Food and Chemical Toxicology 83 (2015) 151e163

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Hepatotoxic effect of ochratoxin A and citrinin, alone and in

combination, and protective effect of vitamin E: In vitro study in
HepG2 cell
Loganathan Gayathri a, b, Rajakumar Dhivya b, Dharumadurai Dhanasekaran a,
Vaiyapuri S. Periasamy c, Ali A. Alshatwi c, Mohammad A. Akbarsha b, c, *
a

Department of Microbiology, Bharathidasan University, Tiruchirappalli 620024, India

Mahatma Gandhi-Doerenkamp Center, Bharathidasan University, Tiruchirappalli 620024, India
Nanobiotechnology and Molecular Biology Research Laboratory, Department of Food Science and Nutrition, College of Food Science and Agriculture, King
Saud University, Riyadh 11451, Kingdom of Saudi Arabia
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 23 February 2015
Received in revised form
2 June 2015
Accepted 8 June 2015
Available online 23 June 2015

Ochratoxin A (OTA) and citrinin (CTN) are the most commonly co-occurring mycotoxins in a wide variety
of food and feed commodities. The major target organ of these toxins is kidney but liver could also be a
target organ. The combined toxicity of these two toxins in kidney cells has been studied but not in liver
cell. In this study HepG2 cells were exposed to OTA and CTN, alone and in combination, with a view to
compare the molecular and cellular mechanisms underlying OTA, CTN and OTA CTN hepatotoxicity.
OTA and CTN alone as well as in combination affected the viability of HepG2 cells in a dose-dependent
manner. OTA CTN, at a dose of 20% of IC50 of each, produced effect almost similar to that produced by
either of the toxins at its IC50 concentration, indicating that the two toxins in combination act synergistically. The cytotoxicity of OTA CTN on hepatocytes is mediated by increased level of intracellular
ROS followed/accompanied by DNA strand breaks and mitochondria-mediated intrinsic apoptosis. Cotreatment of vitamin E (Vit E) with OTA, CTN and OTA CTN reduced the levels of ROS and the cytotoxicity. But the genotoxic effect of OTA and OTA CTN was not completely alleviated by Vit E treatment
whereas the DNA damage as caused by CTN when treated alone was obviated, indicating that OTA induces DNA damage directly whereas CTN induces ROS-mediated DNA damage and OTA CTN combination induces DNA damage not exclusively relying on but inuenced by ROS generation. Taken together,
these ndings indicate that OTA and CTN in combination affect hepatocytes at very low concentrations
and, thereby, pose a potential threat to public and animal health.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Mycotoxin
Citrinin
Ochratoxin A
Hepatotoxicity
Reactive oxygen species

1. Introduction
Mycotoxins are secondary metabolites of fungi that frequently
contaminate long-time stored food and feed commodities (Reddy

Abbreviations: AO, acridine orange; bp, base pairs; CTN, citrinin; cyt c, cytochrome c; DCFH-DA, 20 ,7 -dichlorouorescein diacetate; DCFH, 20 ,70 -dichlorouorescein; EB, ethidium bromide; IC50, the concentration at which 50% of cells are
dead; JC-1, 5,50 ,6,60 -tetrachloro-1,10,3,30 -tetraethyl-imidacarbocyanine iodide; MTT,
3-4, 5-dimethylthiazole-2-yl, 2,5-diphenyl tetrazolium bromide; OTA, ochratoxin A;
PBS, phosphate-buffered saline; ROS, reactive oxygen species; Vit E, vitamin E;
DJm, mitochondrial trans-membrane potential.
* Corresponding author. Mahatma Gandhi-Doerenkamp Center, Bharathidasan
University, Tiruchirappalli 620024, India.
E-mail address: mgdcaua@yahoo.in (M.A. Akbarsha).
http://dx.doi.org/10.1016/j.fct.2015.06.009
0278-6915/ 2015 Elsevier Ltd. All rights reserved.

et al., 2010). Possibly, upon natural contamination, more than one

mycotoxin is produced by a fungal species or several mycotoxins
are produced by different fungal species which co-infest the food/

et al., 2005; Streit et al., 2012). As a
feed commodity (Molinie
consequence, often more than one mycotoxin is found in contaminated food/feed commodities (Reddy et al., 2010). Humans and/or
animals may be exposed to mycotoxins through inhalation, ingestion or skin contact (WHO, 2001). Exposure to multiple mycotoxins
may lead to synergistic, additive or antagonistic interactions (Reddy
et al., 2010). Thus, depending on the concentration and duration of
exposure mycotoxins may cause acute or chronic toxicity disorder
to humans and animals (Speijers and Speijers, 2004). Therefore,
consumption of mycotoxins as a mixture can potentially produce a
greater adverse health effect than when either one alone is

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L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

consumed. In this context, very little information about the toxicological risk of concomitant exposure to mycotoxins, acting at the
molecular level, is available (Grosse et al., 2004; Bouaziz et al.,
sch-Saadatmandi et al., 2012).
2008; Bo
Among different mycotoxins, ochratoxin A (OTA) citrinin
(CTN) is one of the most frequently occurring combinations in a
wide veriety of food and feed commodities (Nguyen et al., 2007;
Reddy et al., 2010; Klari

c et al., 2013). Experimental evidences for
the carcinogenic potential of OTA led to its classication as a potential human carcinogen under group 2B by the International
Agency for Research on Cancer (IARC) whereas CTN is classied
under group 3 in view of inadequate evidences of its in vivo carcinogenicity (IARC, 1993). Several studies have shown frequent cooccurrence of OTA and CTN in the same agricultural crops, food
and feed commodities. Actually, both are produced by Aspergillus
and Penicillium family members which are worldwide in distribution (Park et al., 2002; Nguyen, 2007). Both OTA and CTN are known
to cause several toxic effects to humans and animals, but mainly
affect kidney. Epidemiological studies have shown that OTA and
CTN are responsible for pathogenesis of the human Balkan Endemic
Nephropathy, associated with urinary tract tumor (Schwerdt et al.,
1999; Pfohl-Leszkowicz et al., 2002). Experimental evidences are
also available for hepato-, terato- and immune toxicity of these two
mycotoxins (Vesela et al., 1983; Chan and Shiao, 2007; Chen and
Chan, 2009; Zhang et al., 2009; Chopra et al., 2010; Islam et al.,
2012; Anninou et al., 2014). Most of the previous studies have
elucidated the nephrotoxic potential of both OTA and CTN separately as well as in combination and identied additive/synergistic
llmann et al.,
interaction between the two (Knecht et al., 2005; Fo
2007; Bouslimi et al., 2008a,b; Golli-Bennour et al., 2010; Klaric
et al., 2012; Kumar et al., 2014). There are also quite a few studies
showing OTA and CTN, separately and/or in combination, as toxic to
llmann et al.,
lung broblast cells suggesting pulmonary toxicity (Fo
2000, 2014; Behm et al., 2012)). Apart from kidney and lung other
organs including liver are also targets for mycotoxin pathology. This
is important because liver is the major organ concerned with

et al.,
biotransformation and detoxication of mycotoxins (Lura
2004; Chen and Chan, 2009; Anninou et al., 2014; Huang and
Chan, 2014). Therefore, it is important to look into the mechanism underlying hepatotoxicity caused by OTA and CTN in combination, to evolve strategies to address the harmful effects on human
and animal health.
It has been reported that OTA-induced cytotoxicity is mediated
by increase of lipid peroxidation, oxidative stress, adduct formation with DNA and interference with protein biosynthesis (Kamp
et al., 2005; Ringot et al., 2006; Bouaziz et al., 2008; Chopra
et al., 2010; Klari

c et al., 2013) whereas CTN interferes with
electron transport system, Ca2 uxes and membrane permeability of mitochondria (Da Lozzo et al., 1998; Yu et al., 2006). ROS
play a major role in CTN-induced hepatotoxicity in Wistar rat
(Singh et al., 2013). On the other hand in mouse skin (Kumar et al.,
2011), HepG2 (Chen and Chan, 2009) and HL-60 (Yu et al., 2006)
cells CTN induces ROS-mediated mitochondria-dependent
apoptosis. Thus, considering the prevalence of fungal contamination and the imminent concurrent exposure to OTA and CTN,
the present study was designed essentially to investigate if the
combination of OTA and CTN enhances the hepatotoxic potential
of these toxins.
Cell lines of liver origin are widely used in toxicological research
involving xenobiotic metabolism, genotoxicity and cytoprotective
studies (Knasmller et al., 1998; Majer et al., 2004). The HepG2 is
one of the most often used cell systems in xenobiotic research,
including biochemical and physiological characterization. This cell
line is also qualied for the detection of environmental and dietary
genotoxicants (Knasmller et al., 2004; Mersch-Sundermann et al.,

2004). For example, safrol (Natarajan and Darroudi, 1991; Uhl et al.,
2000), fumonisin B1 (Ehrlich et al., 2002a,b), and isatidine (Uhl
et al., 2000) were tested for genotoxic effect in HepG2 cells.
Therefore, we used this cell to assess the cell proliferation,
morphological characterization, mitochondrial membrane depolarization, generation of ROS, DNA damage and induction of
apoptosis to understand and compare the molecular mechanisms
of hepatotoxicity caused by OTA and CTN alone and in combination.
In addition, the bio-antioxidant vitamin E (Vit E) has been used to
nd if it alleviates the cytotoxic effects of OTA and CTN alone and in
combination in hepatocytes not only to nd the protective effect
but also to understand the connection between ROS generation and
genotoxic potential of the two mycotoxins.

2. Materials and methods

2.1. Chemicals
OTA, CTN and DMEM were obtained from Sigma Chemical
Company (St. Louis, MO, USA). Phosphate-buffered saline (PBS) and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) were purchased from HiMedia Laboratories (Mumbai, India).
Fetal bovine serum (FBS), trypsineEDTA, penicillin and streptomycin were obtained from Invitrogen (USA). All other chemicals
and reagents were of analytical grade.

2.2. Cell culture

Human hepatocarcinoma cell, HepG2, was obtained from National Center for Cell Science (NCCS), Pune, India. The cells were
maintained in DMEM medium supplemented with 10% FBS, and
with 100 U/ml each of penicillin and streptomycin as antibiotics in a
humidied atmosphere of 5% CO2 and 95% air, in a CO2 incubator
(Thermo Scientic, USA).

2.3. Cell viability assay

The MTT tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] colorimetric assay (Mosmann,
1983) was performed to measure the viability of cells. Cells were
seeded in 96 well plate at 5  103 cells/well and treated with
different concentrations of OTA, CTN and combination of the two,
dissolved in minimum quality of dimethyl sulfoxide (DMSO) for
24 h, at 37  C. DMSO was used as the solvent control. After 24 h
incubation, 20 ml of MTT solution (5 mg/ml in PBS) was added to
each well, and incubated for 3 h at 37  C. The medium was then
removed and the purple formazan product was dissolved in 100 ml
of DMSO. The absorbance was measured at 570 nm (measurement)
and 630 nm (reference) using a 96-well plate reader (Bio-Rad,
Hercules, CA, USA). Data were collected for triplicates and used to
calculate the respective means and the standard deviations. The
percentage inhibition was calculated from this data using the
following formula:

Percentage inhibition Mean OD of untreated cells control

 Mean OD of treated cells=Mean OD
of untreated cells control  100:
From the values thus obtained, the IC50 was calculated. IC50 is
dened as concentration of the test substance at which cell viability
is decreased to 50%.

L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

2.4. Acridine orange (AO) and ethidium bromide (EB) uorescent

assay for morphological assessment of cell death
AO/EB staining (Spector et al., 1998) was performed to assess the
morphological characteristics of cells in apoptosis and necrosis. The
cells were cultured in 6-well plates and treated with IC50 concentrations of OTA and CTN alone and in combination at 20% of the
respective IC50 values for 24 h. The treated and untreated cells were
centrifuged (3000 rpm for 5 min) and incubated with AO and EB
solutions (1 part of 100 mg/ml each of AO and EB in PBS) and
examined in a uorescent microscope (Carl Zeiss, Jena, Germany)
using a UV lter (450e490 nm). Three hundred cells per sample
were counted, in duplicate, and scored as viable or dead, and if dead
whether by apoptosis or necrosis as judged from nuclear
morphology and cytoplasmic organization. The percentages of
apoptotic and necrotic cells were then calculated. Morphological
features of interest were photographed.
2.5. Hoechst 33528 staining for assessment of nuclear features
To assess the nuclear morphological features Hoechst 33528
staining (Latt et al., 1975) was adopted. HepG2 cells were cultured
in 6-well plates and treated with OTA and CTN alone, at their
respective IC50 concentrations, and the two in combination at 20%
of respective IC50 values, for 24 h. After incubation, the treated and
control cells were harvested and stained with Hoechst 33258
(1 mg/ml in PBS) for 5 min at room temperature. The cell suspension was placed on a glass slide, and a cover slip was laid over to
reduce light diffraction. At random 300 cells, in triplicate, were
observed at 400 in the uorescent microscope tted with a
377e355 nm lter, and the percentage of cells reecting pathological changes was calculated.
2.6. JC1 staining for assessment of mitochondrial trans-membrane
potential
Mitochondrial trans-membrane potential (DJm) was measured
using the uorescent probe JC-1 (5,50 ,6,60 -Tetrachloro-1,10,3,30 -tetraethyl-imidacarbocyanine iodide), which exhibits membrane
potential-dependent accumulation in mitochondria as indicated by
a specic uorescence emission (Reers et al., 1991). In healthy cells
with high physiological mitochondrial DJm, JC-1 spontaneously
forms complexes known as J-aggregates, with intense orange-red
uorescence. Under condition when DJm breaks down, as during apoptosis, JC-1 remains in the monomeric form and uoresces
green and so the entire cell uoresces green. The cells were grown
in glass cover slips placed in the wells of 6-well plates and treated
with IC50 concentrations of OTA and CTN alone and in combination
at 20% of the respective IC50 values, for 24 h. The cells were stained
with JC-1 dye after 12 and 24 h exposure. The mitochondrial depolarization patterns of the cells were observed in the uorescent
microscope and the uorescence pattern of cells (red, normal;
green, apoptotic) were observed and recorded.

153

(Wang and Joseph, 1999). To evaluate mycotoxins-induced DCF

oxidation, 1  106 cells were seeded in each well of 6 well plate and
treated with the positive control H2O2 and IC50 concentration of
OTA and CTN alone and the two in combination at 20% of the
respective IC50 values for 6 h, 12 h and 24 h. After that, the wells
were loaded with 5 mM of DCFH-DA and incubated for 30 min at
37  C. ROS generation was measured in a spectro-uorimeter with
an excitation wavelength at 485 nm and emission wavelength at
520 nm. Results are expressed as the ratio mycotoxin-induced DCF
uorescence/control uorescence.
2.8. Single-cell gel electrophoresis (comet assay) for genotoxicity
assessment
DNA damage was detected adopting the comet assay (Singh
et al., 1988). The technique combines DNA gel electrophoresis
with orescent microscopy to visualize migration of DNA from individual agarose-embedded cells. In case of breaks in DNA, DNA
supercoils relax and the broken ends migrate towards the anode
during the electrophoresis, forming the tail of a comet. In case of
undamaged DNA, the tail is not formed. The extent of DNA damage
can be assessed from the shape and area of the comet tail (Olive and

th, 2006). The cells were treated with the IC50 concentration of
Bana
OTA and CTN alone and in combination at 20% of the respective IC50
values, for 24 h. The harvested cells were suspended in low melting
point agarose in PBS and pipetted out to microscope slides precoated with a layer of normal melting point agarose. Slides were
immersed in pre-chilled lysis buffer (2.5 M NaCl, 100 mM Na2EDTA,
10 mM Tris, 0.2 mM NaOH [pH 10], and Triton X-100) and incubated
overnight at 4  C in order to lyse the cells and to permit DNA unwinding. Thereafter, the slides were exposed to alkaline buffer
(300 mM NaOH, 1 mM Na2-EDTA, [pH > 13]) for 20 min at 20 V to
allow DNA unwinding. The slides were washed with buffer (0.4 M
Tris, pH 7.5) to neutralize excess alkali before staining with EB. The
slides were observed in the uorescent microscope. One hundred
and fty cells, in triplicate, from each treatment group were digitized and analyzed using CASP software. The images were used to
determine the DNA content of individual nuclei and to evaluate the
degree of DNA damage representing the fraction of total DNA in the
tail.
2.9. DNA laddering assay for the analysis of DNA fragmentation
The procedure adopted for analyzing DNA fragmentation was
according to Bossu (1999). Apoptosis is clearly associated with

2.7. Reactive oxygen species (ROS) assay for the assessment of

oxidative stress
The production of intracellular ROS was measured using the
uorescent probe 20 ,70 -dichlorouorescein diacetate (DCFH-DA).
DCFH-DA diffuses through the cell membrane and is enzymatically
hydrolyzed by intracellular esterases to form the non-uorescent
compound 20 ,70 -dichlorouorescein (DCFH), which is then rapidly
oxidized to form the highly uorescent 20 ,70 -dichlorouorescin
(DCF) in the presence of ROS. The DCF uorescence intensity is
believed to be parallel to the extent of ROS formed intracellularly

Fig. 1. Cytotoxic effect of CTN and OTA individually on HepG2 cells after exposure for
24 h. Data are expressed as mean SD of three independent experiments for each dose
point. *p < 0.05 compared between dose values as well as time points.

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L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

reached about 3 cm from the end of the gel. The DNA on gel was
photographed in Geldoc (Bio-Rad Laboraties Inc., USA).
2.10. Western blot analysis of caspases, Bax and cleaved PARP

Fig. 2. Combined cytotoxicity of OTA and CTN on HepG2 cells after exposure for 24 h.
Each data point is mean SD of three independent experiments for each dose point.
*p < 0.05 compared to control as well as any two experimental groups.

fragmentation of DNA into oligonucleosomal fragments, each about

180 bp long. When DNA is subjected to electrophoresis in agarose
gel, the fragments separate out according to their molecular weight
and form a ladder. Intact DNA does not form this ladder. Each well
of 6 well plate was seeded with 1  105 cells and treated with IC50
concentration of OTA and CTN alone and in combination at 20% of
the respective IC50 values, for 24 h at 37  C. The cells were collected
after the treatment, washed with TTE solution and incubated
overnight with 0.1 ml of ice-cold 5 M NaCl along with 0.7 ml of icecold isopropanol. Precipitation was allowed to proceed overnight
at 20  C, after which the pellet was rinsed in ice-cold 70% ethanol.
The DNA was dissolved by adding 20e50 mL of TE solution. The
samples were run in 1% agarose gel containing 0.5 mg EB/ml.
Appropriate DNA molecular weight marker was also run separately.
The gel was run at 100 V for 45 min and stopped when the dye

For Western blotting, cells were treated with the IC50 concentration of OTA and CTN alone and in combination at 20% of the
respective IC50 values, for 24 h and appropriate amounts of cell
lysates (40 mg protein) were resolved over 10% Tris-glycine polyacrylamide gel and then transferred onto the PVDF membrane. The
blots were blocked using 5% nonfat dry milk and probed using proand cleaved caspases-9, -8 and -3, Bax and cleaved PARP primary
monoclonal antibodies in blocking buffer overnight at 4  C. The
membrane was then incubated with appropriate secondary
antibody-alkaline phosphatase conjugate (Merck, India) followed
by detection using NBIT/BCI kit (Merck, India). To ensure equal
loading of protein, the internal control anti-b-actin antibody (Santa
Cruz, US) was used and compared.
2.11. Statistical analysis
All results were expressed as mean SD of three independent
experiments. Difference between groups was analyzed by paired
sample t-test from GraphPad Prism-6.0, and p < 0.05 was considered statistically signicant.
3. Results
3.1. Effect of OTA and CTN alone and in combination on HepG2 cell
viability
Cytotoxic effect of OTA and CTN alone and in combination, on
HepG2 cells after 24 h incubation was measured by MTT assay, and
the data are shown in Figs. 1 and 2. OTA decreased cell viability in a

Fig. 3. Morphological assessment of apoptosis and necrosis. (A) Effect of CTN and OTA (individually and in combination) on the morphological features after 24 h treatment as
observed adopting AO/EB staining. (B) Percentage of normal, apoptotic and necrotic cells. Data are expressed as mean SD of three independent experiments. *p < 0.05 compared to
control.

L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

155

at 10%, 20%, 30%, 40% and 50% of the respective IC50 concentrations
and the cells were treated for 24 h. The combination of OTA and
CTN at 20% of the respective IC50 (OTA 42 mM CTN 31 mM) reduced
cell viability to 50%. Thus, on concomitant exposure OTA and CTN
showed to be synergistically interacting to exert cytotoxicity in
HepG2 hepatocytes.

3.2. Microscopic evidence for apoptosis and/or necrosis in cells

treated with OTA and CTN alone and in combination

Fig. 4. Assessment of changes in cell nuclei as observed in Hoechst treatment. (A)

Effect of CTN and OTA (individually and combined) on the nuclear features as observed
adopting Hoechst staining after 24 h treatment. (B) Percentage of cells with normal
and abnormal nuclei. Data are expressed as mean SD of three independent experiments. *p < 0.05 compared to control.

dose-dependent manner at concentration range 0e300 mM and the

measured IC50 was 210 mM. Exposure to CTN at concentration range
0e300 mM resulted in reduction of cell viability, again in dosedependent manner, and the IC50 was 155 mM.
Based on the individual IC50 values OTA and CTN were combined

Microscopic evidence for apoptosis and/or necrosis was examined adopting AO/EB staining. In general, dead cells are permeable
to EB and uoresce orange-red, whereas live cells are permeable to
AO only and thus uoresce green. The uorescence pattern depends on the viability and membrane integrity of the cells. Based on
the uorescence emission the morphological changes observed in
the treated cells were as follows: i) viable cells having highly
organized nuclei uoresced green; ii) early apoptotic cells which
showed nuclear condensation uoresced orangeegreen uorescence; iii) late apoptotic cells with the chromatin highly condensed
or chromatin fragmented uoresced orange to red; and iv) necrotic
cells uoresced orange to red with no indication of chromatin
fragmentation. Data on cells indicating apoptotic and necrotic
morphologies, induced on treatment with the IC50 concentration of
OTA and CTN separately and the two in combination at 20% of the
IC50 of each, as collected from manual counting of cells, are presented in Fig. 3A and B which revealed that CTN, alone as well as in
combination with OTA, brings about higher incidence of apoptosis,
and also necrosis to a certain extent, but OTA alone induced greatly
apoptosis, and necrosis only to a very small extent.

3.3. Effect of OTA and CTN alone and in combination on nuclei of

HepG2 cell
Changes in the nucleus as caused by treatment with the mycotoxins were revealed by Hoechst 33528 staining. The cells treated
with IC50 concentration of OTA and CTN alone and in combination
at 20% of the respective IC50 concentrations, revealed changes in
nuclear morphology. In the control cells the nuclear chromatin was
intact while after treatment with the mycotoxins for 24 h changes
such as chromatin marginalization, condensation and fragmentation were noticed. These observations suggest that exposure of OTA
and CTN alone and in combination, led to chromatin fragmentation
which is a characteristic feature of apoptosis. Data collected from
manual counting of cells with normal and abnormal nuclear features are shown in Fig. 4A and B.

Fig. 5. Photomicrographs of cells showing the effect of CTN and OTA (individually and
combined) on the change in mitochondrial trans-membrane potential at 12 h and 24 h
as revealed in JC-1 staining. Results are representative of three independent
experiments.

Fig. 6. Intracellular ROS of HepG2 cells treated with CTN, OTA and mixture the two for
6, 12 and 24 h. ROS level is expressed as fold change in the uorescence in comparison
with control. Data are expressed as mean SD of three independent experiments.
*p < 0.05 compared to the respective controls.

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L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

3.5. Effect of OTA and CTN alone and in combination on induction of

reactive oxygen species (ROS) in HepG2 cell
ROS have been suggested as possible mediators of apoptosis
induced by mycotoxins through oxidative stress. ROS production is
associated with mitochondria-mediated apoptosis. Therefore, induction of intracellular ROS was studied in HepG2 cell exposed for
6, 12 and 24 h to OTA and CTN at the respective IC50 concentrations
as well as the combination of the two at 20% of the respective IC50
concentrations. H2O2 was used as the positive control for ROS
generation, and the increase of ROS level was compared with data
for test mycotoxins. Duration-dependent increase in ROS was
observed in all treatments (Fig. 6). OTA-treated cells suffered with
ROS more than CTN-treated cells. But, the combined treatment of
OTA and CTN induced greater amount of ROS at 24 h.
3.6. Genotoxic effect of OTA, CTN and the combination of both as
revealed in comet assay
Fig. 7. Assessment of DNA damage adopting comet assay. (A) DNA damage induced by
CTN and OTA (individually and combined) after 24 h of treatment as revealed by comet
assay. (B) DNA damage as dened according to the DNA in the tail. The multiple parts
of each column (from the bottom to the top): intact (0e20%), slightly damaged
(20e40%), damaged (40e60%), highly damaged (60e80%), and dead (80e100%). Data
are expressed as mean SD of three independent experiments.

3.4. Effect of OTA and CTN alone and in combination, on

mitochondrial trans-membrane potential (DJm)
Since mitochondria play critical roles in apoptosis, the effect of
OTA and CTN alone and in combination (20% of the respective IC50),
on DJm was studied. JC-1 is a probe which indicates loss of DJm
by shift in uorescence emission from red to green. As shown in
Fig. 5, the untreated cells uoresced red but after 12 h exposure to
OTA and CTN alone and in combination there were red as well as
green emissions indicating loss of DJm to a certain extent, and
after 24 h the cells uoresced only green indicating that DJm was
lost completely. The results illustrated that OTA and CTN alone and
in combination induced loss of DJm which suggested that the
apoptotic cell death was mediated by mitochondria.

Fig. 8. Agarose gel electrophoresis of fragmented DNA extracted from HepG2 cells
incubated for 24 h with individual and combination of toxins, Lane 1: MW marker
100bp; Lane 2: control; Lane 3: CTN (IC50 155 mM); Lane 4: OTA (IC50 210 mM);
Lane 5: OTA (42 mM) CTN (31 mM); Lane 6: OTA (21 mM) CTN (15.5 mM); Lane 7:
OTA (42 mM); Lane 8: CTN (31 mM). Results are representative of three experiments
each.

Single cell gel electrophoresis assay (comet assay) was adopted

to detect the cellular DNA lesions or genotoxicity. This method is
very sensitive for identication of strand breaks in DNA. In this
experiment, HepG2 cells were exposed to the respective IC50
concentrations of OTA and CTN alone and in combination at 20% of
the respective IC50 concentrations. DNA damage was analyzed
based on DNA tail size, shape and migration pattern. The CASP
image analysis software is of help in (i) analysis of a variety of
geometric and densitometry parameters, and (ii) estimation of the
amount of DNA in the head (intact DNA) and the tail (DNA with
strand breaks) regions. As shown in Fig. 7A, both OTA and CTN
caused damage to DNA wherein the impact was more with OTA
than CTN, whereas the combination of OTA and CTN caused only
moderate damage to DNA. Since the tail length and density reected the extent of strand breaks in DNA, the percentage of DNA
in the tail provided a quantitative measure of DNA damage as
shown in Fig. 7B.
3.7. Effect of OTA and CTN aloe and in combination on DNA
fragmentation
From the comet assay we could conrm that OTA CTN at 20%
of their respective IC50 concentrations induced genotoxicity. But it
is important to nd if even at lesser concentrations the combination of toxins would still be genotoxic. Therefore, HepG2 cells were
treated with IC50 concentrations of OTA and CTN separately and as a
combination of OTA and CTN at 10% and 20% of their respective IC50
concentrations. The treatments induced double strand DNA breaks
resulting in DNA ladder. Results of DNA fragmentation assay, as
evidenced by agarose gel electrophoresis, are presented in Fig. 8.
Since OTA CTN at 10% of the respective IC50 concentrations could
induce DNA double strand breaks, we were interested to check
whether the damage inicted on DNA is because of combinative
effect or individual genotoxic effect of OTA and CTN. Therefore, OTA
and CTN were individually tested at their respective 20% IC50 concentrations, in which DNA fragmentation was not observed with
either of the toxins. This shows that the synergistic interaction
between OTA and CTN is important to cause DNA damage at low
concentrations.
3.8. Evidence at molecular level for induction of apoptosis by OTA
and CTN alone and in combination
In order to understand the mechanism underlying the induction
of apoptosis, caspases-9, -8 and -3, Bax and PARP protein expression levels were determined after 24 h treatment with the IC50

L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

concentration of OTA and CTN, alone and in combination, (20% of

the IC50 concentration of each). Pro-caspases-9 and -3 expression
decreased and expression of cleaved-caspases-9 and -3, Bax and
PARP increased in toxin-treated cells. Expression of pro-caspase-8
was unchanged and cleaved caspase-8 decreased in the treated
cells. Taken together, the present data suggest that exposure to OTA
and CTN, alone and in combination, leads to caspase-dependent
mitochondria-mediated intrinsic apoptosis (Fig. 9).

Fig. 9. Caspase induction by CTN and OTA (individually and combined) in HepG2 cell
as revealed by Western blot after 24 h of treatment. (A) Pro- and cleaved caspases-9,
-8, and -3, Bax and cleaved PARP expressions. (B) Scanning densitometry of pro- and
cleaved caspase-9, and caspase-8 in fold-change compared with control. (C) Scanning
densitometry of pro- and cleaved caspase 3, Bax and PARP in fold-change compared
with control. Data are expressed as mean values SD of three independent experiments. Values are statistically signicant compared to control, at p < 0.05.

157

3.9. Protective effect of vitamin E on OTA and CTN alone and in

combination-induced cytotoxicity
To evaluate the protective effect of vitamin E against the cytotoxicity caused by the mycotoxins, we concomitantly treated the
HepG2 cells with OTA and CTN alone, at the respective IC50 concentration and in combination at the respective 20% IC50 concentrations of each, and increasing concentrations of Vit E, i.e., 5 mg,
10 mg and 20 mg (Fig. 10). The Vit E co-treatment offered protection
against toxin-induced cytotoxicity in all cases in concentrationdependent manner. Therefore, Vit E has the potential to protect

Fig. 10. Protective effect of Vit E against CTN and OTA (individually and combined)
-induced cytotoxicity after 24 h treatment as revealed by MTT assay. (A) % viability of
HepG2 cells treated with Vit E alone, CTN alone at IC50 concentration and co-treatment
with VitE at indicated concentrations for 24 h. (B) % viability of HepG2 cells treated
with Vit E alone, OTA alone at IC50 concentration and co-treatment with VitE at
indicated concentrations. (C) % viability of HepG2 cells treated with Vit E alone,
combination of OTA CTN at 20% of IC50 concentration and co-treatment with VitE at
indicated concentrations. Data are expressed as mean SD of three independent experiments. *p < 0.05 compared to untreated as well as Vit E alone treated controls.

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L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

Fig. 11. Antioxidant effect of Vit E against CTN and OTA (individually and combined)
-induced ROS generation as revealed by ROS assay after 6 h, 12 h, 24 h of treatment.
Data are expressed as mean SD of three independent experiments. ROS levels
*p < 0.05 compared to positive control; @ p < 0.05 signicantly lesser than the untreated as well as positive controls.

the cells from cytotoxicity caused by OTA and CTN alone and in
combination.
To assess the antioxidant potential of Vit E against the oxidative
stress caused by OTA and CTN alone at the respective IC50 concentrations as well as combination of both toxins at 20% of the
respective IC50 concentrations, we co-treated the toxin-exposed
HepG2 cells with Vit E at 20 mg concentration for different time
points viz., 6 h, 12 h and 24 h (Fig. 11). The intracellular ROS triggered by OTA and CTN alone and in combination was signicantly
decreased by Vit E treatment in time-dependent manner. Thus, the
results revealed that ROS clearly has a link in the cytotoxicity
caused by the test mycotoxins.
Comet assay was adopted to elucidate the protective effect of Vit
E against the genotoxicity caused by the mycotoxins (Fig. 12). Cotreatment of HepG2 cells with 20 mg Vit E and OTA at its IC50
concentration did not totally alleviate the toxin induced DNA

Fig. 12. Assessment of DNA damage, after Vit E co-treatment, adopting comet assay. (A) Anti-genotoxic effect of Vit E on DNA damage induced by CTN and OTA (individually and
combined) as revealed by comet assay after 24 h treatment. (B) DNA damage in HepG2 cells as dened according to the DNA in the tail. The multiple parts of each column (from the
bottom to the top): intact (0e20%), slightly damaged (20e40%), damaged (40e60%), highly damaged (60e80%), and dead (80e100%). Data are expressed as mean SD of three
independent experiments.

Table 1
Summary of in vitro studies on toxic effects of Ochratoxin A on HepG2 and other cell models.
Cell type

IC50 values/time/assay

Toxic effect

Reference

Human hepatocellular carcinoma (HepG2)

HepG2
HepG2
HepG2
HepG2
HepG2
HepG2
Human neuroblastoma cells (SKeN-MC)
Madin Darby canine kidney cells (MDCK)
Mouse liver hepatocytes-12 (AML-12)
Pig kidney cells (LLC-PK1)
Bovine mammary epithelial cells (BME-UV1)
Neuroblastoma cells (SH-SY5Y)
Chinese hamster lung broblast cells (V79)
Procine urinary bladder epithelial cells (PUBEC)
Procine kidney epithelial cells (PK15)
Green monkey kidney vero cells

90 mM/24 h/uorescein diacetate assay

35 mM/48 h/neutral red assay
250 nM/24 h/neutral red assay
360 mM/24 h/MTT assay
90 mM/24 h/uorescein diacetate assay
5 mg/ml/24 h/micronucleus assay
5 mg/ml/24 h/micronucleus assay
26.8 mg/ml/24 h/MTT assay
14.5 mg/ml/24 h/MTT assay
40 mg/ml/24 h/MTT assay
26.7 mg/ml/24 h/MTT assay
0.8 mg/ml/24 h/MTT assay
0.5 mM/24 h/neutral red assay
19 mM/48 h/neutral red assay
1.2 mM/48 h/neutral red assay
14 mM/24 h/MTT assay
37 mM/48 h/MTT assay

Apoptosis
Cytotoxicity
Cytotoxicity
Cytotoxicity
Cytotoxicity; Apoptosis
Genotoxicity
Genotoxicity
Cytotoxicity
Cytotoxicity
Cytotoxicity
Cytotoxicity
Cytotoxicity
Neurotoxicity
Cytotoxicity
Cytotoxicity
Cytotoxicity
Cytotoxicity

El GolliBennour et al. (2009)

Hundhausen et al. (2005)
sch-Saadatmandi et al. (2012)
Bo
Corcuera et al. (2011)
Bouaziz et al. (2008)
Ehrlich et al. (2002a,b)
Knasmller et al. (2004)
Baldi et al. (2004)
Baldi et al. (2004)
Baldi et al. (2004)
Baldi et al. (2004)
Baldi et al. (2004)
Zhang et al. (2009)
Behm et al. (2012)
Follmann et al. (2000)
Klaric et al. (2012)
Bouslimi et al. (2008a, 2008b)

L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

159

Table 2
Summary of in vitro studies on toxic effects of citrinin on HepG2 and other cell models.
Cell type

IC50 values/time/assay

Toxic effect

Reference

Human hepatocellular carcinoma (HepG2)

HepG2
Green monkey kidney vero cells
Procine urinary bladder epithelial cells (PUBEC)
Chinese hamster lung broblast (V79)
Peripheral blood sample
HL60
Human embryonic kidney cells (HEK 293)
Mouse embryonic stem cells

35 mM/24 h/modied MTT assay

2.5 mg/ml/24 h/micronucleus assay
220 mM/48 h/MTT assay
5 mM/48 h/Neutral red assay
53 mM/48 h/neutral red assay
40 mM/48 h/micronucleus assay
50 mM/24 h/MTT assay
100 mM/24 h/MTT assay
30 mM/24 h/MTT assay

Cytotoxicity; apoptosis
Genotoxicity
Cytotoxicity
Cytotoxicity
Cytotoxicity
Genotoxicity
Cytotoxicity
Cytotoxicity
Cytotoxicity

Chen et al. (2009)

Knasmller et al. (2004)
Bouslimi et al. (2008a, 2008b)
llmann et al. (2000)
Fo
Follmann et al. (2000)
nmez-Altuntas et al. (2007)
Do
Yu et al. (2006)
Chang et al. (2011)
Chan (2007)

damage as seen in comet-shaped tails still produced whereas cotreatment of Vit E with CTN at its IC50 concentration alleviated
the DNA damage completely since no comet tail was observed.
Interestingly, cells exposed to the combination of toxins co-treated
with Vit E did not reveal complete alleviation of the genotoxic effect
but the DNA tail length was reduced signicantly.
4. Discussion
Exposure to concomitantly produced mycotoxins appeals for
concern because multiple toxins can affect certain targets which
initiate more than one complicated cellular response and alter the
homeostasis (Speijers and Speijers, 2004). However, little information about the toxicity and safety assessment of concomitantly
occurring mycotoxins is available. Therefore, in this study one of the
frequently co-occurring mycotoxins, OTA and CTN, were employed
alone as well as in combination, to investigate if the toxic effects of
both the toxins would be enhanced by their combination as
compared to their effects when treated alone. The mechanism of
action of chemicals may vary even among compounds of the same
chemical class for reasons that are not always obvious. In addition,
knowledge about the mechanisms underlying toxicity is perhaps
the only hope for suggesting rational therapy for toxic symptoms of
compounds already in our environment (McKinney, 1985). Hence,
this study aimed at further distinguishing the impact of OTA, CTN
and their combination on induction of cytotoxicity, genotoxicity,
ROS generation, alteration in DJm and apoptosis in HepG2 cells so
as to assess the ability of the toxins to induce hepatotoxicity.
The cytotoxic potential of OTA and CTN was assessed individually as well as in combination adopting MTT assay, in the background that the cytotoxic effect of OTA is stronger than that of CTN
llmann et al., 2000, 2007, 2014; Knecht et al., 2005; Behm et al.,
(Fo
2012; Klaric et al., 2012; Kumar et al., 2014). Our results show that
at increasing concentrations OTA as well as CTN were moderately
cytotoxic to HepG2 cells in which OTA (IC50 210 mM) was found to
be a weak toxicant compared to CTN (IC50 110 mM) which suggests that different cells representing different organs are differently susceptible to OTA and CTN (Tables 1 and 2). On the other
hand, combination of OTA and CTN at 20% (OTA 42
mM CTN 31 mM) of the respective IC50 concentrations led to an
increased cytotoxicity compared to individual toxins at their
respective IC50 concentrations. Therefore, OTA and CTN act synergistically in producing hepatotoxicity. Bosulimi et al. (2008a) have
shown that OTA and CTN in combination produced higher magnitude of cytotoxicity, in dose-dependent manner, than the individual
exposures on Vero cells that represent the kidney. But, in that study,
OTA and CTN were combined at equimolar concentrations (OTA
24 mM CTN 24 mM) without taking into consideration their individual IC50 concentrations. In our combinatorial toxicity study we
reduced the concentration to similar percentile values of the
respective individual IC50 values and obtained best results at 20% of

the respective IC50 values. Quite a few previous studies also reported synergistic/additive/antagonistic interaction between OTA
and CTN in different models and for different end points (Sum llmann et al., 2014; Also, Siraj et al., 1981; Vesela et al.,
marized by Fo
1983; Mayura et al., 1984; Manning et al., 1985; Brown et al., 1986;
nninou et al., 2014). But in nature mycotoxins can co-occur in any
ratio (Bosulimi et al., 2008a), and the effect of mixtures is often
dependent on the concentrations wherein an effect can change
from additive to synergistic with increasing concentrations
llmann et al., 2014). In our study the combinatorial concentra(Fo
tion of the mycotoxins, at the test doses acted synergistically to
induce cytotoxicity in HepG2 cell model.
Since both OTA and CTN are cytotoxic, the mode of cell death
induced by the toxins was assessed. Cell death can be categorized as
apoptosis and/or necrosis because dying cells are engaged in processes such as compaction, loss of integrity of plasma membrane
and fragmentation of chromatin/nucleus in the case of apoptosis
and cell swelling and lysis in the case of necrosis. Staining with AO
& EB after treatment with the test toxins showed that at its IC50
concentration OTA brought about nuclear and morphological
changes of apoptosis in the HepG2 cells whereas CTN and combination of OTA and CTN induced greatly apoptosis, and to a certain
extent necrosis. This shows that the synergistic interaction between OTA and CTN inuences the combinative toxicity in inducing
apoptotic death of hepatocytes.
Since mitochondrial membrane depolarization is an early event
of apoptosis, we assessed the mitochondrial trans-membrane potential in HepG2 cells treated with OTA and CTN alone and in
combination. The tested mycotoxins individually as well as in
combination altered the mitochondrial trans-membrane potential
which indicates that the mitochondria are a key intra-cellular
target to mycotoxin attack and evidenced an early event in
apoptosis.
Alteration in DJm can cause imbalance in redox environment of
the cell (Deavall et al., 2012). As a consequence, production of ROS
inside the cell could be induced which would interrupt multiple
signaling pathways. So, we were guided to measure the level of
intracellular ROS induced by OTA and CTN alone and in combination. OTA and CTN, individually, induced oxidative stress in the
cells, which was dependent on duration of exposure. Remarkably,
as a combination OTA and CTN, each at 20% of the respective IC50
concentrations, exhibited potency to increase the level of ROS equal
to that by individual toxins at their respective IC50 values. This
revealed that ROS have a link in the cytotoxic effect of the toxins in
HepG2 cell.
ROS, once generated, readily react with the macromolecules of
the cell such as DNA, proteins and lipids to cause lethal cellular
effects (Murphy, 2009). Since DNA is the most crucial molecule in
the cell it is important to study the genotoxicity induced by the
toxins in the same treatment protocol. We evaluated the extent of
single- and double-strand DNA damage caused by OTA and CTN

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L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

alone and in combination, adopting comet assay and DNA ladder

assay. Comet assay revealed that OTA induced severe damage to
DNA but CTN could cause only lesser damage. These results are
consistent with the earlier studies on genotoxicity of OTA and CTN
(Ehrlich et al., 2002a,b; Grosse et al., 2004; Donmez-Altuntas et al.,
2007; El GolliBennour et al., 2009; Corcuera et al., 2011). The
combination of OTA and CTN at the respective 20% IC50 concentrations induced DNA damage but not to the extent as OTA alone at
its IC50 concentration. As evidenced by DNA ladder assay OTA and
CTN, alone and in combination, at 10% and 20% of the respective
IC50 concentrations could induce double-strand breaks in DNA. But
OTA (42 mM) and CTN (31 mM) separately at the concentrations in
the combination treatment could not induce DNA damage. Thus,
our results clearly demonstrated that OTA and CTN when combined
as low doses act synergistically to exert genotoxicity in HepG2 cells.
Apparently, DNA damage, stress signaling pathways, DJm and
anti-proliferative cellular responses lead to triggering of apoptotic
cell death. Pro-apoptotic stimuli trigger activation of the initiation
phase that in turn would activate the effect or phase that would
induce apoptotic changes in nuclei (Saraste and Pulkki, 2000). Only
after this degradation phase apoptosis becomes morphologically
evident. Apoptosis can take to either or both of two pathways, caspase-8-associated death receptor pathway (or) extrinsic pathway and
caspase-9-associated mitochondria-mediated intrinsic pathway
(Bouaziz et al., 2008; Kumar et al., 2011). In both pathways
procaspase-3 is cleaved to active caspase-3, the executioner caspase,
which in turn cleaves PARP protein that is involved in the DNA repair
mechanism (Yu et al., 2006; Kumar et al., 2011). Therefore, cleaved
PARP expression is considered a hallmark of apoptosis (Kumar et al.,
2011). OTA and CTN, alone as well as in combination, decreased the
expression of procaspase-9 where upon expression of cleaved
caspase-9 was enhanced whereas procaspase-8 expression was unchanged and cleaved caspase-8 was not at all expressed. These results
indicate that the mycotoxins do not trigger the extrinsic pathway,
which involves caspase-8, but trigger the mitochondria-mediated
intrinsic pathway, which involves caspase-9. Since the test toxins
induced mitochondria-mediated caspase-9-dependent apoptosis, we
were led to check the downstream events viz., expression of executioner caspase-3, pro-apoptotic protein Bax, and also PARP cleavage.
We noticed that procaspase-3 expression was reduced and cleaved
caspase-3 expression was increased in the treated cells which
represent an irreversible point in the process of apoptosis. The proapoptotic protein Bax and cleaved PARP expressions were signicantly enhanced in the treated cells which conrm the involvement
of mitochondria in apoptotic cell death induced by the test mycotoxins individually as well as in combination.
A few reports on in vitro (Schwerdt et al., 1999; Bouaziz et al.,
2008; Chopra et al., 2010; Zhang et al., 2009; El GolliBennour
et al., 2009) and in vivo (Atroshi et al., 2000) models to evidence
the hepatotoxic effect of OTA which identied that OTA induced
oxidative DNA damage and caused activation of P53-caspasedependent (Bouaziz et al., 2008, 2011) and caspase-independent
mitochondria-mediated intrinsic, and FAS ligand-mediated
extrinsic pathways of apoptosis (Chopra et al., 2010). Oxidative
stress-mediated DNA damage or the vice versa remains inconclusive. Our results pertaining to OTA-induced hepatotoxicity on
HepG2 cells match the previous ndings.
CTN has been reported to cause apoptosis in vitro (Yu et al.,
2006; Chan, 2007; Chan and Shiao, 2007; Chan, 2008; Chen and
Chan, 2009; Kumar et al., 2011) and it was suggested that the
genotoxic property and induction of oxidative stress of CTN remain
controversial but the basis of induction of apoptosis could be ROSmediated DNA damage and mitochondria-mediated apoptotic
caspase cascade activation. Our results concur with these reports.
Even though the individual mechanisms of action of the two

toxins differ, as a combination at low concentration the toxins

behave differently to exert toxicity on target organs. When either
one is present in the body it could affect the susceptible organ at
high concentrations but together the toxins can be toxic at a lower
ratio. Altogether, we hypothesized that OTA CTN induced ROS
and DNA damage, in that sequence, followed by loss of mitochondrial membrane potential leading to release of cyt C, and facilitated
the apoptotic cell death along the intrinsic pathway. Since ROS is
involved in the induction of cell death, we tested Vit E, a natural fatsoluble anti-oxidant, which preserves the cellular and sub-cellular
membrane functions and reduces the lipid peroxidation (Pfohl sch-Saadatmandi et al.,
Leszkowicz, 1997; Baldi et al., 2004; Bo
2012) to nd if it would protect the cells from mycotoxinsinduced cellular damage. Treatment of Vit E at 20 mg concentration along with OTA and CTN individually could reduce the cytotoxicity moderately whereas co-treatment of Vit E with OTA and
CTN mixture reduced the cytotoxicity to a great extent and thus
increased the viability of HepG2 cells. The level of ROS was reduced
in time-dependent manner after the co-treatment of Vit E with OTA
and CTN individually as well as in OTA CTN cobination. The results suggest ROS as the cause of cellular damage in OTA and CTNinduced hepatotoxicity and the results are consistent with the
previous ndings (Baldi et al., 2004; Hundhausen et al., 2005; Chen
sch-Saadatmandi
and Chan, 2009; El GolliBennour et al., 2009; Bo
et al., 2012). In order to clarify the link between induction of ROS
generation and DNA damage we treated the cells with OTA and CTN
individually as well as in combination along with Vit E, and the
results revealed that the genotoxicity caused by OTA was not
reversed by the antioxidant treatment. Therefore, it is clear that in
the present context DNA damage caused by OTA is not mediated by
ROS, and ROS is an event later to DNA damage (Hundhausen et al.,
2005) although the view that OTA-induced genotoxicity involves
oxidative DNA lesions coupled with direct DNA adducts is alive
(Pfohl-Leszkowicz and Manderville, 2007). Further investigation is
needed to nd how precisely OTA-treatment causes DNA damage.
On the other hand, CTN-induced genotoxicity was greatly reversed
by Vit E treatment which shows that CTN-induced DNA damage is
mediated by ROS. Interestingly, the genotoxicity induced by the
combination of OTA and CTN was also not completely reversed
since there was still a slight damage to DNA. Although the cytotoxicity and level of ROS were reduced, the genotoxic property of
OTA and CTN as a combination was not completely alleviated by the
anti-oxidant Vit E. Apparently, the individual genotoxic properties
of OTA and CTN take different courses, but together they become
strong genotoxic agents.
OTA and CTN co-occurrence was noticed after the outbreak of
Balkan endemic nephropathy in Bulgarian villages (PetkovaBocharova et al., 1990; Vrabcheva et al., 2000). The concentration
of OTA and CTN found in cereals varies between <0.5 and 140 mg/kg
and <5e420 mg/kg, respectively (Vrabcheva et al., 2000). According
to our results, as a combination OTA 42 mM CTN 31 mM (corresponding to OTA 16.9 ppm CTN 7.7 ppm) was found to be cytotoxic and genotoxic, and at OTA 21.0 mM CTN 15.5 mM
(corresponding to OTA 8.4 ppm CTN 3.8 ppm) concentration
inict genotoxicity in the HepG2 cells. Therefore, as a combination
OTA and CTN act synergistically and can be potentially hepatotoxic
to humans and animals. However, there are two major issues to
consider at this point. First, the use of HepG2 as the hepatocyte
model. We are fully conscious of a few limitations in this cell model
as a hepatocyte. It is in fact a hepatocarcinoma cell, the expression
of quite a few genes concerned with liver metabolism having been
compromised. Our choice of this cell was based on authors who use
this cell as normal in the context of hepatotoxicity studies (Tables 1
and 2) and, also, we did not have access to normal or closer-tonormal human liver cells. Having realized these limitations in our

L. Gayathri et al. / Food and Chemical Toxicology 83 (2015) 151e163

161

Table 3
Summary of plasma levels of OTA and CTN in the human.
Toxin

Human population

Technique

Liquid chromatography with uorescence

detection (LCeFLD)
University students in Bangladesh
High pressure liquid chromatography (HPLC)uorescence analysis
Canadian
Liquid chromatography with uorescence
detection (LCeFLD)
Scandinavian blood donors from Oslo, Norway, and HPLC
from Visby, Sweden)
German adults
LCeMS/MS-based analysis

Ochratoxin Valencia (Spain)

Citrinin

present study, we intend to extend this study to cryopreserved

human primary hepatocyte. The second issue is concerned with
in vitroein vivo correlation. It is difcult to correlate the toxin
concentration applied in cell culture to the dose used in whole
animals in view of potential toxicokinetic variations. This issue is
confounded by the potential toxicokinetic variation between animal models and humans. Generally, the doses used for in vitro investigations are invariably several times higher than real time
in vivo exposure (Glden and Seibert, 2006). Nevertheless, this
study which is only in vitro, clearly shows that OTA and CTN if
happen to occur concurrently act synergistically and this inuence
can as well be extended to humans and animals in vivo unless
future in vivo/clinical studies prove otherwise. Another perspective
of in vitro-in vivo correlation pertains to the very low plasma levels
of OTA and CTN in humans (Table 3) which are orders of magnitude
lower than in experimental animals or concentrations applied in
llmann et al., 2014).
in vitro studies for hazard characterization (Fo
Therefore, in future studies on combinatorial toxicity of such mycotoxins, it would be pertinent to correlate the in vitro data with the
plasma levels of the concerned mycotoxins.
5. Conclusion
The present study dwelt upon the cytotoxicity and mechanism
of cell death caused by OTA and CTN individually and in combination in HepG2 cells to elucidate the hepatotoxicity with special
reference to the binary exposure. It is revealed that OTA-induced
cytotoxicity is mediated by direct DNA damage whereas CTN
caused ROS-mediated DNA damage. Cells suffering from DNA
damage directly or through ROS take to intrinsic pathway of
apoptotic cell death. For the rst time the present study reveals that
OTA and CTN as a combination has the ability to induce cytotoxicity
at a dose 20% of the respective IC50 concentrations, accompanying
greatly ROS-independent DNA damage and mitochondriamediated caspase-dependent intrinsic pathway of apoptotic cell
death. Therefore, combination of OTA and CTN poses a signicant
health risk to humans and/or animals and calls for further assessment of combinative toxicity of mycotoxins. Further studies on
hepatic-metabolism and metabolism-dependent toxicity of OTA
and CTN, individually as well in combination, including in coculture and in vivo models, is relevant to explain the basis of the
hepatotoxicity since while undergoing biotransformation reaction
the action of CYP enzymes on OTA and CTN individually as well as
in combination may vary and could produce either toxic or nontoxic metabolites which in turn would target the organ (liver)
that metabolizes the parent toxin or other target organs like kidney.
Thus, it is important to consider the hepatic metabolism of OTA and
CTN, especially in combination, to understand their synergism in
the toxic manifestations. OTA and CTN only were considered in this
study but possibly in nature several combinations of mycotoxins
may be present in food and feed commodities. Therefore, there is

Levels in blood plasma

Reference

0.15e5.71 mg/l

Medina et al. (2010)

0.20e6.63 ng/ml

Ali et al. (2014)

0.29e2.37 ng/ml

Scott et al. (1998)

0.18 ng/ml in Oslo and 0.21 ng/ Thuvander et al. (2001)

ml in Visby
0.11e0.26 ng/ml
Meinolf Blaszkewicz
et al. (2013)

need to identify the minimum/tolerance dose of combinatorial

mycotoxins in foodstuffs to avoid the health risk in humans and/or
animals.
Acknowledgment
The nancial assistance from Doerenkamp-Zbinden Foundation,
Switzerland, is heartily acknowledged. The Visiting Professorship to
M. A. Akbarsha from King Saud University, Riyadh, KSA, is also
acknowledged.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.fct.2015.06.009.
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