimize health
risks associated with aflatoxin intake.
In Greece, the regulations concerning aflatoxins in food are conformed to
the European Commission regulation (EC, 2006), according to which the
maximum limits of AFB1 and total aflatoxins (sum of AFB1, AFB2, AFG1
and AFG2) were established in specific foodstuffs ranging from 2.0 to 12.0
g/kg for AFB1 and 4.015.0 g/kg for total aflatoxins. Considering that
imported foods are likely to be major sources of aflatoxins as well as
aflatoxigenic moulds, special food control measures have been implemented in
order to minimize risk of aflatoxin occurrence in foodstuffs imported from
other countries (EC, 2009a and EC, 2009b). Products exceeding the maximum
levels should not be placed on the markets of the EU. Directive 2002/32/EC
lays down maximum levels for aflatoxins B1 in feed materials.
The present chapter focuses on reviewing the status of aflatoxins in
Greece and carries out a basic comparison with other Mediterranean countries.
In this context, it is essential to point out the critical factors which raise
concern such as human health impact due to aflatoxin accumulation. The daily
intake of aflatoxins depends on the concentration in food, the amount
consumed and the frequency of the consumption. Risk assessment will provide
a link between hazards in the food chain and the actual risks to human health.
2. CRITICAL POINTS FOR THE DETERMINATION

OF AFLATOXIN IN AGRICULTURAL PRODUCTS
Monitoring for aflatoxins is performed by national certified laboratories

such as the General Chemical State Laboratory, which is reporting to the
Hellenic Food Authority (EFET), by private ones and by university
laboratories for research purposes. In previous years results from National
laboratories, were reported to European Food Safety Authorities as part of the
food safety and quality control program complied with the EU 1881/2006
regulation. Private laboratories perform analysis for aflatoxins in different
substrates mainly for food industries as a prerequisite for certification

purposes.
There are several analytical methods and procedures for the detection of
aflatoxins in agricultural products. Thin-layer chromatography (TLC) was one
of the first methods used to detect aflatoxins in agricultural products
(Fernndez-Ibaez et al., 2009). Usually immunological methods i.e. enzyme
linked immunosorbent assays (ELISA) are used for monitoring purposes while
several methods based on high performance liquid chromatography (HPLC)
and gas chromatography (GC) are being used as well (Muscarella et al., 2007)
The great majority of proposed methods for the detection of aflatoxins suggest
two steps including isolation from the substrate with extraction procedures and
quantification using different analytical methods and techniques i.e. liquid-
liquid extraction, supercritical fluid extraction and solid-phase extraction
(Tripathi and Mishra, 2009). HPLC and ELISA are highly sensitive methods
compared to the rest of the suggested methods and are characterized by low
detection limits, a very important asset in risk assessment studies. The
disadvantage of these methods is the fact they are time consuming, require a
well trained personnel and use non-environmentally friendly solvents.
Moreover, ELISA may present pseudo-positive results and unacceptable
quantification accuracy (Wang et al., 2011). Due to the fact that aflatoxins are
not the only mycotoxins infecting agricultural products, liquid
chromatography coupled to mass spectrometry (LC-MS) or tandem MS (LC-
MS/MS) methods are developed for simultaneous detection of different
mycotoxins in foodstuffs and feeds. While conducting risk assessment the co-
occurrence of several mycotoxins in the same sample is crucial since it is
affecting their mode of action and consequently their adverse health effects
(Ibaez-Vea et al., 2012). During the last years rapid methods for the detection
of aflatoxins on site have been developed. Additionally HPLC and GC require
expensive analytical instruments and this reduces their suitability for screening
purposes. Near infrared reflectance spectroscopy (NIRS) is one of the most
promising methods mainly due to rapid and low-cost characteristics. With the
exception of NIRS all previously mentioned techniques have been used in
studies performed in Greece.
The analyzed agri-food products for aflatoxins residues may be divided
into the following categories: 1) Cereals (primary production and processed
products) and small grains such as wheat, barley and rice 2) Milk and dietary
products i.e. butter, yogurt 3) Nuts and dried fruits 4) Feeds and 5) Other i.e.
olives, olive oil, bee pollen. The most important factors affecting aflatoxin
contamination are biological and environmental while harvesting, storage and
distribution-processing conditions of agricultural products are also crucial

(Paterson and Lima, 2010). Sampling procedures are also of great importance
during aflatoxins analysis. The proposed sampling plans, techniques and
approaches should ensure that the samples are representative which in some
cases is really difficult mostly due to the quantity of initial batches i.e.
containers with cereals.
As the consumption of food is increasing and climate is changing the
detection of aflatoxins in agricultural products is a major challenge and
concern for food safety. Due to globalization, agricultural products with
aflatoxins may travel all over the world. That in combination with the fact that
there is an increasing number of new products in the food market, results in
need for less time consuming methods, but still reliable in order to monitor
food within the tight time limits set by commercial needs (Stroka and Anklam,
2002). Fast methods requiring less specialized personnel for screening
purposes as well as more precise methods might be a part of future food safety
strategy against aflatoxins.
3. AFLATOXINS IN THE GREEK PRODUCTS

3.1. Milk and Dairy Products
As already mentioned milk and dairy products are of great importance for
the agricultural economy and in the same time are characterized by high
nutritional value. Almost 32% of EU milk production is consumed as fresh
milk while cheese, and butter represent the 37% and 16% of milk used
(http://ec.europa.eu /agriculture/publi/fact/milk/2007_en.pdf). Beside cows,
ewes and goats milk, feta cheese and yogurt are the most important dairy
products produced and consumed in Greece. It is well known up to know that
the Greek dairy industry is accounting for 17% of the total food and drink
production in sector (http://gain.fas.usda.gov/Recent%20GAIN%20 Publi
cations/Greece%20Dairy%20Semi-Annual%202012_Rome_Greece_6-8-20
12.pdf). Based on the recent literature, it is concluded that aflatoxin M1
(AFM1) is one the major xenobiotics affecting milk and dairy products
worldwide, in terms of safety and quality (Tsakiris et al., 2013b). Aflatoxin B1
found in animal feeds is metabolized to M1 and secreted in to milk.
Consumption of M1 contaminated milk and dairy products is of great concern
for consumers mainly because of the severe impact on human health and
especially for sensitive human groups such as neonates and children (Oveisi et
al., 2006). Greece and other Mediterranean countries are characterized by

climate conditions, which potentially favor the occurrence of AFM1 in milk
and milk products indicating a high-risk geographical area. Despite the fact of
the importance of milk and dairy products in the local economy and diet the
number of surveys in literature are limited.
Feta is one the most well known Greek cheese, daily consumed as one the
major diet components. During 1985 Penicillium and Aspergilus seem to be
the most common fungus species found on teleme cheese (similar to feta)
while no residues of aflatoxins were detected in 94 commercial teleme cheese
samples analyzed (Zefiridis, 1985). AFM1 was also not detected in a similar
study performed during 1989 in feta and teleme cheese samples
(Karaioannoglou et al., 1989). Another monitoring study was performed
during 2002, from March to June (Kaniou-Grigoriadou et al., 2005). More
specific, a total of 162 samples of ewes milk, curd and feta cheese were
monitored for residues of AFM1. All milk samples presented residues lower
than 50 ng l-1, while in curd, which is an intermediate product and is not
consumable, the levels ranged from non-detectable up to 84.1 ng kg-1. The
concentration ratio of AFM1 from initial milk samples to corresponding curd
present values from 4.3 up to 5.6. In the final products, produced after two
months of ripening, no residues of AFM1 were detected. The detection of
AFM1 in selected samples was based on ELISA.
The surveys referring to the occurrence of AFM1 in yoghurt are even
more limited compared to milk and feta. To the best of our knowledge the
occurrence of AFM1 during the production process of yoghourt alone, has
been evaluated in Greece, using a liquid chromatographic system with a
spectrofluorometric detector (Govaris et al., 2002). In general, levels of AFM1
seem to be decreased during yoghurt production and only Streptococcus
thermophiles (from lactic acid bacteria) presented a lower increase in yoghurt
with high AFM1 concentration. Additionally pH 4.0 seems to have a negative
effect on the stability of AFM1 especially after fermentation compared to pH
4.6 in which AFM1 seems to be more stable.
Karaioannoglou in 1989 presented the first survey of AFM1 on raw and
pasteurized milk. The levels of AFM1 in raw milk varied from 100 up to 130
ng l-1 in four out of 99 samples while no residues were detected in pasteurized
milk. In another monitoring study in Greece during 1995 and 1996, 81 samples
of commercial pasteurized milk from Athens market were analyzed for the
presence of AFM1. From 81 samples analyzed, only 3 presented residues in
levels exceeding the current MRLs (160, 170 and 177 ng l-1). The rest of the
samples, with an exception of 9 samples in which no AFM1 was detected,
were found to contain less than 5 ng l-1 (Markaki and Melissari, 1997). The
analytical methods applied for the detection of AFM1 in milk in both studies
were ELISA and HPLC (for quantification in samples contained AFM1 above
5 ng l-1).
Another monitoring study in raw and pasteurized milk was performed
from 1999 until 2001 from December up to May for each year. A total of 114
of pasteurized, ultrahigh temperature-treated and concentrated milk (UHT)
samples were analyzed for AFM1 residues. In the same study another 52 raw
milk samples from cow, sheep and goat were monitored (Roussi et al., 2002).
More than 80% of pasteurized, UHT and concentrated milk samples had
residues of AFM1 (though lower than the MRL) while for raw milk the levels
were ranging from 40% up to 73% of the total samples. Only 2 raw cow
(3.4%), 1 raw sheep (3.7%) and 2 concentrated (13.3%) samples presented
residues of AFM1 above the limit of 50 ng l-1. n the specific study it was
concluded that during the collection of milk in milk industries the
contamination of raw milk samples in the bulk tank results in higher frequency
and lower concentration of AFM1 residues. A liquid chromatographic system
equipped with a spectrofluorometer was used for the detection of AFM1 in the
selected milk samples.
Nowadays the objective of monitoring studies is to evaluate human
exposure to AFM1 and potential risk via dietary consumption. From
November 2009 until June 2010, 196 milk samples from the Greek market
(conventional, organic and kids milk) with different lipid content were
monitored for AFM1 residues using an ELISA method. Only two milk
samples were found above MRLs while risk assessment scenarios developed
for ages 1, 3, 5, 7 and 12 presented Hazard Index (HI) ratio less than one, with
highest HI values during ages of 1-3 (Tsakiris et al., 2013). In another study
243 samples of ewes and goats milk, conventional and organic, where
collected from December 2010 until July 2011, directly from farms, and
monitored for AFM1 residues (Malissiova et al., 2013). Only 1.7% of milk
samples, which were organic, presented AFM1 residues above MRLs. Organic
milk samples presented higher contamination levels when compared with
conventional ones.
Comparing the reported results in milk with recent related studies
performed in other Mediterranean countries we may say that similar mean
values are presented. In Italy 316 pasteurized samples were monitored during
2003 to 2005 for Aflatoxin M1 and the reported mean value was 27 ng/L while
the reported range of detection was 10-90 ng/L (Nachtmann et al., 2007). In
Spain 72 samples were monitored for aflatoxin M1 with reported mean value
of 9.69 ng/Kg and maximum reported value of 13.61 ng/kg (Cano-Sancho et

al., 2010). In Portugal 40 samples of pasteurized milk were monitored for
AFM1 during 2011 with reported mean value 23.4 ng/L (Duarte et al., 2013).
Since feta is a traditional Greek cheese protected by domination of origin it is
not possible to compare the results with other Mediterranean countries. As far
as yogurt is concerned results for AFM1 residues are very limited. Only one
study from Portugal presented details from ninety-six samples of yogurt
monitored for AFM1 residues during 2001. AFM1 was detected in 18.8% of
samples ranging from 19 to 98 ng/kg (Martins, 2004).
Based on the presented results we may conclude the AFM1 is still present
in milk and dairy products but the severity and frequency of detection are
reduced within the last decades. Monitoring results should be followed by risk
assessment studies in order to access the potential adverse health effects in
humans (especially in sensitive groups such as children).
3.2. Olive and Olive Oils
Olive and its derivatives, especially olive oil, are key components of the
Mediterranean diet as well as one of the basic products of the Mediterranean
basin. Greece is the worlds third larger producer of olive oil coming after
Spain and Italy. Despite having the highest annual per capita consumption in
the world (16 kg), one third of annual production (135.000 t) goes to export,
thus rendering Greece the leading exporter of extra virgin olive oil (IOC).
Greece also supports a large production of table olives, with many varieties
which include Konservolia (dual-purpose variety), Kalamon (another dual-
purpose variety) and lastly Chalkidiki. Through the last decade, table olive
production has remained above 100.000 t a year (IOC).
The Mediterranean diet features olive oil as the primary source of fat, due
to its beneficial health effects. "Extra-virgin" and "virgin" olive oils in
particular, contain a high level of polyphenols, which act like antioxidants.
The medicinal and nutritional value of olives and olive oil though, may be
significantly reduced by the development of fungi on olives, because they can
disturb the synthesis of fatty acids (El Adlouni et al., 2006) as well as lead to
the production of aflatoxins.
Mold growth and mycotoxin production are generally related to weather
extremes. As far as olives are concerned, inadequate storage conditions, such
as prolonged contact with the ground and weakly ventilated places both favor
the toxinogenic moulds colonization (Ferracane et al., 2007). This may result
in the contamination of the olive, with a strong possibility to transfer to the

olive oil. Olives which are held under conditions of high humidity and
temperature are often contaminated by toxigenic fungi (Pardo et al., 2005),
although toxigenic molds do not grow equally well on all varieties of Greek
olives (Ghitakou et al., 2006). Most storage fungi belong to the genus
Aspergillus which is adapted to low moisture conditions. However, mold
growth often occurs in products due to recontamination (Leontopoulos et al.,
2003).
Nevertheless, the studies concerning aflatoxin production on olives are
few in number. Reports from several authors show that olives may be
contaminated with a wide variety of molds including Aspergillus parasiticus
(Ghitakou et al., 2006), as well as that the possible consequent presence of
aflatoxins in olives, can lead to their transfer in oil (Cavaliere et al., 2007;
Ferracane et al., 2007). Some incidences of aflatoxin occurrence in olive oil
have been reported from Greece (Daradimos et al., 2000; Leontopoulos et al.,
2003; Papachristou and Markaki, 2004; Ghitakou et al., 2006), Spain
(Cavaliere et al., 2007), Morocco (Cavaliere et al., 2007), and other
Mediterranean countries. The leading factor which promotes mold growth and
mycotoxin production in olives seems to be the inadequate storage practices
(Cavaliere et al., 2007; Papachristou and Markaki, 2004; Ferracane et al.,
2007). However, reports on aflatoxin contamination in olives and their
products are sometimes contradictory.
Daradimos et al., 2000 analyzed 50 samples of Greek olive oil for
aflatoxin B1, and found traces in 72% of samples tested, within a range
generally very low (2.8 15. 7 ng/kg) with only one sample to be
contaminated with 46.3 ng/kg. The levels were lower than levels allowed even
by the strictest regulation existing for aflatoxins, such as for aflatoxin M1 in
liquid milk with the advisory level in some countries being 0.05 mg/kg or 0.5
mg/kg. Previous reports state that olive oil samples originating from Greece
and Spain were found to contain AFB1 at levels of 510 mg/kg (Toussaint et
al., 1997). Therefore, the study presented by Daradimos et al., 2000 indicates a
big reduction of the contamination level in the olive oil of Greek origin during
the last decades.
Other data on the aflatoxins occurrence in olive and olive oil samples from
Greece reveal that the presence of AFB1 is infrequent and rather limited. The
Papachristou and Markaki, 2004 study showed the presence of aatoxin B1
(60 ng/kg) in only one out of 50 olive oil samples from Southern Greece.
Besides these results (2004), data from Leontopoulos et al., 2003 confirm that
olives are a feeble substrate for the biosynthesis of AFB1. Leontopoulos et al.,
2003 also stated that in black olives and olive oil produced in Greece,
occurrence of AFB1 is limited and not dangerous even though among all kinds
of olives, the black Greek style is the most exposed to mold contamination
(Tantaoui-Elaraki and Mannioui, 1996; Ghitakou et al., 2006). Ghitakou et al.,
2006 revealed AFB1 presence ranging between 0.151.13 ng AFB1 /g in all
the tested olives from Greek retail market. AFB1 production in two different
varieties of black olives after inoculation by A. parasiticus was found to be not
signicantly higher compared with control samples.
In the majority of studies conducted in Greece since the year 2000, HPLC
technique with fluorescence detector was applied following aflatoxin
extraction and purification by immunoaffinity columns clean up step.
Recovery levels and sensitivity have proven to be quite satisfactory (Ghitakou
et al., 2006; Daradimos et al., 2000; Leontopoulos et al., 2003; Papachristou
and Markaki, 2004). More sophisticated techniques such as liquid
chromatographytandem mass spectrometry (LC-MS/MS) have been applied
to olive oils but although suitable for confirmatory analysis the sensitivity was
less than that of HPLC-fluorescence detection. Furthermore, the equipment is
expensive and requires considerable operator expertise (Mahoney and
Molyneux, 2010).
Aflatoxin occurrence in olives and olive oils has been under investigation
in other Mediterranean countries as well, mostly Spain, Italy, Morocco and
Egypt, since olive and olive oil consumption constitute a major part of the
Mediterranean diet as aforementioned. Table black olives Greek style
produced in Egypt, found to be the most exposed to mold contamination
(Yassa 1994). In that study, a total of 40 mold strains were isolated from black
table olives produced in Egypt, whilst nine out 40 were strains of A. flavus and
five strains of A. parasiticus which were found to produce AFB1 on culture
media and olive paste. In a more recent study, presence of AFB1 has been
confirmed in four out of ten olive samples, bought at retailer and at
supermarket in Morocco and ranged between 0.5 and 5 g/kg (El Adlouni et
al., 2006).
Aflatoxin contamination of olive oil has received the most attention but
the results obtained have been somewhat inconsistent. Total aflatoxin levels of
0.0060.04 ppb were found in 46% of 28 Sicilian olive oil samples examined
(Finoli et al., 2005). In a later study, Cavaliere et al., 2007, out of 20
experimental and 15 commercial olive oil samples analyzed by LC-MS/MS
only three of the latter were contaminated.
Ferracane et al., 2007 analyzed 30 samples of virgin oils from southern
Italy and Morocco, and reported 0.54 to 2.50 ng/g (ppb) AFB1 in 10% of
samples. AFB1 was also found in three out of four samples from North Africa
(up to 2.4 ng/g). Contamination levels of AFB1 seem to be rather similar
among the Mediterranean countries including Greece and were generally
found to be low.
The presence of aflatoxins can be limited during the refining steps that
virgin and extra virgin olive oils are produced. Stringent conditions for seed
cleaning, extraction, high-temperature heating, degumming, bleaching and
deodorizing can lead to the elimination of aflatoxins in highly refined oils
(Bao et al., 2010). Although the refining step prior the analysis of oil partially
removes aflatoxins from contaminated oil (Le Tutour et al., 1983; Parker and
Melnick 1996), pressed olive oil has been shown to contain 1847% of the
aflatoxins originally present in the contaminated olives (Mahjoub and
Bullerman 1990). Moreover, olive oils processed through cold-pressed
methods allow for antioxidants to be retained therefore special attention must
be given to proper harvesting and preservation of the olives so that they
remain uncontaminated.
In some other cases, limited AFB1 production in damaged olives was
supported by the presence of antimicrobial constituents such as caffeic acid,
coumarins, avones, catechin and phenolic compounds (Ghitakou et al., 2006).
Furthermore, Aziz et al., 1998 reported that compounds extracted from olive
fruits, such as oleuropein, inhibited aatoxin production. Another important
factor is microbial competition. Leontopoulos et al., 2003 mentioned
previously that treatment does not eliminate totally the natural microora
present in olives. In the Ghitakou et al., 2006 study microbial competition
seems to restrain high AFB1 production in selected kinds of olives. However,
AFB1 production is different depending on the substrate. AFB1 levels
produced in all three kinds of olives were dependent a) on the variety of
substrates and b) on the time of incubation. Finally, there doesnt seem to be a
correlation between mycotoxin occurrence and conventional qualitative
parameters such as peroxide number, spectrophotometric evaluation and acid
values (Ferracane et al., 2007).
It is essential that olives are stored and handled properly to prevent the
growth of aflatoxigenic mold and AFB1 production. Conditions during storage
can be controlled to a greater extent than in the eld. In order to achieve high
quality and safety of final products, optimization of storage conditions
(temperature, salt content, packaging) could inhibit contamination from
toxigenic molds and mycotoxin production (Ghitakou et al., 2006).
Furthermore, application of strict regulation for the AFB1 in olive oil is
essential in order to fortify the safe AFB1 daily intake. Current tolerance
levels set by the European Community for most food products are 2 ppb
aflatoxin B1 and 4 ppb total aflatoxins, although edible oils are not specifically
addressed (EC, 2006). Therefore, it is not possible to assess the risk due to the
AFB1 occurrence in olives since to our knowledge an AFB1 Tolerable Daily
Intake is not established for humans yet.
Although the levels of Aflatoxin detected in olives and olive oils
originated from aflatoxins in Greek products in general do not reach alarming
levels but instead they are well below normal levels. However, the frequent
consumption even at low levels can pose a hazard to public health.
Considering that Greece has the highest consumption per capita, the daily
consumption of olive oil containing low level aflatoxins signicantly
contributes to the total daily intake of aflatoxins. The possibility of synergism
with other mycotoxins present in other food commodities must also be taken
under consideration.
3.3. Feeds
Aflatoxin contamination constitutes a major issue not only in food but also
in feeds. Measures for the contamination of feed are essential since they have
an enormous health and economic significance. There are two types of feeds.
Feeds of animal origin that are usually commercial compound feeds and feeds
of vegetable origin. Common feeds of vegetable origin used are maize grain,
barley grain, wheat grain and cottonseed meal. Feeds also often contain corn,
oat and field pea.
In order to ensure the safety of food products deriving from animals it is
important to maintain a good level of feed hygiene. The moisture content of
feeds is a crucial factor for the development of moulds and the subsequent
production of aflatoxins. The initial mould population is indicative of the
hygienic status of feedstuff. Factors promoting aflatoxins in feed are high
moisture and temperature, the type of feed and the quality of feed storage
(Vlachou et al., 2004; Malissiova et al., 2013; Oluwafemi et al., 2009). Feed
contamination with aflatoxins occurs on a global scale but the relative impact
of toxins can vary depending on the different geographical characteristics. In
Greece and the Mediterranean area, the humid conditions and warm climate
favor toxin production. Contamination can occur during processing of
products pre- and/or post- harvest whenever conditions allow the development
of spoilage fungi. Inadequate pre-harvest and post-harvest conditions and
practices (e.g. improper drying of grains after harvest), poor storage, insect
attack, non-use of mold inhibitors, are all factors that facilitate aflatoxin
contamination in feed ingredients (Boudra and Morgavi, 2005). The
application of Good Agricultural Practice (GAP) programs as well as the
establishment of Hazard Analysis and Critical Control Point (HACCP)
systems is considered essential for the control of aflatoxin contamination in
feeds. As an additional control method, feed millers are encouraged to add
toxin binders of fungal growth inhibitors to their feeds. However, in the case
of organic farming the restriction of fungicide use may affect the quality of the
feed ingredients used (Malissiova et al., 2013).
Vlachou et al., 2004 investigated the presence of AFB1 in feed samples
collected from storage areas of animal farms from all provinces of Greece. The
samples corresponded to the most common ingredients used in feeds at the
time: maize grain, barley grain, wheat grain and cottonseed meal. Samples of
commercial compound feeds were also examined. TLC technique was used to
measure AFB1. AFB1 was detected in 7 out of 183 samples containing levels
less than the maximum permitted EU limit for feeds (20-50 ppb). AFB1 was
only detected in maize grain and cottonseed meal. Maize grain presented the
highest moisture levels while cottonseed meal the lowest. At the time of the
investigation, even the maize sample that was found containing 90 ppb AFB1
could have been used as feed by diluting it with uncontaminated maize grain
according to a provision of EU law that stated that raw materials containing up
to 200 ppb of AFB1 could be used by Recognized Feed Manufacturers.
However, this directive changed in 2003 due to certain food crises scandals
and the possibility to dilute contaminated feeds was banned (EU, 2002; EU,
2003). Generally, aflatoxin B1 does not constitute a problem for feeds in
Greece.
In general, studies on the presence of aflatoxins in animal feeds are
limited. A couple of papers are reported from Morocco, which is surrounded
by the Mediterranean Sea and the Atlantic Ocean and thus presents a similar
climate with the Southern Greece as it is characterized by high temperature
and humidity. Kichou and Walser (1993) analyzed 315 samples of poultry
feeds using a semi-quantitative ELISA and TLC methods. The feed
ingredients included corn, wheat, soybean meal, sunflower meal, cottonseed
meal and sorghum. Contamination levels ranged from 20-200 g/kg apart from
4 samples that reached AFB1 levels from 2000-5625 g/kg. The latter highly
contaminated feed samples were linked to clinical aflatoxicosis in chickens. In
another survey, Zinedine et al. (2007b) analysed 21 samples from Rabat. The
results exposed a 66.6% of aflatoxin contamination while AFB1 contamination
levels of poultry feeds ranged between 0.05 and 5.38 g/kg. In both studies
from Morocco, the contamination levels often exceeded the MRLs set by the
European Regulations in feedstuff. This can be attributed to the fact that food
safety and quality standards practices such as GAP, Good Manufacturing
Practices and HACCP system are often not applied by the Moroccan food
production units (Zinedine and Manes, 2009).
AFM1 as discussed earlier has been traced in the milk of sheep, cows,
goats, camels and buffalos in different proportion ranges (Battacone et al.,
2003; Battacone et al., 2005). The relationship between the ingested AFB1 and
the AFM1 excreted in milk varies according to the animal breed, the
production of milk and the frequency of the daily milking. Because of AFM1
toxicity, the European Union has set the maximum limit of detection at 50 ppt
(EC, 2006).
The environmental conditions in Greece favor AFB1 production in feeds.
Therefore the presence of AFM1 in milk is possible as it was pointed out by
Roussi et al. (2002). Since an important part of the Greek economy relies on
farming of goats, a few studies have investigated the AFM1 carryover in the
milk of Greek goats. Kourousekos et al. (2012) divided 30 greek goats into 3
groups of ten goats each. One group was used as control and the other two
groups were treated. Each goat received 50 or 100 g of AFB1 per day for 35
days. According to the results, after AFB1 administration, AFM1 was
detectable in quite high concentrations in the milk of goats from the two
treated groups. AFM1 concentrations increased with the increase of the
administrated AFB1 dosage. 59.38% of milk samples from the group
administered with 50 g AFB1 and 83.33% from the group administered with
100 g AFB1 presented AFM1 concentrations >80 ppt. AFM1 concentrations
in most milk samples from the two treated groups exceeded the maximum
limit of 50 ppt set by the European Union. This practically shows that doses of
even 50 g AFB1 included in feeds may render the milk unsafe. The excretion
of AFM1 appears to be direct and depends on the AFB1 dose.
Malissiova et al. (2013) as already reported performed another study in
Greece involving the monitoring of AFM1 levels in ewe's and goat's milk. The
results suggest the use of safe feeds by the majority of the monitored farms as
well as secure production practices. Risk factors potentially related to AFM1
contamination include winter season, the use of warehouse for feed storage
and feeding pea. Moreover differences noticed in levels of AFM1 in milk in
different countries may be attributed to the different climatic conditions and
the feed storage practices (Zinedine et al., 2007a; Montagna et al., 2008;
Magan et al., 2011).
In general, AFB1 does not constitute a problem for feeds in Greece.

However, appropriate measures to prevent contamination should never be
ignored. It is essential to follow a HACCP system as well as a GAP program.
Finally, feed manufacturers should comply with the regulations and EU
legislation to ensure feed and consequently food safety.
3.4. Aflatoxins in Minor Crops and Food Commodities
Aflatoxins can be found as well on other food commodities that are

otherwise considered beneficial for the human health. Bee pollen, pistachio
nuts and breakfast cereals are food items that could be consumed daily by the
inhabitants of Greece.
Bee pollen is known for its medical properties (antibiotic, antioxidant,
antineoplastic, antidiarhoeic) since ancient times and it is nowadays used as a
food supplement. It consists mainly of protein, but also contains vitamins,
minerals, carotenoids, sugars, lipids as well as flavonoids and phenolic acids,
which is why it has such a strong antioxidant effect (Graikou et al., 2011).
There is only one study about the AFB1 in pollen of Greek origin (Pitta and
Markaki, 2010). In this study, a routine method was developed and validated
to determine AFB1 in bee pollen, using a simple extraction step,
immunoaffinity column clean-up and finally determination by HPLC with a
fluorescence detector. The main aim of the study was to examine if the bee
pollen can act as a substrate for the AFB1 production. There were three groups
of samples (15g pollen/flask) in the study: a) Bee pollen without any treatment
containing natural microbiota., b) Treated bee pollen, in order to eliminate the
natural microbiota and then inoculated with A. parasiticus., c) Treated, non-
inoculated bee pollen (control samples). The study revealed that in the first
group, where the pollen still had its natural micro biota, there was no
production of AFB1 throughout the incubation and observation time (at 30o C
for 20 days). Moreover, no mycelial growth was apparent, which could be
explained if the microbial competition is taken into account. On the contrary,
AFB1 production was observed in the other two groups with treated bee
pollen, inoculated with A. parasiticus as well as the control group (maximum
values: 79.29 ng/flask and 32.44 ng/flask, respectively, with the inoculated
group having significantly higher values, p0.05). These values occurred
during the 12th day of incubation and after that peak the concentration of
AFB1 started to decline. Furthermore, it is apparent that the treatment did not
eliminate all the aflatoxigenic molds of the bee pollen, but the production of
AFB1 in the inoculated group started on the 3rd day of the incubation, whereas
the control group appeared to have a delayed effect (7th day). In a previous
study it was shown, that in ready-to-eat bee pollen produced in Spain, A.
parasiticus was isolated, which was able to produce AFB1 (Gonzlez et al.,
2005). Furthermore, a study on 20 samples of Spanish bee pollen yielded no
quantifiable amounts of five different mycotoxins, including AFB1 (Medina et
al., 2004).
Another food commodity known for its beneficial health properties
(especially for the cardiovascular system) are pistachio nuts. They are a rich
source of carotenoids, selenium and flavonoids, as well as other minerals,
sugars and fibers, proteins, several vitamins and fatty acids (Tsantili et al.,
2011). However, pistachio nuts as well have been found to be a substrate for
the production of AFB1. In a study conducted on samples of Greek pistachio
nuts of the variety Aegina (P. vera cv Aegina), five different steps of the
pistachio nuts cultivation and processing were examined (early maturity,
maturity, harvest, drying and storage), in order to detect where the highest
production of aflatoxins occurs and which factors affect it (Georgiadou et al.
2012).
The pistachio trees were from four different orchards and producers,
where the cultivation and processing of the nuts varied. However all the trees
were of the same variety and grew under the same weather conditions. The
results showed that the aflatoxins could occur at any stage of the procedure,
with AFB1 being the most abundant aflatoxin in all stages (its value was much
higher than G1, B2, and G2). However, maturity appeared to be the most
critical stage for aflatoxin contamination in pre-harvest stages (only one
orchard appeared to have a small amount of AFB1: 3.1 g/kg). Higher levels
of aflatoxins were observed during maturity in the case where there was high
insect infestation (1191.47148.34 g/kg AFB1) compared to the orchards that
were well irrigated and where plant protection was applied (6.62.0, 15.03.7
and 33.7 9.5 g/kg AFB1). Furthermore, improper sun drying led to higher
AFB1 values (105831 g/kg AFB1) compared to mechanical drying in a hot
air dryer (0.240.17 g/kg AFB1). Finally, poor storage conditions of room
temperature and relatively high humidity also contributed to the production of
aflatoxins (92681 and 37137 g/kg AFB1), while storing the pistachio nuts
under controlled conditions (5-7 C and 45-60% RH) prevented the occurrence
of aflatoxins (0 g/kg AFB1). These findings are in accordance to other
studies in pistachios from other countries. In addition, imports of nuts in
Greece are constantly controlled through the national market surveillance
program according to which sampling of cargos in Greek ports is

implemented.
Finally breakfast cereals were a minor food commodity and only one
paper in Greek literature provides details about analytical techniques and
levels of AFB1 in selected samples from Greek market (Villa and Markaki,
2009).
An HPLC method with fluorescence detector was used for the
determination of AFB1 in 55 cereal samples during 2006 and 2007 collected
randomly from super-markets in Athens. 56.3% of samples presented residues
of AFB1 while seven samples were found to be contaminated at levels higher
than the EU limit of 2 ng/g.
CONCLUSION
Aflatoxin M1 (AFM1) is one the major xenobiotics affecting milk and
dairy products in terms of safety and quality.
AFM1 residues were not detected in feta samples during the last
years. The concentration ratio of AFM1 from initial milk samples to
corresponding curd present values from 4.3 up to 5.6.
AFM1 is still present in milk in Greece and other Mediterranean
countries but the severity and frequency of detection are reduced
within the last decades. Monitoring results should be followed by risk
assessment studies in order to access the potential adverse health
effects in humans (especially in sensitive groups such as children).
The leading factor which promotes mould growth and mycotoxin
production in olives seems to be inadequate storage practices.
Studies presented within the last decades indicating a recent reduction
of the contamination level in the olive oil of Greek origin.
Contamination levels of AFB1 seem to be rather similar among the
Mediterranean countries including Greece and were generally found
to be low.
In Greece, as far as feeds are concerned, AFB1 was only detected in
maize grain and cotton seed meal but in general, aflatoxin B1 does not
constitute a problem for feeds in Greece. However, appropriate
measures to prevent contamination should never be ignored. It is
essential to follow a HACCP system as well as a GAP programme.
In minor crops such as pistachio, breakfast cereals and bee pollen

more monitoring studies are needed for safe conclusions.
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Chapter 10
AFLATOXINS AS SERIOUS THREATS

TO ECONOMY AND HEALTH
Lipika Sharma1, Bhawana Srivastava2, Shelly Rana2,

Anand Sagar2 and N. K. Dubey3
1
G.B. Pant Institute for Himalayan Environment and Development
Himachal Unit, Mohal- Kullu, Himachal Pradesh, India
2
Mycology & Plant Pathology Laboratory, Department of Bio-Sciences,
Himachal Pradesh University, Shimla, India
3
Laboratory of Herbal Pesticide, Centre of Advanced Study in Botany,
Banaras Hindu University, Varanasi, India
ABSTRACT
This review deals with the aflatoxins especially with their food
sources, wide occurrence and toxicological effects on animals and
humans. Aflatoxins are highly oxygenated, heterocyclic,
difuranocoumarin compounds and are an important group of mycotoxins
produced by the fungi. There are almost 20 different types of aflatoxins
identified till now; among these AFB1 is considered to be the most toxic.
Aflatoxins persist to some extent in food even after the inactivation of the
fungi by food processing methods, such as ultra-high temperature
Corresponding Author: Dr. Bhawana Srivastava, (Dr. D.S. Kothari postdoctoral fellow, UGC),
Mycology & Plant Pathology Laboratory, Department of Bio-Sciences, Himachal Pradesh
University, Shimla, India-171005, Tel: 91-9889206615, 91-177-2621692, e-mail:
bhawana.bhu@gmail.com.
260 Lipika Sharma, Bhawana Srivastava, Shelly Rana et al.
products, due to their significant chemical stability. Aflatoxins can affect

a wide range of commodities including cereals, oilseeds, spices, and tree
nuts as well as milk, meat, and dried fruits. Twenty five percent of the
worlds crops are affected with mycotoxins. On a worldwide scale, the
aflatoxins are found in stored food commodities and oil seeds. Some of
the foods on which aflatoxin producing fungi grow well include cereals
(maize, sorghum, pearl millet, rice, wheat, corn, oats, barley), oilseeds
(peanut, soybean, sunflower, cotton), spices (chile peppers, black pepper,
coriander, turmeric, ginger), and tree nuts (almond, pistachio, walnut,
coconuts), sweet potatoes, potatoes, sesame, cacao beans, almonds, etc.,
which on consumption pose health hazards to animals, including
aquaculture species of fish, and humans. Food commodities affected by
aflatoxins are also susceptible to other types of mycotoxins and multiple
mycotoxins can co-exist in the same commodity. Various cereals affected
by aflatoxins are also susceptible to contamination by fumonisins,
trichothecenes (especially deoxynivalenol), zearalenone, ochratoxin A
and ergot alkaloids.
More than 5 billion people in developing countries worldwide are at
risk of chronic exposure to naturally occurring aflatoxins through
contaminated foods. Aflatoxin is a potent liver toxin causing
hepatocarcinogenesis, hepatocellular hyperplasia, hepatic necrosis,
cirrhosis, biliary hyperplasia, and acute liver damage in affected animals.
Effects of aflatoxins in animals depend on age, dose and length of
exposure, species, breed and nutritional status of the animal. Health
effects occur in fish, companion animals, livestock, poultry and humans
because aflatoxins are potent hepatotoxins, immunosuppressants,
mutagens, carcinogens and teratogens. Aflatoxin B1 has been shown to
cause significant morphological alterations along with reduced
phagocytic potential in chicken and turkey macrophages. Aflatoxin- B1
exposure to chicken embryos causes significant suppression in
macrophage phagocytic potential in chicks after hatch. Aflatoxin
intercalates into DNA and alkylates the DNA bases through its epoxide
moiety resulting in liver cancer. Other effects include mutagenic and
teratogenic effects. Exposure of biological systems to harmful levels of
aflatoxin results in the formation of epoxide, which reacts with proteins
and DNA leading to DNA-adducts, thus causing liver cancer. The
primary target of aflatoxins is the hepatic system. Acute effects include
hemorrhagic necrosis of the liver and bile duct proliferation while chronic
effects include hepatocellular carcinoma (HCC). HCC is the sixth most
prevalent cancer worldwide with a higher incidence rate within
developing countries. Preliminary evidence suggests that there may be an
interaction between chronic aflatoxin exposure and malnutrition,
immunosuppression, impaired growth, and diseases such as malaria and
HIV/AIDS. Outbreaks of acute aflatoxin poisoning are a recurrent public
health problem. The discussion of this problem and its remedies must be
Aflatoxins As Serious Threats to Economy and Health 261
held in the context of the associated question of food insufficiency and

more general economic challenges in developing countries. Aflatoxin
constitutes a serious health concern to the entire food chain, necessitating
a multidisciplinary approach to analysis, action, and solution.
Keywords: Aflatoxons, Aspergillus sp., Hepatotoxins, Mycotoxins,

Occurrence, Toxicological Effects
INTRODUCTION
One of the greatest challenges of the world is to produce enough food for
the growing population. The situation is particularly critical in developing
countries, where the rate of net food production is slowing down in relation to
population growth. The world food situation is aggravated by the fact that in
spite of the use of all available means of plant protection, about one-third of
the yearly harvest of the world is destroyed by pests (Varma and Dubey,
1999). Considerable postharvest losses of food commodities are brought about
by infestations caused by different pests. International agencies that monitor
world food resources have acknowledged that one of the most feasible options
for meeting future food needs is reduction of postharvest losses (Kelman,
1984; Tripathi and Dubey, 2004). Fungi are significant destroyers of
foodstuffs during storage, rendering them unfit for human consumption by
retarding their nutritive value and sometimes by producing mycotoxins.
Approximately 2540% of cereals world-wide are contaminated with
mycotoxins produced by different storage fungi (Kumar et al., 2007). Tropical
countries, because of their temperature and congenial environment suffer
severe losses from pests due to conducive atmosphere. Generally, conditions
of these countries such as high temperatures and moisture, unseasoned rains
during harvest and flash floods lead to fungal proliferation and mycotoxins.
Aflatoxin belongs to a group of fungal toxins known as mycotoxins; these
are highly oxygenated, heterocyclic, difuranocoumarin compounds and are an
important group of mycotoxins produced by the fungi Aspergillus flavus, A.
parasiticus and A. nomius (Diaz et al., 2008).Other species of Aspergillus such
as A. bombycis, A. ochraceoroseusand A. pseudotamari mayalso produce
aflatoxins (Bennett & Klich, 2003; Klich et al., 2000; Mishra & Das, 2003;
Akbarsha et al., 2011). These species contaminate various agricultural
commodities either before harvest or at post-harvest stages under favorable
conditions of temperature and humidity. The discovery of aflatoxins dates
back to the year 1961 following the severe outbreak of turkey X disease, in
the England, which resulted in the deaths of more than 100.000 turkeys and
other farm animals. Aflatoxins are toxic secondary metabolites produced by
Aspergillus fungus growing in susceptible agricultural commodities. They can
result in major economic losses and can negatively affect animal and human
health. The name aflatoxins, an acronym, has been formed from the following
combination : the first letter, A for the genus Aspergillus, the next set of
three letters, FLA, for the species flavus, and the noun TOXIN meaning
poison (Rustom,1997; Filazi & Sireli,2013).
Characteristics and Types
Once aflatoxin is produced, it is stable. Heat, cold and light do not affect
it. It is also colorless, odorless and tasteless, and because of the low
concentrations involved and the uneven distribution in grain bins, aflatoxins
are difficult to detect. Aflatoxins persist to some extent in food even after the
inactivation of the fungi by food processing methods, such as ultra-high
temperature products, due to their significant chemical stability (De Viries,
1997; Peraica, 1999). There are almost 20 different types of aflatoxins
identified till now, among these B1, B2, G1 andG2 are more prominent while
AFB1 is considered to be the most toxic (IARC,2002; Mushtaq et al. 2012).
There are four major natural aflatoxins (AFs), AFB1, AFB2, AFG1 and
AFG2. The hierarchy of toxicity of different aflatoxins is in the order
AFB1>AFG1>AFB2>AFG2. There are two additional metabolic products of
aflatoxins B1and B2, viz., M1 and M2. Aflatoxin M1 (AFM1) is a major
metabolite of aflatoxin B1 (AFB1), which is formed when animals ingest feed
contaminated with aflatoxin B1. The AFB1, once ingested by the animal, is
rapidly absorbed by the gastrointestinal tract and is transformed into the
metaboliteAFM1, which appears in the blood after 15 minutes and is then
secreted in the milk by the mammary gland (Van Egmond, 1989; Battacone, et
al. 2003). The amount of AFM1 which is found in milk depends on several
factors, such as animal breed, lactation period, mammary infections etc It
has, anyway, been demonstrated that up to 6% of the ingested AFB1 is
secreted into the milk as aflatoxin M1 (Van Egmond & Dragacci, 2001) and,
because AFM1is relatively resistant to heat treatments (Yousef & Marth,
1989; Galvano et al., 1996), it is almost entirely retained in pasteurized milk,
powdered milk, and infant formula. Moreover,only a limited decrease of
AFM1 content has been verified in UHT milk after long storage(Galvano et
al., 1996; Martins & Martins, 2000; Tekinsen & Eken, 2008). Aflatoxin B1
(AFB1) is the most potent and potentially lethal metabolite and is a known
human carcinogen. The hepatotoxicity and carcinogenic effects of AFB1 have
been clearly demonstrated, thus it has long been classified as a group 1 human
carcinogen by the International Agency on Research on Cancer (IARC, 2002).
Initially, the IARC classified AFM1 as a possible carcinogen for humans
(group 2b) since toxicological data was limited (IARC, 1993). However,
genotoxicity and cancerogenity of AFM1 have been observed in vivo,
although lower than those of AFB1, and its cytotoxicity has been definitively
demonstrated (Caloni et al., 2006). As a result of these and other further
investigations, the IARC moved aflatoxin M1 from group 2B to group 1
human carcinogen (IARC, 2002, Anfossi et al., 2011).
Figure 1. Structure of some major aflatoxins.
Factors That Affect Aflatoxin Contamination
Factors that affect aflatoxin contamination include the climate of the

region, the genotype of the crop planted, soil type, minimum and maximum
daily temperatures, and daily net evaporation (Wilson and Payne 1994; Ono,
Sugiura et al. 1999; Brown, Chen et al. 2001; Bankole and Mabekoje 2004;
Fandohan, Gnonlonfin et al. 2005). Aflatoxin contamination is also promoted
by stress or damage to the crop due to drought prior to harvest, insect activity,
poor timing of harvest, heavy rains at harvest and post-harvest, and inadequate
drying of the crop before storage (Hell, Cardwell et al. 2000; Ono, Sasaki et al.
2002; Hawkins, Windham et al. 2005; Turner, Sylla et al. 2005). Humidity,
temperature, and aeration during drying and storage are major factors behind
contamination.
Table 1. Chemical and physical properties of aflatoxins
Aflatoxin Molecular Molecular weight Melting point

formula (0C )
B1 C17 H12O6 312 268-269
B2 C17 H14O6 314 286-289
G1 C17 H12O7 328 244-246
G2 C17 H14O7 330 237-240
M1 C17 H12O7 328 299
M2 C17 H14O7 330 293
B2A C17 H14O7 330 240
G2A C17 H14O8 346 190
Food Sources and Occurrence
Well-known within the agricultural community, aflatoxins have been

studied for over forty years due to their widespread occurrence and their
significant impact on crops (Eaton and Groopman 1994; Wild and Turner
2002; Shephard 2003; Fung and Clark 2004; Williams, Phillips et al. 2004).
Aflatoxins are toxic secondary metabolites produced by Aspergillus fungi.
Aflatoxins can affect a wide range of commodities including cereals, oilseeds,
spices, and tree nuts as well as milk, meat, and dried fruit. Twenty five percent
of the worlds crops are affected with mycotoxins. Some of the foods on which
aflatoxin producing fungi grow well include cereals (maize, sorghum, pearl
millet, rice, wheat, corn ), oilseeds (peanut, soybean, sunflower, cotton), spices
(chile peppers, black pepper, coriander, turmeric, ginger), and tree nuts
(almond, pistachio, walnut, coconuts). Many of the above mentioned
ingredients are used in human food andin various livestock and poultry feed
rations and so these species are often contaminated with aflatoxin (Mushtaq et
al. 2012). The major sources of exposure are maize and groundnuts as these
are the foods that are most susceptible to contamination and consumed in the
greatest amounts. Aflatoxins are most prevalent in areas located between
latitudes 40N and 40S of the equator. On a worldwide scale, the aflatoxins
are found in stored food commodities and oil seeds such ascorn, peanuts,
cottonseed, rice, wheat, oats, barley, sorghum, millet, sweet potatoes, potatoes,
sesame, cacao beans, almonds, etc., which on consumption pose health
hazards to animals, including aquaculture species of fish, and humans (Abdel-
Wahab et al., 2008;Hussein & Brassel, 2001; Akbarsha et al., 2011). The
greatest risk for health impact lies within developing countries located in
tropical regions, which rely on these commodities as their staple food source.
Food insufficiency and lack of diversity substantially contribute to the
susceptibility of individuals and communities to aflatoxins.
Various approaches exist for the determination of aflatoxin in food and
feed commodities. Generally, all analytical methods follow the basic protocol
of extraction, clean-up, separation, detection, identification and quantification.
However, the most widely used techniques are those which include a
chromatographic step to separate the mycotoxin of interest like minicolumn
chromatography, thin layer chromatography, high performance liquid
chromatography and gas liquid chromatography.
Figure 2. Presentation showing economic loss because of aflatoxin occurrence.

Although immunoassay-based quantitative methods are fast and

promising, for mycotoxin research they have the possibility of producing mis-
leading results because of cross-reaction and interference in the complex
matrixes (Nilu & Boyaciog, 2002; Hu, 2006; Mushtaq et al. 2012).
Toxicological Effects
The health issues related to aflatoxins are equally complex and demand
more research. The ingested aflatoxin undergoes various possible pathways
depending on different parameters like dose quantity, type of species, age,
diet, and immune system of host. Aflatoxin is a potent liver toxin causing
hepato-carcinogenesis, hepatocellular hyperplasia, hepatic necrosis, cirrhosis,
biliary hyperplasia, and acute liver damage in affected animals. Other effects
include mutagenic and teratogenic effects. Large doses of aflatoxin are lethal
and chronic exposure to low levels of aflatoxin can result in cancer and
immunosuppression (Sharma, 1993; Yarru, 2008). Exposure of biological
systems to harmful levels of aflatoxin results in the formation of epoxide,
which reacts with proteins and DNA leading to DNA-adducts, thus causing
liver cancer (Turner et al., 2009; Groopman et al., 2008). Infants are at much
higher risks of health problems compared to adults (Sergent et al., 2008).
Aflatoxins, in general, and AFB1 in particular, can induce DNA damage, gene
mutation, sister-chromatid exchanges and other chromosomal anomalies,
which account for their genotoxic, teratogenic and carcinogenic properties
(Batt et al., 1980; International Agency for Research on Cancer (IARC, 1993;
Ray-Chaudhuri et al., 1980). AFB1 can form adducts with DNA, RNA and
protein, which form the major basis of the health risks (Sun et al., 2001;
Williams et al., 2004). The maximum legal limit allowed for AFB1 in infant
food in the European Union is 0.1 g kg1 (European Commission, 2006). In
developing countries, the majority of the people survive largely on cereal
based diets. Consequently, nutritional deficiencies are very prevalent in
populations consuming high levels of cereals, particularly in children (Bankole
& Adebenjo, 2003). Moreover, poor diet and multiple infectious hazards are
associated with malnutrition and growth faltering in infancy and childhood
(IARC, 2002). More than 5 billion people in developing countries worldwide a
great risk of chronic exposure to naturally occurring aflatoxins through
contaminated food (Shephard, 2003; Williams et al., 2004) and more so in the
tropical regions, where the climatic conditions favour luxurious growth of
Aspergillus spp, and people rely on commodities such as cereals, oilseeds,
spices, tree nuts, milk, meat and dried fruits that are potentially contaminated
by aflatoxins (Strosnider et al., 2006). In July 2005, the United States Centers
for Disease Control and Prevention (US CDC) and the World Health
Organization (WHO) hosted a workshop to create an integrated plan intended
to generate culturally appropriate, long-term, public health strategies to reduce
aflatoxin exposure in developing countries. Participants included 40
internationally recognized scientists from diverse backgrounds (i.e. public
health, agriculture, animal health, trade and social science) and key public
health officials and stakeholders from countries heavily affected by aflatoxins.
Aflatoxins affect many species including humans, dogs, feeder pigs, dairy
cattle, and chickens.
Effects of Aflatoxins on Animal Health
The effects of aflatoxins on animal health have been observed in many

species for over forty years (Patten 1981; Miller and Wilson 1994) beginning
with the documentation of Turkey X disease in 1960. The primary target of
aflatoxins is the hepatic system. Acute effects include hemorrhagic necrosis of
the liver and bile duct proliferation while chronic effects include
hepatocellular carcinoma (HCC). In animals, suppression of immunity, growth
retardation, and increased susceptibility to infectious disease due to aflatoxin
exposure is well-documented (Patten 1981; Miller and Wilson 1994). Trout,
one of the early models for aflatoxicosis is very sensitive to aflatoxin and
develop clinical signs such as hepatoma. Swine at weaning and marketing
stages are resistant to dietary levels of aflatoxins up to300 ppb. Clinical signs
associated with aflatoxicosis in dairy cattle include reduced feed intake, milk
production, weight gain and liver damage. Aflatoxin M1, an aflatoxin
metabolite found in milk, was also found in milk products such as yogurt
(Martins M.L e tal., 2004; Yarru, 2008). Symptoms of aflatoxicosis include
feed refusal, decreased feed efficiency, and stunted growth, decreased milk
production and impaired reproductive efficiency (Diekman& Green, 1992;
Oguz&Kurtoglu, 2000; Pier, 1992; Raju & Devegowda, 2000). Numerous
studies have shown that feed intake is reduced in broiler chicks fed aflatoxin
(Osborn et al., 1992; Ledoux et al., 1998). Reduced feed intake results in
decreased average daily gain (Ledoux et al., 1998). One important thing to be
determined is the effects of aflatoxin on nutrient digestibility and
bioavailability. This can be done by measuring the activity and levels of
various digestive enzymes and by evaluating serum chemistry of affected
animals. Balachandran and Ramakrishnan (1987) studied the influence of

dietary aflatoxin on serum enzyme levels in broiler chicken. They measured
SGPT, SGOT, serum amylase and lipase levels in Cobb broiler chickens fed
3ppm aflatoxin.
Table 2. FDA action levels for aflatoxins in human and animal foods
Commodities . Total aflatoxin

action level (ppb)
Human food 20
Milk 0.5
Beef cattle 300
Swine over 100 lbs 200
Breeding beef cattle, swine, or mature poultry 100
Corn for immature animals and dairy cattle 20
Corn for breeding beef cattle, swine and mature poultry 100
Corn for finishing swine 200
Corn for finishing beef cattle 300
Cottonseed meal (as feed ingredient) 300
All feedstuff other than corn 20
Source: Wu, et al. 2011.
They observed an increase in serum lipase and SGOT levels, and a

decrease in serum amylase levels all of which resulted in alteration in nutrient
digestion, absorption and metabolism. Osborne and Hamilton, (1981) observed
decreased serum lipase, amylase, trypsin, lipase, RNase, and DNase activities
in chickens fed aflatoxin. They did not measure serum calcium and
phosphorus levels which play a role in rubbery leg syndrome, one of the
symptoms associated with aflatoxicosis. Health effects occur in fish,
companion animals, livestock, poultry and humans because aflatoxins are
potent hepatotoxins, immunosuppressants, mutagens, carcinogens and
teratogens. Public health concerns center on both primary poisoning from
aflatoxins in commodities, food and feedstuffs, and relay poisoning from
aflatoxins in milk (Coppock& Christian, 2007). The ability of the immune
system to combat infection and disease is another key component of animal
health. Aflatoxin B1 has been shown to cause significant morphological
alterations along with reduced phagocytic potential in chicken (Neldon-ortiz
and Qureshi, 1991a) and turkey (Neldon-ortiz and Qureshi, 1991b)
macrophages. Aflatoxin-B1 exposure to chicken embryos causes significant
suppression in macrophage phagocytic potential in chicks after hatch (Neldon-

ortiz and Qureshi, 1991c; Yarru, 2008).
Effects of Aflatoxins on Human Health
The effects of aflatoxins on humans, as with animals, are dependent upon

dosage and duration of exposure. Acute exposure can result in aflatoxicosis,
which manifests as severe, acute hepatotoxicity with a case fatality rate of
approximately 25% (Cullen and Newberne 1994). Early symptoms of
hepatotoxicity from aflatoxicosis can manifest as anorexia, malaise, and low-
grade fever.
Figure 3. A picture summarizing main characteristics of aflatoxins.
Acute high level exposure can progress to potentially lethal hepatitis with
vomiting, abdominal pain, jaundice, fulminant hepatic failure, and death.
Outbreaks of acute aflatoxicosis are a recurring public health problem
throughout the world (Krishnamachari, Bhat et al. 1975; Ngindu, Johnson et
al. 1982; Lye, Ghazali et al. 1995; CDC 2004).Hepatocellular carcinoma
(HCC) as a result of chronic exposure has been well documented, generally in
association with hepatitis B virus or other risk factors (Qian, Ross et al. 1994;
Wang, Hatch et al. 1996; Chen, Chen et al. 2001; Henry, Bosch et al. 2002;
Omer, Kuijsten et al. 2004). The International Agency for Research on Cancer
(IARC) first recognized aflatoxins as carcinogenic in 1976 and has
subsequently reaffirmed naturally occurring mixtures of aflatoxins and
aflatoxin B1 as Group 1 carcinogens (carcinogenic to humans) (IARC 2002).
Additional effects of chronic exposure have not been widely studied but are
thought to include immunologic suppression, impaired growth, and nutritional
interference (Patten 1981; Cullen and Newberne 1994; Fung and Clark 2004;
Williams, Phillips et al. 1994).
Health Impact and Burden of Disease
HCC is the sixth most prevalent cancer worldwide with a higher incidence
rate within developing countries (Parkin, Bray et al. 2005), however, the
burden of HCC attributable to aflatoxins when accounting for other co-
morbidities, such as hepatitis B (HBV), is not known. Several studies in China
have indicated combined exposure to HBV and aflatoxins is associated with a
much higher risk of HCC (Qian, Ross et al. 1994; Wang, Hatch et al. 1996).
This interaction has not been studied in other high risk areas such as sub-
Saharan Africa and the molecular mechanism of the interaction between HBV
and aflatoxins is not known (Turner, Sylla et al. 2002; Wild and Turner 2002).
Quantifying the proportion of HCC attributable to aflatoxin exposure, to HBV,
and to the interaction of aflatoxin exposure and HBV will help identify the
best public health strategy to reduce HCC, including the benefits and limits of
widespread HBV vaccination. Additional health effects associated with
chronic aflatoxin exposure have not been well studied. Without knowing the
relationship between chronic exposure and health, the true human health
impact and the resulting burden of disease in developing countries are not
known. Preliminary evidence suggests that there may be an interaction
between chronic aflatoxin exposure and malnutrition, immunosuppression,
impaired growth, and diseases such as malaria and HIV/AIDS. Experimental
animal evidence suggests that chronic exposure to aflatoxins may lead to
impaired immunity, reduced uptake of nutrients from the diet, and growth
retardation (Hall and Wild 1994; Miller and Wilson 1994). Several studies of
children in Benin and Togo have shown an association between aflatoxin
albumin adduct levels and impaired growth (Gong, Cardwell et al. 2002;
Gong, Egal et al. 2003; Gong, Hounsa et al. 2004). In a recent study in Ghana,
higher levels of aflatoxin B1-albumin adducts in plasma were associated with
lower percentages of certain leukocyte immune phenotypes (Jiang, Jolly et al.

2005). A study in Gambian children found an association between serum
aflatoxin-albumin levels and reduced salivatory secretory IgA levels (Turner,
Moore et al. 2003). While the effects on immunity suggest the possible
influence of aflatoxins on susceptibility to infectious disease, further
investigation is needed.
Aflatoxin associated health effects pervade the developing world despite
the fact that these effects could be mitigated or prevented with the current state
of agricultural knowledge and public health practice. The discussion of this
problem and its remedies must be held in the context of the associated
question of food insufficiency and more general economic challenges in
developing countries. Outbreaks of acute aflatoxin poisoning are a recurrent
public health problem. In 2004, one of the largest, most severe aflatoxicosis
outbreaks occurred in Kenya followed by another outbreak in 2005 (CDC
2004). Given that diseases in the developing world often go unreported, the
Kenya outbreaks are likely to be an underestimation of the problem;
furthermore, the burden of disease attributable to chronic aflatoxin exposure
(e.g. hepatocellular carcinoma, impaired growth, immune suppression)
remains undefined. These outbreaks emphasize the need to quantify and
control aflatoxin exposure in developing countries and highlight the potential
role of public health.
A broad range of signs and symptoms can be used to diagnose
aflatoxicosis based on the level of exposure. The signs and symptoms of this
condition include vomiting, abdominal pain and hemorrhaging, pulmonary
edema, acute liver damage, loss of digestive tract function, convulsions,
cerebral edema, and coma. Hepatitis B vaccinations, education through
awareness campaigns, and chemoprevention measures such as competitive
displacement, plant extract application, and methyl eugenol spray have proven
to be effective interventions in controlling and preventing the adverse health
effects of aflatoxin exposure (Right Diagnosis, 2011). Aflatoxin constitutes a
serious health concern to the entire food chain, necessitating a
multidisciplinary approach to analysis, action, and solution. To maximize
resources, a targeted monitoring and surveillance system for high-risk areas
and their populations should collect and analyze appropriate specimens
(usually food, urine, and serum) (Strosnider, 2006). Aflatoxicosis is a disease
caused by aflatoxin poisoning. The disease can be acute, meaning it is caused
by the short-term exposure to high levels of aflatoxin, or chronic, meaning that
it has been caused by long-term exposure to low to moderate levels of
aflatoxin. Symptoms differ between the acute and chronic forms of the disease
and have been outlined in this section.
Acute Aflatoxicosis
Acute aflatoxicosis, associated with extremely high doses of aflatoxin, is

characterized by hemorrhaging, acute liver damage, edema, and high mortality
rates in humans. Acute aflatoxicosis is associated with sporadic outbreaks of
the consumption of highly contaminated foods. Early symptoms of acute high
level exposure to aflatoxin include diminished appetite, malaise, and low
fever; later symptoms, which include vomiting, abdominal pain, and hepatitis,
can signal potentially fatal liver failure (Barret, 2005). Acute aflatoxicosis in
animals was first documented in 1960, after more than 100,000 turkeys died
following an outbreak in the United Kingdom (Wu et al., 2011.)
Chronic Aflatoxicosis
Chronic aflatoxicosis is associated with long-term exposure to low to

moderate levels of aflatoxin in the food supply. Chronic low-level exposure to
aflatoxin, particularly aflatoxin B1, is associated with an increased risk of
developing hepatocellular carcinoma, or liver cancer, as well as impaired
immune function and malnutrition and stunted growth in children. Aflatoxin
B1 is the most potent liver carcinogen and is found in greater concentrations
than any other naturally occurring aflatoxin (Wu et al., 2011; Bhat, &
Vasanthi, 2003). According to the World Health Organization (WHO),
hepatocellular carcinoma is the third leading cause of cancer deaths globally
(WHO., 2008). Approximately 83 percent of cancer fatalities in East Asia and
Sub-Saharan Africa are due to liver cancer (Parkin et al., 2002; Strosnider et
al., 2006; Kirk et al., 2006). Hepatocellular carcinoma, as a result of chronic
aflatoxin exposure, presents most often in persons with a chronic hepatitis B
virus and/or chronic hepatitis C virus infections (Groopman & Kensler, 2005;
Wu & Khlangwiset, 2010; Liu & Wu, 2010). This indicates that exposure to
aflatoxin and hepatitis binfection, key risk factors for liver cancer, are
particularly prevalent in developing nations in which people subsist largely on
grains (Liu & Wu, 2010). Chronic aflatoxicosis also increases the risk of
developing impaired immune function and malnutrition, a concern already
prevalent in populations consuming high levels of cereals (Wu et al., 2011;
Bhat & Vasanthi, 2003; Bankole & Adebanjp, 2003). Cancer risk assessments
and acute toxicity studies acrossspecies show that adult humans are relatively
tolerant of aflatoxin; however, data reviewed in earlier sections indicate that
there is evidence that aflatoxin exposure affects early development, as well as
some aspects of human immunity and nutritional processes (Williams et al.,
2004). Maize (Zeamayis L.) and peanuts (Arachishypogaea L.) form the staple
food of many African and Asian diets. As these two crops are highly
susceptible to aflatoxin infection, the incidence of aflatoxin exposure is closely
related to the subsistence diet of populations in developing countries (Liu &
Wu, 2010). From 2001 to2003, developing countries produced 46 percent of
the global maize crop (Wu et al., 2011). Poor harvesting and storage practices
and weak regulations of mycotoxin contamination in developing countries
exacerbate rates of aflatoxin exposure (Wild & Gong, 2010).
Links to HIV and TB
It has been suggested that the immune suppression and nutritional effects
of chronic aflatoxin exposure may be linked to the high prevalence of HIV.
This possible link, however, is not conclusive, as research targeting the cancer-
causing effects of aflatoxin has generally overshadowed research focusing on
nutrition and immunity (Shekhar et al., 2009). Aflatoxin exposure has been
shown to cause immune suppression, particularly in cell-mediated responses
(Safarac et al., 2010).
The correlation between aflatoxin-albumin levels and CD4 counts in HIV
positive individuals has recently been studied. CD4 interacts with cells that act
as the gateway for HIV infection. CD4 proteins that have been weakened by
aflatoxin exposure may correlate positively with HIV infection (Nyandieka et
al., 2009). In addition, for the first time, new research has linked high aflatoxin
levels with an increased risk of developing tuberculosis (TB) in HIV positive
individuals. TB transmission associated with aflatoxin exposure raises a new
health concern among HIV positive individuals, in addition to concerns related
to increased susceptibility to liver disease (Allameh et al., 2005). Persons who
are exposed to aflatoxin and are HIV positive have decreased plasma vitamin
A and vitamin E in the blood, although there was no interaction detected
between aflatoxin and HIV infection (Safamehr, 2008). Nevertheless, other
mechanisms have been proposed to explain the link between HIV and
aflatoxin exposure. Williams et al. hypothesized that HIV infection is likely to
increase aflatoxin exposure by two possible routes: (1) HIV infection
decreases the levels of antioxidant nutrients that promote the detoxification of

aflatoxin, or (2) the high degree of co-infection of HIV-infected people with
hepatitis B also increases the biological exposure to aflatoxin. Although no
specific studies on humans have yet been conducted, the evidence suggests a
decrease in animal immune systems as a result of aflatoxin exposure
(Dixon et al., 2008)
Co-Existence and Interactions of Multiple Mycotoxins
Food commodities affected by aflatoxins are also susceptible to other

types of mycotoxins and multiple mycotoxins can co-exist in the same
commodity (Bankole and Mabekoje 2004; Fung and Clark 2004; Speijers and
Speijers 2004).
Various cereals affected by aflatoxins are also susceptible to
contamination by fumonisins, trichothecenes (especially deoxynivalenol),
zearalenone, ochratoxin A and ergot alkaloids. Maize can be contaminated
with aflatoxins, fumonisin, trichothecenes, zearalenone and, rarely,
ochratoxin-A, while wheat can be contaminated with aflatoxins,
trichothecenes, ochratoxin-A, ergot alkaloids and zearalenone. Therefore
individuals may be exposed to various combinations of mycotoxins (CAST
2003). The health effects associated with exposure to multiple mycotoxins are
not well documented. Related mycotoxins are thought to have an additive
effect while unrelated mycotoxins may have a synergistic effect (Speijers and
Speijers 2004). A better understanding of exposure to multiple mycotoxins and
the health effects associated with the interactions of multiple mycotoxins
would clarify the true health impact of mycotoxins.
Future Prospective
As it has been mentioned before, most aflatoxicosis results from eating

contaminated foods. Unfortunately, except for supportive therapy (e.g., diet
and hydration) there are almost no treatments for aflatoxin exposure. In Figure
4, we reproduce an overview for preventing acute aflatoxicosis in developing
countries (Strosnider et. al., 2006). Methods for controlling aflatoxin exposure
are largely prophylactic. In a primary prevention trial, the goal is to reduce
exposure to aflatoxins in the diet. A range of interventions includes planting
pest-resistant varieties of staple crops, attempting to lower mold growth in
harvested crops, improving storage methods following harvest, and using

trapping agents that block the uptake of unavoidably ingested aflatoxins. In
secondary prevention trials, one goal is to modulate the metabolism of
ingested aflatoxin to enhance detoxification processes, thereby reducing
internal dose and subsequent risk (Groopman, 2008,).
Figure 4. Overview of preparedness, surveillance and response activities for preventing

acute aflatoxicosis (Strosnider et. al., 2006).
Further, more studies and researches are needed in order to develop

appropriate technology for treatment of aflatoxin exposure and to minimize
their resulting health effects. Aflatoxins are not only a big problem at crop
production level, but also it has become a global health issue because of the
consequences that the consumption of this toxin generates in animals and
human beings. Diverse worldwide established groups have the challenge of
identifying public health strategies, which complement the agricultural ones in

order to reduce aflatoxin exposure, especially in developing countries.
Although there have been documented some researches about how to prevent
and control aflatoxicosis, but a little is known about aflatoxin exposure and the
resulting health effects. Efforts to reduce aflatoxin exposure require the
commitment of sufficient resources and the collaboration between the
agriculture and public health communities as well as local, regional, national,
and international governments.
Because of the recent investigations conducted, it is important to take
actions to prevent damage and diseases; thats why, at first, governments
supported by scientific research groups should report publicly the risks that
aflatoxins consumption means by quantifying the human health impacts and
the burden of disease due to the toxin exposure; then, they should compile
inventory and worldwide statistics in order to evaluate the efficacy of the
current intervention strategies. It is also important to increase disease
surveillance, food monitoring, laboratory detection of mycotoxins and public
health response capacity of affected regions. Public health services should
offer immediate attention to aflatoxicosis diagnoses and opportunistic diseases
caused by them in order to reduce mortality rates in humans and animals.
Finally, it is important to develop response protocols to be used in an event of
an outbreak of acute aflatoxicosis, which could become in an epidemic stage.
ACKNOWLEDGMENTS
The authors are thankful to the University Grants Commission, New Delhi
for providing financial assistance in the form of Dr. D.S. Kothari postdoctoral
fellowship and to Food and Public Health Branch of the Food and
Environmental Hygiene Department of HKSAR Government for their report.
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INDEX
age, xv, 5, 80, 85, 128, 129, 135, 138, 141,

A 142, 147, 155, 194, 202, 203, 204, 217,
237, 260, 266
access, 159, 219, 242, 251
agencies, 194, 209, 215, 219, 261
accounting, 209, 239, 270
agribusiness, xi, 108, 113
acetonitrile, 16
agricultural market, 215
acetylcholinesterase, 200
agriculture, ix, 28, 30, 32, 64, 222, 239, 267,
acid, 11, 12, 16, 23, 25, 28, 50, 51, 54, 56,
276
57, 59, 85, 133, 143, 160, 185, 186, 190,
AIDS, 100
195, 236, 245, 283
albumin, 80, 81, 94, 98, 99, 101, 103, 105,
activity level, 178
136, 137, 151, 201, 227, 270, 273, 278,
acute aflatoxin poisoning, xv, 260, 271
280
adaptation, 181
alkaloids, xv, 260, 274
additives, 83, 253
allele, 149
adenine, 151
almonds, xiv, 5, 69, 70, 76, 78, 84, 112,
adenovirus, 155
113, 121, 207, 208, 212, 220, 229, 260,
adjustment, 115
265
adolescents, 278
alveolar macrophage, 96, 103
adsorption, 11, 27
amines, 29, 58, 85, 226, 254, 280
adults, 203, 204, 266
amino, 143, 146, 147, 181, 184
adverse conditions, vii, 1
amino acid(s), 143, 146, 147
adverse effects, ix, 6, 64, 210
ammonia, 12, 200, 277, 283
advisory body, 205
amylase, 268
aetiology, 152
anatomy, 189
aflatoxicosis, xiii, 8, 26, 30, 77, 79, 88, 104,
animal disease(s), 8, 210
105, 192, 196, 198, 200, 223, 247, 267,
animal feeds, xiv, 12, 217, 222, 234, 235,
268, 269, 271, 272, 274, 275, 276, 281,
239, 247, 278
282, 283, 285
animal welfare, 214
Africa, 45, 71, 80, 88, 122, 198, 201, 227,
annealing, 132
272, 278, 280, 284, 285, 286
anorexia, 6, 79, 269
agar, 23, 57, 165, 171
ANOVA, 18
288 Index
antibiotic, 249 barriers, 110

antibody, 10, 99, 100, 131, 201 base, 30, 40, 116, 143, 146, 197, 212
antigen, 104, 129, 206, 278 base pair, 197
antioxidant, 249, 257, 274, 281 BCG immunotherapy, 156
apoptosis, 51, 151, 197, 200 BD, 150
apoptotic pathways, 96 beef, 75, 78, 217, 268
appetite, 6, 272 benefits, 120, 270
appropriate technology, 275 benign, 197
aquaculture, xv, 260, 265 beverages, 203
aqueous solutions, 14 BI, 155
ARC, 254 bias, 148
Argentina, vi, 42, 57, 78, 157, 159, 160, bile duct, xv, 260, 267
161, 162, 163, 164, 166, 181, 183, 184, bioavailability, 12, 267
185, 186, 187, 188, 189, 221 biochemical processes, 80
Argentinean peanut, xi, 157, 159, 166, 184 biochemistry, 277, 283
arthropods, 166, 185 biodiversity, 162
ascites, 79 biological activity(s), 19, 24, 182
Asia, viii, 35, 41, 71, 198, 201 biological consequences, 104, 283
Asian countries, 75 biological control, 31, 53
aspergillosis, 52 biological samples, 29
Aspergillus terreus, 57, 60 biological systems, xv, 260, 266
assessment, 32, 83, 89, 109, 113, 118, 193, biomarkers, xiii, 80, 82, 85, 86, 88, 102,
202, 204, 205, 208, 209, 227, 228, 237, 136, 143, 144, 145, 192, 222, 223
238, 252, 253, 257, 284 biomonitoring, 82
atmosphere, 45, 261 biosynthesis, 37, 45, 46, 47, 48, 49, 50, 54,
attachment, 40 55, 56, 57, 59, 61, 62, 66, 86, 102, 186,
attribution, 211 195, 198, 236, 243, 280
authority(s), xiii, 9, 112, 114, 116, 192, 209, biotic, 36
211, 214, 215, 219, 230 birds, 195, 199, 200
autopsy, 79 birth weight, 198, 224
avoidance, 4 bladder cancer, 145, 146, 153
awareness, 45, 271 bleaching, 245
bleeding, 79
blood, 80, 97, 103, 104, 136, 139, 151, 196,
B 225, 262, 273
body fluid, 80
B1 (AFB1), vii, ix, xi, 1, 3, 64, 92, 110,
body weight, 195, 206
126, 127, 159, 206, 235, 262
Bolivia, 108, 110, 114, 115, 121
Bacillus subtilis, 11
Bosnia, 219, 221
bacteria, viii, 2, 4, 11, 23, 24, 25, 27, 28, 29,
Botswana, 42
30, 32, 50, 52, 53, 59, 240
brain, 200, 226
bacterial cells, 53
brain chemistry, 200
bacterial strains, 23, 53
Brazil, v, x, 42, 58, 59, 68, 69, 76, 78, 82,
Bahrain, 78, 220
84, 107, 108, 109, 110, 111, 112, 113,
ban, 212
114, 115, 116, 118, 119, 120, 121, 122,
Bangladesh, 42
Index 289
123, 163, 181, 182, 184, 202, 203, 213, cattle, 4, 6, 8, 9, 25, 32, 38, 75, 76, 78, 79,
219, 220, 228, 282 217, 267, 268
Brazil nuts, x, 68, 69, 76, 78, 82, 84, 107, CCA, 131
108, 109, 110, 111, 112, 113, 114, 115, CD8+, 98
118, 119, 120, 121, 122, 123, 213, 220 CDC, 216, 267, 269, 271, 278
breakfast cereals, xiv, 202, 234, 236, 249, cell cycle, 96, 197
251, 252, 257 cell death, 197, 226
breast cancer, 143, 152, 153, 154 cell killing, 12
breast milk, 37, 57, 81, 225 cell line, 96, 153
breeding, 78, 217, 268 cell surface, 96, 102
Britain, 92 cellular immunity, 98
buffalo, 73, 86, 89 central nervous system, 95
Burkina Faso, 185 cerebral edema, 79, 271
businesses, 67 certificate, 111, 112
by-products, 11, 119 certification, 111, 112, 114, 118, 119, 219,
238
CFR, 26
C challenges, xv, 261, 271
cheese, 24, 76, 84, 235, 239, 240, 242, 254,
Ca2+, 226
258, 284
cacao, xiv, 260, 265
chemical(s), ix, xiv, 2, 10, 11, 12, 14, 29,
calcium, 12, 32, 268, 281
53, 64, 85, 87, 89, 155, 159, 217, 226,
calibration, 15, 17
235, 260, 262, 280
Cameroon, 38, 53
chemical stability, xiv, 260, 262
campaigns, 271
chemokines, 95
cancer, xv, 77, 79, 85, 88, 94, 111, 113, 130,
chemoprevention, 271
143, 146, 147, 151, 153, 154, 156, 197,
chemotaxis, 98, 105
208, 223, 260, 266, 270, 272, 273, 282
Chicago, 135
cancer death, 272
chicken, xv, 260, 268, 281, 282
CAP, 210
chicken embryos, xv, 260, 268
capillary, 16
childhood, 155, 198, 266
carbohydrate(s), 184, 196, 197
children, xiii, 33, 69, 71, 76, 77, 79, 81, 85,
carbon, 45, 47, 48
88, 100, 105, 192, 193, 194, 196, 198,
carbon dioxide, 45
201, 202, 203, 204, 207, 214, 221, 224,
carcinogen, ix, 3, 9, 29, 64, 79, 127, 148,
227, 239, 242, 251, 257, 266, 270, 272,
235, 263, 272
279, 285
carcinogenesis, 28, 105, 127, 195, 266, 279
China, 42, 71, 75, 84, 95, 125, 126, 127,
carcinogenicity, 31, 127, 193, 194, 198,
131, 150, 152, 154, 155, 185, 203, 219,
205, 208
221, 228, 270, 283
carcinoma, 126, 155, 156, 195, 197, 269,
chloroform, 130
272
cholesterol, 51
cardiovascular system, 250
chromatid, 266, 277
carotenoids, 151, 249, 250
chromatographic technique, 87
case study, 32, 144
chromatography, 10, 29, 37, 54, 234, 238,
catalysis, 225
265
290 Index
chromosome, 45, 143, 145, 146, 147, 277 composition, 11, 180, 181, 188, 235
chromosome map, 45 compounds, viii, ix, xiii, xiv, 2, 3, 11, 12,
chronic diseases, 198 26, 29, 35, 37, 63, 92, 181, 184, 192,
chronic fatigue syndrome, xiii, 192, 193 205, 235, 245, 259, 261
cirrhosis, xv, 129, 226, 260, 266 concordance, 17
City, 91, 125, 184, 224 conditioning, xii, 158, 171, 173
cleaning, 108, 245 confinement, 38
cleanup, 9 conflict, 120
climate(s), viii, xiii, xiv, 2, 6, 8, 18, 36, 39, conflict of interest, 120
43, 44, 45, 56, 59, 64, 66, 81, 82, 192, conformity, 110, 118
234, 239, 240, 246, 247, 255, 256, 263 confounders, 129, 135
climate change, xiii, xiv, 44, 45, 192, 234, congress, 218, 231
256 consensus, 46, 67
climatic factors, 7 consent, 129
cloning, 62 conservation, x, 107, 108, 110
clusters, 46, 48, 49, 60 constituents, 29, 58, 85, 245, 254, 255, 280
cocoa, 81, 203 consumers, xi, 8, 108, 110, 113, 119, 194,
coding, 112, 143, 146, 152 203, 204, 207, 208, 209, 210, 211, 215,
codon, 131, 138, 139, 143, 144, 147, 148, 239
149, 152, 154, 156, 197, 222 consumption patterns, 207
coffee, 256 containers, 239
cognitive development, 198 contaminant, vii, viii, 1, 8, 35, 41, 127, 168,
cognitive function, 200 205, 212, 216
collaboration, 219, 276 contaminated food, ix, xiii, xv, 5, 11, 63, 92,
colleges, 48 113, 192, 194, 208, 260, 266, 272, 274
colonisation, 40, 41, 49 control group, 249
colonization, 166, 172, 187, 189, 242 control measures, 237
color, 11, 109 controversial, 199
colorectal cancer, 153, 154, 156 conversion rate, 6
coma, 79, 271 cooking, 159
commerce, 215 cooperation, 194
commercial, 8, 12, 25, 30, 73, 159, 207, coordination, 54, 58, 200
239, 240, 244, 246, 247, 255, 256 copper, 12
commodity, xv, 108, 113, 119, 202, 204, correlation, 93, 101, 138, 147, 174, 178,
208, 250, 251, 260, 274 179, 183, 198, 201, 245, 273
communication, 50, 211 cost, 9, 10, 11, 45, 70, 238, 286
community(s), xii, 44, 158, 163, 172, 185, Costa Rica, 33, 59
264, 265, 276, 285 cotton, viii, xiv, 5, 35, 39, 40, 56, 58, 185,
compatibility, 188 251, 260, 264
competition, 11, 66, 174, 245, 249 coumarins, 127, 245
compilation, vii covalent bond, 93
complement, 82, 200, 276 covering, 8
complexity, xi, 107 CPT, 195
compliance, 119, 194, 209, 215, 216, 218, cracks, 41
219, 230 crises, 247
Index 291
Croatia, 1, 13, 18, 19, 22, 23 denaturation, 131

crop(s), viii, xiii, xiv, 3, 4, 6, 7, 31, 35, 36, Department of Agriculture, 216
37, 38, 39, 40, 41, 44, 49, 52, 53, 59, 62, Department of Health and Human Services,
66, 67, 73, 116, 159, 163, 164, 166, 189, 231
192, 194, 236, 252, 255, 256, 260, 263, depth, 8
264, 273, 274, 275, 280, 282 derivatives, 64, 80, 81, 202, 242
crop production, 275 destruction, 19, 201, 217
crop residue, 67 detectable, 81, 100, 118, 145, 206, 207, 240,
cross sectional study, 198, 224, 279 248
CT, 133, 137, 138, 140, 141, 144, 149, 155 detection, x, 9, 10, 13, 15, 18, 25, 29, 31,
cues, 47 34, 81, 86, 87, 107, 131, 132, 238, 239,
cultivars, 181, 190, 257 240, 241, 242, 244, 248, 251, 253, 254,
cultivation, vii, xiv, 2, 4, 22, 67, 163, 164, 255, 257, 265, 276
202, 203, 234, 236, 250 detection system, 131, 132
cultural conditions, 48 detention, 219
cultural practices, 53, 164 detoxification, xiii, 5, 10, 11, 25, 29, 81,
culture, 45, 57, 165, 176, 225, 244 192, 195, 274, 275, 283
culture conditions, 165 developed countries, 81, 116, 117, 220
culture media, 244 developing countries, xv, 2, 33, 57, 79, 81,
culture medium, 176 87, 88, 105, 117, 119, 193, 209, 210,
current limit, 119 220, 223, 260, 261, 265, 266, 270, 271,
CV, 14 273, 274, 276, 279, 285
cycles, 131 developing nations, 272
cytochrome, xi, 93, 98, 126, 197 diarrhea, 79, 199
cytokines, x, 92, 95, 96, 98 diet, 2, 80, 97, 99, 194, 201, 202, 203, 204,
cytoplasm, 22 222, 225, 236, 240, 242, 244, 266, 270,
cytotoxicity, 263, 278 273, 274
Czech Republic, 200 dietary habits, 202, 204
dietary intake, 80, 89, 206
digestibility, 199, 225, 267
D digestion, 130, 195, 268
digestive enzymes, 197, 199, 267, 282
dairy industry, 239
disease progression, 101
dairy products, xiv, 8, 9, 32, 38, 60, 75, 203,
disease rate, 100
234, 235, 239, 242, 251, 286
diseases, xv, 6, 36, 38, 53, 80, 98, 101, 198,
database, 81, 143, 146, 208
201, 210, 223, 224, 260, 270, 271, 276
deaths, 57, 66, 84, 94, 262
disorder, 204
decay, 21, 71
dispersion, 280
decontamination, 31, 32, 218
displacement, 271
defects, 50
disposition, 226
defence, 52
distillation, 11
defense mechanisms, ix, 64
distilled water, 22
deficiency(s), 146, 197, 215
distribution, 8, 65, 129, 132, 138, 160, 194,
degradation, 4, 11, 12, 19, 49, 286
204, 207, 239, 262
degumming, 245
diversity, 172, 265
demographic data, 129, 135
292 Index
DNA damage, xi, 102, 126, 127, 139, 147, energy, 5, 6, 159
150, 151, 226, 266 enforcement, 81, 194, 214, 215, 216
DNA ligase, 143, 152 engineering, 7
DNA polymerase, 152 England, 3, 193, 262
DNA repair, xi, 93, 126, 127, 128, 132, 133, environment(s), 6, 36, 45, 53, 65, 109, 166,
137, 138, 139, 140, 141, 143, 145, 146, 183, 214, 261
147, 148, 150, 153, 154, 155, 156 environmental conditions, viii, 33, 36, 39,
DNA-adducts, xv, 139, 260, 266 43, 48, 52, 60, 160, 165, 183, 248
DNase, 268 environmental factors, ix, 5, 25, 47, 63, 66,
documentary evidence, 111 187, 235
dogs, 5, 267 environmental protection, 110
dominance, 174 environmental stress, 48, 183
dopamine, 200 enzyme(s), xi, 10, 94, 98, 99, 126, 130, 197,
dosage, 97, 248, 269 198, 200, 225, 226, 238, 268, 277
dosing, 97, 103 enzyme-linked immunosorbent assay, 130
dough, 228 epidemic, 276
draft, 120 epidemiologic studies, 80
drought, 6, 7, 39, 40, 52, 56, 66, 67, 71, 165, epidemiology, 88, 103, 280
166, 185, 197, 264 epilepsy, 200
drugs, 55, 215 epithelial cells, 283
drying, xii, 6, 11, 39, 58, 66, 67, 74, 108, epithelium, 199
130, 158, 159, 171, 246, 250, 264, 279 equipment, 10, 244
ethanol, 22, 130
ethnicity, 129
E eukaryote, 59
Europe, 71, 72, 102, 110, 113, 194, 200,
East Asia, 272
203, 209
ecology, 39, 41, 47, 61, 62
European Commission, 70, 113, 114, 207,
economic losses, 116, 262
210, 211, 229, 230, 237, 255, 266, 279
economic problem, viii, 2, 10
European Community, 12, 110, 246
economics, ix, 64
European Parliament, 27, 28, 83, 253
ecosystem, xi, 157, 180
European policy, 110
edema, 79, 196, 272
European Social Fund, 82
education, 271
European Union, ix, x, 8, 27, 63, 64, 67, 68,
egg, 6, 79
69, 70, 74, 82, 107, 113, 121, 122, 159,
Egypt, 42, 83, 244, 276
185, 205, 210, 229, 248, 253, 266, 279
electron, 224
evaporation, 263
ELISA, vii, 2, 4, 9, 13, 14, 16, 17, 18, 21,
evidence, xiv, xv, 8, 79, 80, 81, 82, 94, 127,
24, 26, 30, 34, 130, 234, 238, 240, 241,
129, 148, 198, 199, 205, 206, 215, 222,
247, 255
234, 260, 270, 273, 274
ELISA method, 13, 14, 16, 21, 24, 241
evolution, 29, 195
elucidation, 139, 148
examinations, 129
e-mail, 259
excision, 143, 146, 156
encephalopathy, 200, 226
exclusion, 209
encoding, 143
excretion, 34, 248
endocrine, 6
Index 293
exons, 143, 145, 146, 147 fluorescence, 10, 86, 193, 244, 249, 251,
expertise, 244 255
exploitation, 110 food additive(s), 223
export control, 82, 214 Food and Drug Administration, xiii, 192,
export market, 109 209, 215, 231
exporter(s), 119, 214, 231, 242 food chain, xii, xv, 28, 59, 66, 67, 84, 158,
exports, 109, 110, 118, 209 202, 211, 235, 237, 261, 271
expressed sequence tag, 62 food industry, 194, 209
extraction, 10, 11, 16, 57, 110, 129, 130, food intake, 77
238, 244, 245, 249, 265 food production, viii, 2, 10, 67, 81, 211,
extracts, 15, 26, 256 248, 261
extrusion, 224 food products, 12, 28, 84, 202, 203, 207,
214, 218, 238, 246
food safety, vii, x, xiii, 1, 2, 8, 30, 45, 64,
F 74, 83, 88, 119, 122, 194, 209, 210, 211,
216, 218, 220, 224, 229, 231, 233, 237,
farms, 3, 7, 58, 241, 247, 248
239, 248, 249
FASAY, 151
food security, 210, 220
fat, 2, 198, 242
foodborne illness, 216, 218
fatty acids, 49, 51, 52, 55, 56, 186, 242, 250
force, 8, 212
FDA, xiii, 5, 28, 192, 215, 216, 217, 218,
formation, viii, xi, xv, 2, 3, 18, 19, 25, 48,
219, 231, 268
52, 56, 60, 62, 93, 98, 126, 127, 139,
feces, 92
144, 159, 188, 197, 236, 260, 266
federal agency, 215
formula, 76, 221, 262, 264
feedstock, 5
France, 29, 58, 85, 202, 203, 254, 280
feedstuffs, 8, 9, 75, 78, 194, 209, 214, 268
free radicals, 19, 104, 195
fermentation, 11, 204, 240
freezing, 66
fetal development, 257
fruits, ix, xiv, 63, 66, 69, 77, 81, 88, 108,
fetus, 201
109, 120, 159, 202, 238, 245, 260, 267,
fever, 79, 269, 272
280, 284
fibers, 250
Functional Analysis of Separated Alleles in
fibroblasts, 146, 155
Yeast, 151
filament, 144
functional food, 119
financial, 101, 150, 276
funding, 219
financial support, 101
fungal infection, 3, 57
fish, xv, 6, 260, 265, 268
fungus, xii, 36, 39, 40, 41, 47, 49, 53, 54,
flagellum, 224
60, 64, 92, 163, 165, 173, 181, 183, 189,
flavonoids, 249, 250, 257
192, 197, 199, 240, 262
flavor, x, 107, 108
furan, 19
floods, 261
fusion, 127
flora, 5, 25
flotation, 11
flour, 43, 73, 77, 86, 87, 182 G
flowers, 64, 159
fluid, 30, 45, 238 gamma radiation, viii, 2, 26, 33, 54
fluid extract, 238 gastrointestinal tract, 225, 262
294 Index
gene expression, 47, 99, 195, 286 haplotypes, 154

gene regulation, 50 harmful effects, viii, 2, 8, 25
genes, vii, ix, xi, 36, 45, 46, 47, 48, 49, 50, harmonization, 74, 222
53, 61, 62, 126, 127, 128, 132, 133, 137, harvesting, xiv, 3, 4, 6, 7, 10, 53, 74, 159,
138, 139, 140, 141, 148, 150, 151, 153, 234, 238, 245, 256, 273
155, 163, 195 hazards, vii, xiv, 30, 31, 71, 199, 204, 210,
genetic defect, 199 235, 237, 258, 260, 265, 266
genetic engineering, 53 HBV, 94, 101, 128, 129, 135, 136, 138, 141,
genetic mutations, 148 142, 156, 206, 270
genetics, vii, ix, 36, 45, 46 HBV infection, 94, 101, 138
genome, vii, viii, 36, 46, 53, 57, 102 HCC, xi, xv, 93, 94, 101, 126, 127, 128,
genomics, 62 129, 130, 132, 135, 136, 137, 138, 139,
genotype, 61, 132, 135, 138, 139, 263 141, 143, 144, 145, 146, 147, 148, 149,
genotyping, 132 197, 260, 267, 269, 270
genus, vii, ix, xii, 1, 3, 18, 42, 63, 64, 71, health care, 37, 116
158, 168, 171, 172, 243, 262 health effects, x, 64, 68, 81, 193, 194, 195,
Georgia, 61 201, 204, 211, 215, 224, 226, 238, 242,
Germany, 13, 14, 15, 188 251, 270, 271, 274, 275
germination, 19, 21, 40, 159, 163, 182, 256 health problems, 237, 266
gerontology, 155 health risks, 74, 211, 237, 266
ginger, xiv, 69, 92, 203, 260, 264 health services, 276
gland, 262 hematemesis, 79
global scale, 246 hemorrhage, 79
globalization, 209, 239 hepatic encephalopathy, 281
glutamate, 200, 226 hepatic failure, 269
glutathione, 151, 225, 284 hepatic necrosis, xv, 260, 266
government intervention, 215 hepatitis, 6, 79, 80, 82, 103, 129, 152, 206,
governments, 108, 119, 207, 276 207, 227, 269, 270, 272, 274, 278, 281,
grants, 184 282, 284
Great Britain, 188, 189 hepatitis a, 79
Greece, vi, xiii, 191, 233, 235, 236, 237, hepatitis b, 272
238, 239, 240, 242, 243, 244, 245, 246, hepatitis c, 207, 282
247, 248, 249, 250, 251, 255, 256, 257 hepatocarcinogen, 92
green olives, xiv, 234, 236, 254 hepatocarcinogenesis, xv, 103, 260, 284
growth rate, 6, 21 hepatocarcinoma, ix, 64
Guangdong, 147 hepatocellular carcinoma, xi, xv, 6, 33, 57,
guanine, 93, 99, 102, 103, 151, 197 80, 82, 93, 94, 105, 126, 127, 128, 149,
guidance, 194, 215, 216, 230 151, 152, 154, 155, 156, 197, 260, 267,
guidelines, 210 271, 272, 276, 282, 284, 285
Guinea, 284 hepatocytes, 79, 152, 196, 222, 254
Guyana, 108 hepatoma, 267
hepatotoxicity, 79, 222, 263, 269
hepatotoxins, xv, 260, 268
H herbal medicine, 186, 280
heterogeneity, 102
half-life, 21, 80
Index 295
heterozygote, 135 impairments, 6

histone(s), 46, 49, 99 import restrictions, 113
history, 129, 164, 211, 225 imported products, 114, 219
HIV, xv, 80, 98, 100, 101, 260, 270, 273 imports, 71, 112, 209, 214, 250
HIV/AIDS, xv, 80, 260, 270 improvements, 3
homeostasis, 95, 96, 199 in utero, 81
homozygote, 137, 138, 144, 145, 149 in vitro, 21, 23, 95, 96, 97, 98, 100, 104,
hormone, 198 189, 193, 206, 278, 281
host, ix, x, 36, 38, 49, 50, 53, 54, 56, 58, 92, in vivo, 23, 95, 97, 100, 104, 206, 263
101, 266 incidence, ix, xv, 2, 64, 70, 71, 73, 79, 80,
human body, 195, 202 82, 94, 101, 110, 114, 118, 147, 160,
human exposure, 68, 80, 88, 201, 204, 241 163, 171, 172, 180, 181, 182, 186, 196,
human health, viii, 8, 9, 10, 35, 38, 87, 212, 206, 260, 270, 273
214, 215, 223, 224, 235, 237, 239, 249, income, 70
258, 262, 270, 276 incubation period, 172, 180
human immunodeficiency virus, 103 incubation time, 57
humidity, 2, 7, 8, 18, 45, 56, 65, 67, 109, independent variable, 135
173, 202, 243, 247, 250, 261 India, 33, 43, 77, 182, 189, 203, 220, 221,
humoral immunity, x, 91, 100, 201 223, 230, 259, 281
hydrogen, 19, 95 indirect effect, 109
hydrogen peroxide, 19, 95 individuals, x, 6, 92, 98, 101, 138, 145, 206,
hydrolysis, 93 265, 273, 274
hydrophobicity, 55 Indonesia, 76, 173
hygiene, 111, 119, 246 inducer, 49, 51, 55
hyperplasia, xv, 260, 266 induction, ix, 61, 64, 77
industry(s), 11, 25, 88, 110, 121, 186, 211,
216, 238, 241
I infancy, 266
infants, 37, 69, 76, 77, 214, 221
icterus, 196
infection, x, 7, 36, 40, 41, 44, 55, 56, 58, 88,
ID, 103, 123, 145, 146, 231
92, 94, 96, 98, 100, 101, 129, 135, 138,
identification, 46, 53, 62, 111, 168, 193,
156, 163, 164, 165, 166, 173, 201, 206,
204, 223, 265
227, 268, 272, 273
IFN, 95, 97
infectious agents, 80
immune function, 33, 105, 227, 272, 285
infertility, 198, 224
immune response, x, 92, 95, 100, 103
infestations, 261
immune system, 6, 82, 101, 199, 266, 268,
inflammation, x, 92
274
informed consent, 129
immunity, x, xiii, 79, 91, 99, 100, 192, 193,
infrastructure, 119
194, 195, 200, 201, 225, 267, 270, 273
ingest, 262
immunoglobulin, 153
ingestion, ix, 4, 6, 63, 64, 78, 79, 193, 204,
immunomodulatory, x, 91, 100
225
immunosuppressants, xv, 260, 268
ingredients, 8, 38, 54, 78, 217, 247, 264
immunosuppression, x, xv, 91, 101, 260,
inhibition, 19, 22, 23, 198, 255, 256
266, 270
initiation, 7
impaired immune function, 272
296 Index
injury, 172 kidneys, 79

inoculation, 39, 41, 244 kinetic parameters, 225
inoculum, 3, 39, 40, 41, 59, 163, 168, 183, Korea, 76, 77
186 Kuwait, 78, 84, 220
insects, 6, 36, 40, 44, 182, 187
insomnia, 200
inspections, 216 L
integrity, 112
lack of confidence, 117
inter kingdom communications, ix, 36
lactation, 83, 89, 262
interference, 36, 193, 200, 266, 270
lactic acid, viii, 2, 4, 11, 23, 24, 27, 28, 29,
international standards, 110
32, 240
international trade, 68, 108, 210
Lactobacillus, 23
intervention, 115, 116, 276, 285
laws, 216
intervention strategies, 276
LC-MS/MS, vii, 2, 4, 13, 15, 16, 17, 54,
intoxication, 6, 116, 195
123, 234, 238, 244
introns, 143, 145, 146, 147
lead, 3, 4, 41, 49, 53, 66, 79, 100, 143, 151,
investment, 119
155, 197, 198, 211, 242, 243, 245, 261,
ionization, 12, 16
270
ionizing radiation, 30, 33
learning, 200
ions, 17
legislation, vii, 32, 67, 74, 111, 113, 114,
Iowa, 278
115, 182, 194, 210, 211, 214, 215, 219,
Iran, 37, 60, 116, 122, 199, 252, 253, 256
230, 249, 258
Ireland, 91, 101
legs, 79
irradiation, 11, 12, 19, 20, 21, 27, 31
Lepidoptera, 61
irrigation, 52, 56
lesions, 93
ischemia, 226
leukemia, 155, 156
isolation, xii, 41, 157, 160, 163, 168, 171,
leukocytes, 136, 137
176, 238
light, 12, 47, 48, 54, 61, 82, 85, 92, 200,
issues, 8, 210, 216, 219, 220, 225, 266
215, 224, 262
Italy, 28, 45, 73, 87, 191, 222, 228, 241,
linoleic acid, 51, 57, 236
242, 244, 255, 256
lipases, 197, 236
lipid oxidation, 236
J lipid peroxidation, 198
lipids, 79, 249
Japan, xiii, 58, 61, 76, 155, 192, 203, 220, liquid chromatography, vii, 2, 4, 10, 34, 54,
228 73, 86, 234, 238, 244, 252, 255, 256,
jaundice, 79, 269 257, 265, 280
justification, 110 liver cancer, ix, xv, 5, 64, 78, 80, 82, 85,
102, 128, 151, 152, 197, 205, 206, 207,
222, 223, 260, 266, 272, 279, 280, 283
K liver cells, 196
liver cirrhosis, ix, 64, 135, 196
Kenya, 30, 57, 58, 71, 73, 78, 83, 84, 86,
liver damage, ix, xv, 64, 79, 80, 195, 260,
166, 182, 183, 187, 196, 199, 221, 223,
266, 267, 271, 272
271, 278, 282
liver disease, 129, 148, 273
Index 297
liver failure, 197, 272 materials, 7, 12, 13, 19, 21, 28, 62, 66, 237
livestock, xiii, xv, 6, 65, 192, 260, 264, 268, matrix(s), 15, 266, 280
278 MB, 155
local government, 129 measurement(s), 15, 25, 31, 80, 81
localization, 29 meat, xiv, 4, 23, 26, 29, 30, 74, 83, 85, 86,
loci, 128 216, 260, 264, 267
logistics, 67 media, 24, 28, 44, 227
longitudinal study, 85, 224, 279 median, 196
Louisiana, 125 medical, 69, 73, 81, 129, 221, 249
lovastatin, 49, 51, 57, 60 medical history, 129
low risk, xi, 108 medicine, 154
LSD, 178 Mediterranean, 236, 237, 240, 241, 242,
lung cancer, 80, 153 243, 244, 245, 246, 247, 251, 253
lymph node, 155 Mediterranean countries, 237, 240, 241,
lymphocytes, 95, 97, 99, 101, 102, 103, 146 243, 244, 245, 251
lymphoid, 99 melon, 43
lysine, 93, 99 memory, 200
lysis, 100, 198 meristem, 40
meta-analysis, 144, 152, 153, 154
metabolic, 94
M metabolic disorder(s), 30
metabolism, ix, xiii, 31, 36, 48, 50, 52, 64,
Macedonia, 191, 233
85, 103, 148, 192, 193, 195, 196, 198,
macrophages, xv, 95, 96, 100, 102, 104,
206, 254, 268, 275, 279, 280
200, 260, 268, 281
metabolites, vii, ix, x, xii, 1, 3, 5, 11, 38, 47,
magnitude, 196
51, 63, 64, 80, 81, 91, 92, 94, 96, 99,
majority, 194, 204, 238, 244, 248, 266
102, 105, 123, 158, 159, 174, 192, 193,
malaise, 79, 269, 272
196, 235, 262, 264
malaria, xv, 80, 199, 201, 227, 260, 270
metabolized, xi, 98, 126, 197, 239
Malaysia, 43, 76, 200, 281
metals, 48
malignancy, 197
metastasis, 129, 155
malignant tumors, 143, 145, 146
methanol, 14
malnutrition, xv, 80, 116, 197, 199, 260,
methodology, 8, 180, 204, 205, 218
266, 270, 272
Mexico, 39, 58, 71, 78, 219, 221
mammalian cells, 154
mice, 98, 151, 198
mammals, 3
microbiota, 60, 79, 199, 249
management, 39, 43, 57, 88, 117, 119, 150,
micronutrients, xiii, 192, 193
218
microorganism(s), 6, 11, 21, 37, 172
manufacturing, 3, 74, 114, 118, 221
microscopy, 61
market access, 183
minicolumn, 265
marketing, 81, 110, 267
mission, 110, 111, 112
marketing strategy, 110
Missouri, 40, 286
mass, vii, 2, 4, 10, 16, 54, 87, 234, 238, 244,
misuse, 222
252
mitochondria, 198
mass spectrometry, vii, 2, 4, 10, 16, 54, 87,
mitosis, 197
234, 238, 244, 252
298 Index
modelling, 44, 208 native population, 163

models, 43, 103, 267 near infrared spectroscopy, 253
modifications, xiv, 46, 49, 234 nebulizer, 16
moisture, 6, 7, 11, 13, 31, 36, 44, 67, 70, 71, necrosis, xv, 79, 196, 197, 260, 267
159, 173, 182, 186, 187, 243, 246, 247, neonates, 239
261, 282 Nepal, 43
moisture content, 7, 11, 13, 31, 44, 70, 159, nerve, 200
173, 246, 282 nervous system, 200
mold(s), 31, 60, 83, 87, 89, 159, 182, 184, Netherlands, 72, 187, 189, 209
197, 226, 243, 244, 245, 247, 249, 257, neurodegeneration, 226
258, 274 neurogenesis, 146
molecular weight, 23 neurons, 146
molecules, ix, 36, 37, 51, 52, 53, 94, 96, 99, neurotoxicity, 193
101 neurotransmitter(s), 200
monoclonal antibody, 29, 30, 130 neutrophils, 97, 104
Moon, 95, 96, 104 New Zealand, 78, 200, 203, 219, 220
morbidity, 198 Nigeria, 26, 43, 78, 219, 220, 224, 230, 256,
Morocco, 243, 244, 247, 258 277
morphogenesis, 50 NIR, 257
morphology, 51, 52, 151, 189, 198, 199, nitric oxide, 95, 104
283 nitrite, 83
mortality, 198, 272, 276 nitrogen, 45, 48, 56, 129
mortality rate, 272, 276 NMDA receptors, 200
motif, 46 nodes, 129
mould spores, 21 North Africa, 245
Mozambique, 95 North America, 71, 81
MR, 101, 105, 156 nucleoprotein, 144
mRNA, 95, 97, 98, 102, 156 nucleus, 166
multilateralism, 210 nutrient(s), 6, 11, 33, 36, 40, 159, 174, 199,
mung bean, 77, 220 225, 267, 268, 270, 274
mutagenesis, 48, 156 nutrition, 2, 82, 198, 199, 211, 222, 253,
mutant, xi, 50, 52, 126, 132, 137, 139, 146, 273
148 nutritional deficiencies, 266
mutation(s), xi, 12, 93, 102, 105, 126, 127, nutritional status, xv, 5, 260
128, 131, 135, 138, 139, 143, 145, 146,
147, 148, 149, 151, 152, 153, 156, 197,
222, 223, 266 O
mycelium, 22, 168, 170, 171
officials, 267
mycology, 61
oil, xiv, 22, 32, 43, 75, 89, 119, 242, 243,
myeloid cells, 99
244, 245, 246, 260, 265, 280
oil samples, 243, 244
N oilseed(s), viii, xiv, 27, 35, 39, 49, 60, 66,
68, 69, 213, 230, 235, 260, 264, 266
naphthalene, 186 oleic acid, 49
native isolates, 163
Index 299
olive oil, 236, 238, 242, 243, 244, 245, 246, phenolic compounds, 245, 252
251, 252, 253, 254, 256, 257 phenotype(s), xii, 50, 157, 160, 164, 271
operations, 3 Philippines, 39, 41, 43, 58, 60
optimization, 245 phosphate, 14, 131
organ(s), 79, 127, 195, 197, 210, 225, 226, phosphorus, 268
283 physical properties, 171, 178, 264
organism, 48, 196 physiology, 49
ox, xiv, 49, 52, 259, 261 pigs, 5, 26, 75, 76, 79, 96, 99, 100, 227, 267
oxidative stress, 154 pistachios, ix, 5, 30, 63, 66, 68, 69, 70, 71,
oxygen, 45 76, 78, 82, 84, 92, 113, 116, 121, 202,
oxylipins, ix, 36, 49, 50, 51, 52 207, 208, 212, 220, 222, 229, 235, 250
ozone, 123 placenta, 139, 151, 201
placental barrier, 199
plant growth, 40, 190
P plants, viii, 4, 26, 32, 35, 36, 37, 40, 41, 42,
44, 49, 50, 53, 54, 58, 64, 73, 87, 159
p53, 127, 138, 139, 143, 151, 152, 197, 222,
plasma membrane, 198
223
platinum, 153
pain, 79, 196, 269, 271, 272
point mutation, 153
Pakistan, 58, 73, 83, 86, 87, 89, 281
poison, 262
parasite(s), 4, 80
Poland, 173, 184
parasitic infection, 200
polarity, 16
participants, 98
policy, 210, 211, 215, 216, 218, 220
pasta, 72
pollen, 40, 236, 238, 249, 252, 256
pasteurization, 38
pollutants, 257
pasture, 8, 38
polymerase, 149, 195
pathogenesis, ix, 36, 49, 54, 280
polymerase chain reaction, 149
pathogens, 36, 50, 53, 86
polymorphism(s), xi, 126, 127, 132, 133,
pathology, 32, 280
138, 143, 144, 145, 146, 147, 148, 150,
pathways, 49, 52, 99, 266
151, 152, 153, 154, 155, 156, 284
PCR, 87, 131, 132, 133, 134, 138, 147, 149,
polyphenols, 242
174, 180, 188, 195
polypropylene, 173
peanut meal, 3, 12, 19, 33, 75, 193
population density, 52
penicillin, 49, 51
population group, xiv, 207, 234
peptide, 53
population growth, 261
peripheral blood, 128, 129, 130, 136, 137,
population structure, 188
139
Portugal, 107, 242, 253, 255, 281
peripheral nervous system, 200
positive correlation, 44, 179, 183
peroxide, 14, 245
positive relationship, 40
Peru, 108
poultry, xv, 3, 6, 74, 78, 79, 86, 201, 217,
pesticide, 53
247, 254, 256, 258, 260, 264, 268, 286
pests, 61, 261
poverty, 110
pH, 14, 23, 44, 47, 48, 59, 66, 67, 92, 130,
poverty reduction, 110
131, 171, 240
pregnancy, 228
phagocytosis, x, 92, 98, 100, 200
preparation, 13, 81
phenol, 130
300 Index
preparedness, 275
present value, 240, 251
R
preservation, 245
race, 128, 129, 135, 138, 141, 142
prevention, viii, 2, 10, 19, 25, 28, 32, 33, 85,
radiation, 4, 12, 19, 21, 25, 26, 28, 89
121, 148, 152, 218, 274, 280, 284
radicals, 19
principles, 118, 211, 212, 229
rain forest, 108
private sector, 116
rainfall, 38, 160, 164
probability, xi, 44, 157, 204, 205, 228
rainforest, 108
probe, 131, 132
raw materials, 4, 247
probiotic, 23, 24, 29
RE, 103
producers, 36, 37, 42, 44, 70, 108, 109, 118,
reactions, 151, 195
162, 164, 165, 168, 176, 179, 182, 211,
reactive oxygen, 96, 105
216, 218, 250
reagents, 9
productive efficiency, 26
reasoning, 194
progenitor cells, 97
recall, 219
pro-inflammatory, 95, 100
recognition, 147
proliferation, xv, 21, 89, 97, 195, 260, 261,
recombination, 144, 153, 154, 155
267
recovery, 9, 13, 14, 15, 16
promoter, 46
recurrence, 156
prophylactic, 274
regions of the world, 8, 209, 235
prostate cancer, 152, 155
regression, 135, 139
protection, xi, 22, 25, 52, 108, 111, 113,
regression analysis, 139
194, 211, 218, 250, 261
regression model, 135
protein synthesis, 6, 198, 199, 200
regulations, vii, x, 8, 28, 64, 70, 74, 79, 82,
proteinase, 130
87, 88, 113, 119, 159, 182, 194, 204,
proteins, xv, 47, 48, 49, 52, 93, 99, 146,
209, 210, 214, 216, 218, 219, 237, 249,
196, 250, 260, 266, 273
273
public concern, 53
regulatory framework, xiii, 192
public health, xv, 32, 84, 111, 112, 113,
rejection, 115, 116, 119
116, 121, 194, 205, 208, 209, 212, 214,
relative size, 144
215, 219, 221, 246, 260, 267, 269, 270,
relaxation, 113
271, 276, 285
relevance, 201
pulmonary edema, 79, 271
reliability, 112
purification, 244
repair, xi, 102, 126, 127, 138, 139, 143, 145,
P-value, 135
146, 148, 149, 150, 151, 152, 154, 155,
pyridoxine, 197
156
repression, 60
Q reproduction, 52, 58, 278
requirements, 10, 199, 211, 229
quality control, 122, 131, 132, 237 researchers, 42, 139, 147, 173
quality standards, 218, 248 residues, xiv, 12, 53, 93, 99, 146, 203, 227,
quantification, 9, 10, 17, 87, 180, 188, 238, 234, 238, 240, 241, 242, 251, 257
241, 265 resistance, 53, 55, 56, 83, 163, 181, 184,
questionnaire, 129, 130 278
resolution, 10
Index 301
resources, 116, 205, 261, 271, 276 security, 27, 210

respiration, 6 seed, viii, 19, 35, 39, 40, 49, 51, 52, 54, 58,
response, 77, 95, 96, 97, 99, 101, 103, 104, 61, 108, 115, 159, 167, 170, 172, 173,
114, 154, 155, 201, 205, 208, 211, 225, 175, 178, 180, 181, 185, 187, 189, 230,
275, 276 245, 251
response capacity, 276 seeding, 164
restrictions, 112 selectivity, 186
retail, 211, 244 selenium, 119, 151, 250
retardation, 267, 270 semen, 198
reticulum, 195 sensations, 200
retina, 226 sensing, ix, 36, 48, 50, 51, 52, 53, 60, 61
RH, 173, 250 sensitivity, 10, 98, 199, 244
Rhizopus, 168, 169, 170, 171, 173, 182 sequencing, vii, viii, 36, 46, 132
risk assessment, vii, xiii, 8, 113, 118, 122, Serbia, 203, 228
192, 194, 204, 205, 208, 210, 228, 235, serine, 94, 99, 102
238, 241, 242, 251, 257, 273 serotonin, 200, 226
risk factors, 255, 269, 272 serum, 80, 81, 98, 105, 129, 136, 201, 206,
risk management, xiii, 192 267, 268, 271, 277, 283
risk perception, 220 services, 231
RNA, 195, 200, 266 sex, 80, 128, 129, 135, 138, 141, 142, 147,
rodents, 6, 182 151, 195, 198
room temperature, 250 sexual development, 55
roots, 40 SGOT, 268
routes, 273 SGPT, 268
rubber, 110 sheep, 4, 24, 79, 241, 248, 252
rules, 212, 214, 219, 230 showing, 127, 163, 171, 172, 175, 214, 265
sibling, 59, 87
signalling, ix, 36, 47, 49, 50, 52, 94, 99, 101
S signals, 50, 92
signs, 79, 267, 271
safety, xiv, 12, 67, 74, 119, 120, 209, 210,
silk, 39, 40, 41
211, 212, 215, 218, 221, 231, 234, 239,
Singapore, 75
245, 246, 251
sister chromatid exchange, 283
saliva, 201
Slovakia, 200
salts, 283
small intestine, 193, 222
samplings, 175, 178, 179
SNP, 143, 146, 153
Saudi Arabia, 25, 78, 220
socioeconomic status, 198
schistosomiasis, 151
sodium, 12, 32, 281
schizophrenia, 156
software, 15
school, 71
soil type, 263
science, 210, 223, 267
solution, xv, 13, 15, 16, 19, 23, 119, 131,
scope, 10, 211, 216
261, 271
secondary metabolism, vii, ix, 36, 45, 46,
solvents, 10, 11, 238
47, 48, 49, 50, 53, 54, 55, 58, 60, 62
South Africa, 71, 78, 220, 221, 228
secretion, x, 92, 95, 96, 97, 98, 100, 102,
South America, 39, 71
201
302 Index
sowing, xii, 158, 160, 165, 186 suppliers, 211, 219

soy bean, 8, 127 supply chain, 27
soybeans, 77 suppression, xv, 6, 52, 80, 260, 267, 269,
SP, 152, 155, 189 270, 271, 273
Spain, 63, 72, 82, 88, 89, 202, 228, 241, surface component, 24
242, 243, 244, 250, 252, 254 surveillance, 207, 250, 271, 275, 276
specific gravity, 109 survival, 22, 26, 184, 279
spectroscopy, 234, 238 susceptibility, ix, x, 5, 6, 30, 64, 80, 92, 100,
sperm, 198, 224 104, 143, 147, 152, 153, 154, 155, 200,
spleen, 97 265, 267, 271, 273, 284
spore, 7, 50, 71 sustainability, 122, 214
SS, 105, 156 sustainable development, 210
stability, 84, 240, 254, 279 Sweden, 13, 72
stakeholders, 70, 267 Switzerland, 33, 219
standard deviation, 13, 17 symptoms, xiii, 6, 79, 192, 197, 198, 200,
standard error, 177, 178 268, 269, 271, 272, 283
starvation, 57, 79, 84 syndrome, 80, 268
state(s), 8, 9, 32, 47, 58, 70, 116, 184, 243, synergistic effect, 96, 256, 274
271 synthesis, vii, ix, 36, 46, 47, 81, 185, 195,
statistics, 276, 282 198, 200, 242
stereospecificity, 225
sterigmatocystin, ix, 36, 38, 46, 47, 48, 49,
50, 55, 86, 280 T
sterile, 12, 56, 168, 172
T cell(s), 97, 98, 100, 201
stimulation, 96, 97
T regulatory cells, 98
stress, xii, 39, 52, 56, 66, 71, 158, 165, 166,
Taiwan, 155, 278, 284, 285
168, 264
tannins, 189
stressors, 5
target, xv, 3, 10, 48, 79, 100, 195, 260, 267
structural gene, 46
tariff, 110
structure, 10, 24, 44, 50, 62, 190, 223, 235
TDI, 79
style, 244, 253, 257
teams, 148
styrene, 186, 280
techniques, vii, viii, xiv, 2, 7, 36, 46, 138,
sub-Saharan Africa, 80, 270
195, 202, 204, 234, 236, 238, 239, 244,
subsistence, 273
251, 265
substitution, 143
technology, 25
substrate(s), viii, xii, xiv, 7, 10, 14, 21, 35,
telomere, 45
39, 49, 65, 66, 158, 163, 181, 234, 236,
teratogenic effects, ix, xv, 64, 77, 260, 266
238, 243, 245, 249, 250, 255
testing, 119, 218
succession, xii, 158, 173
testosterone, 80, 195
sucrose, 22
tetrahydrofuran, 92
Sudan, 43
TGA, 131
sulfate, 12
Thailand, 37, 39, 41, 43, 57, 60, 61, 200,
sulfuric acid, 14
203
Sun, 153, 266, 284
therapy, 274
supplementation, 57
thermal stability, 66
Index 303
Third World, xiii, 192 treatment, 12, 68, 69, 95, 96, 97, 98, 197,
threats, 52 212, 224, 226, 245, 249, 257, 275
thymus, 131 tremor, 200
tissue, 58, 95, 128, 129, 135, 138, 139, 168, trial, 257, 274
197 triggers, 96
tissue homeostasis, 95 trypsin, 268
TLR4, 96 tryptophan, 200, 226
TNF-, 95, 97, 98 tuberculosis, 273
tocopherols, 186 Tukey Test, 167
Togo, 81, 85, 198, 224, 270, 279 tumor(s), ix, 64, 129, 135, 139, 143, 152,
total product, 108 155, 197, 200, 222, 223
toxic effect, ix, 5, 38, 64, 93, 102, 143, 147, tumor cells, 155
194, 195, 196, 281, 284 Turkey, 43, 72, 73, 87, 92, 193, 219, 221,
toxic products, 10 267, 284
toxicity, xiii, 3, 5, 19, 25, 32, 33, 37, 64, 77, turnover, 7, 199
79, 80, 97, 99, 102, 151, 192, 193, 194, tyrosine, 200, 226
195, 196, 198, 199, 248, 255, 262, 273,
285
toxicology, vii, 88, 105, 150, 223, 224, 226, U
285
U.S. Department of Agriculture, 231
toxigenic fungi, viii, 35, 36, 42, 55, 60, 86,
United Kingdom (UK), 66, 103, 105, 272,
109, 243
285, 286
toxin, viii, xi, xv, 2, 3, 9, 10, 11, 22, 23, 35,
United Nations, x, 64, 84, 186
37, 38, 44, 53, 55, 66, 73, 89, 126, 127,
United States, xiii, 38, 95, 185, 192, 200,
143, 147, 148, 164, 178, 179, 199, 216,
202, 231, 267, 285
235, 246, 260, 266, 275, 276, 283
urea, 12, 14
TP53, xi, 126, 127, 128, 131, 145, 148, 149,
urine, 5, 27, 34, 80, 81, 92, 93, 102, 103,
151
271
trade, 8, 37, 110, 116, 119, 120, 202, 209,
USDA, 217
210, 215, 220, 267
UV, 29
trade liberalisation, 210
UV light, 37
trade policy, 120
traits, 86, 187
transcription, 47, 48, 50, 59, 60, 146, 195 V
transcription factors, 47
transduction, 104 vaccinations, 100, 271
transformation, 137, 153 vaccine, 100, 104, 201
translation, 195 validation, 13, 14, 15, 103, 253
transmission, 224, 273 variables, 132, 135, 204
transparency, 211, 219 variations, 5, 83, 89, 100, 173, 179, 194,
transplant, 53 204
transport, 4, 7, 47, 66, 74, 111, 159 varieties, 190, 242, 243, 244, 274
transportation, 67 vector, 187
traumatic brain injury, 226 vegetable oil, 27, 68, 81, 230, 256
vegetables, 72, 229, 280, 284
304 Index
Venezuela, 108, 200 wood, 19

ventilation, 67 workers, 80, 194, 209
vertebrates, 193 World Health Organization (WHO), x, 8,
virus infection, 272, 282 12, 28, 29, 32, 33, 42, 62, 64, 85, 86,
viruses, 85, 152, 279, 284 120, 205, 207, 210, 223, 224, 226, 229,
visualization, 37 254, 267, 272, 282, 285
vitamin A, 273 World Trade Organization (WTO), 210,
vitamin E, 223, 273 212, 222
vitamin K, 200 worldwide, ix, x, xiv, xv, 5, 38, 52, 63, 64,
vitamins, 159, 249, 250 73, 74, 79, 82, 84, 94, 159, 202, 214,
vomiting, xiii, 79, 192, 196, 269, 271, 272 239, 260, 265, 266, 270, 275, 276
vulnerability, 81
X
W
xeroderma pigmentosum, 149, 156
Washington, 27, 61
water, xii, 6, 7, 14, 16, 19, 21, 37, 44, 48,
57, 58, 59, 65, 66, 67, 158, 168, 184, Y
185, 188, 235, 253, 256
yeast, 22, 26
weight gain, 267
Yemen, 78, 220
weight loss, 6, 79
yield, 26, 45, 206, 207
welfare, 230
young people, 80
well-being, 37
wells, 14, 131
West Africa, 81, 85, 102, 189, 204, 224, Z
277, 279, 284
wheat germ, 89 zinc, 57
White Paper, 211, 229
wild type, 51, 135

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FOOD SCIENCE AND TECHNOLOGY

Additional books in this series can be found on Novas website

Additional e-books in this series can be found on Novas website

NOTICE TO THE READER

Independent verification should be sought for any data, advice or recommendations

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc. New York

Chapter 6 Polymorphisms of DNA Repair Genes

Progress in understanding the biology of Aspergillus has greatly improved

concentrations higher than permitted was associated with climate conditions

Because of the multiple adverse health effects to humans and animals

contaminated by aflatoxin M1 (main metabolite of B1) as a result of animals

storage) as a result of transitional weather conditions or of poor storage.

aquaculture species of fish, and humans. Food commodities affected by

Jelka Pleadin1,, Ksenija Markov2, Jadranka Frece2,

Corresponding Author: Tel: +38516123626; Fax: +38516123670; E-mail: pleadin@veinst.hr.

possibility of its reduction using different methods, samples of maize,

deoxynivalenol. Maize and maize products are known to be prone to

1.1. Exposure to AFB1 through the Food Chain

mycotoxins than most animals, especially pigs, as ruminal microbial

1.1.1. AFB1-Related Effects Seen in Humans and Animals

1.1.2. Conditions under Which AFB1 Gets to Be Produced in Cereals

1.1.3. The Occurrence of AFB1 in Feed

1.2. Current EU Legislation

Since the discovery of aflatoxins in the 1960s, regulations have been

1.3. Analytical Methods of AFB1 Determination

For the purpose of AFB1 determination, different screening and

either a primary antibody specific for the target molecule or an enzyme

1.4. Methods of AFB1 Reduction

As the presence of moulds and/or mycotoxins in food can be dangerous

1.4.1. Biological Reduction

1.4.2. Physical Reduction

Despite the debate on safety of irradiated foods, food irradiation is

1.4.3. Chemical Reduction

2. SURVEY OF AFB1 BIO-PREVALENCE IN CEREALS

In order to investigate the bio-prevalence, i.e. the occurrence of AFB1 in

2.2. Implementation of the ELISA Method

2.2.1. Validation of the ELISA Method

Table 1. Results of the ELISA method validation

2.2.2. Employment of the ELISA Method

2.2.2.1. Sample Preparation

2.2.2.2. ELISA Assay

concentrations were calculated from a six-point calibration curve and

2.3. The Implementation of LC-MS/MS Method

2.3.1. LC-MS/MS Validation

Table 3. Repeatability (sr), in-house reproducibility (sWR) and recovery

2.3.2. LC-MS/MS Implementation

2.3.2.1. Sample Preparation

2.3.2.2. Conditions under Which LC-MS/MS Was Implemented

spectrometer was operated in the multiple reaction monitoring mode, the

2.4. AFB1 Concentrations Determined in Cereals

Statistical analysis of data on AFB1 concentrations obtained by the two

Table 4. Concentrations of AFB1 in cereals harvested during 2010-2012

Among the investigated cereals, maize was proven to be most

3. INVESTIGATION INTO THE POSSIBILITIES OF AFB1

The use of gamma () radiation to inactive aflatoxins has already been

Range of Number of Mean AFB1 level Dose of 5 kGy Dose of 10 kGy

A novel way of reducing the proliferation of microorganisms and/or the

Table 6. Inhibitory effects (%) of essential oils on mould growth

Table 7. AFB1 binding by lactic acid bacteria

AFB1 bound SDa (%)

Atalla, M. M., Hassanein, N. M., El-Beih, A. A., Youssef, Y. A. (2003)

Bryden, W. L. (2012) Mycotoxin contamination of the feed supply chain:

from deteriorated paper materials by radiation. International