I.

HISTORY AND SCOPE OF TOXICOLOGY

A. Antiquity: Romans, Greeks, Chinese

• Knowledge of drugs and poisons

• Categorized and characterized poisons
• Assassinations, Royal "tasters"…

Socrates (470-399 B.C.):

• most celebrated poisoning victim
• executed by poison hemlock
• active principle: coniine (affects
peripheral nervous system)

Theophrastus (370 -286 B.C.)

• a student of Aristotle,
• most influential botanist of antiquity
• Published De Historia Plantarum with references to poisonous plants

B. Middle Ages: Italians developed poisoning into art

1. Venice's "Council of Ten" (governing body of Republic of Venice)

• Dispensed contracts to poison political enemies

• Council transactions show detailed records with name of victim,

contractor, type and dose of poison, results…

2. Borgia: prominent family "applied toxicologists"

• Cesare, Lucretia
• Victims: husbands, wives, lovers, political opponents, clergy…
• Donizetti’s 1833 Opera “Lucrezia Borgia”

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 3. Catherine de Medici (1519-1589)

• Ruthless wife of Henry II of France, mother of three French kings,

ruler of France – “The Bloodiest Queen”
• Early "experimental toxicologist"
• Experimented on the underclass – homeless, sick
• Killed political enemies for hire…
• Documented signs and symptoms

C. Age of Enlightenment:

1. Paracelsus (Philipus Aurelius Theophastrus Bombastus von Hohenheim-

Paracelsus) (1493-1514)

• Pioneered use of minerals and plants in medicine

 Introduced mercuric compounds for treatment of syphilis

• Instrumental in logical development of toxicology as science

• Developed concept of "dose"

• Action a result of chemical entity—toxicon

• His book On the Miners' Sickness and Other Diseases of Miners

documented silicosis and other occupational hazards of mining

• Challenged Galen’s theory of disease as an “imbalance of the four

body humors”: blood, plegm, black bile and yellow bile.

 Paracelcus: illness from attack by external agents

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Paracelcus’ Toxicological axioms:

1. experimentation essential to determine action of toxic chemicals

2. one can distinguish between therapeutic and toxic actions of
chemicals
3. therapy vs toxicity not always discernible except by dose
4. therapeutic and toxic action of chemicals are often specific

2. Mathieu Orfila (1787-1853) Spanish born French toxicologist and

chemist--personal physician to Louis XVIII "The Father of Modern
Toxicology"

• Established toxicology as science—described toxicant disposition:

 absorption, distribution, and elimination..

• Compiled chemical and biological information on most known poisons

• Proposed necessity of chemical analysis to prove cause-and-effect

D. Modern Era:
• 19th Century: analytical methods developed for poisons like As, Hg..
• Modern toxicology is multi-disciplinary, comprising scientists from
diverse fields, such as:
• biochemistry,
• molecular biology,
• engineering,
• epidemiology,
• chemistry,
• biology,
• genetics,
• microbiology,
• public health
• statistics
• pathology
• nutrition and food science, and others

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 II. Basic Principles Of Toxicology

A. Dose: is the single most important factor of toxicity....

1. Types of doses or exposures:

a. acute: exposure for a duration of less than 24 hr; often a single

exposure...

b. subacute: repeated exposure for a month or less

c. subchronic: exposure duration from between 1-3 months

d. chronic: exposure > 3 months, often lifetime......

oral (p.o.; as in diet), intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.)

dose

acute subacute subchronic chronic

1 day 1 month 3 months

B. Response: assumptions...

• chemical interacts with a molecular receptor to produce a response...

• response is related to the concentration of the compound at receptor...

• the concentration of the compound at receptor related to the dose....

1. Acute: rapidly developing, with a short and relatively severe course

• high dose CN- : immediate loss of consciousness and death in a few

minutes.....

4
 KCN poison pills taken by prominent Nazis Hermann Goering and
Heinrich Himmler at the end of WWII.

2. Subacute: symptoms generally not as severe, but toxic effects often same
as acute ...

3. Chronic: progresses at a slow and varying rate; may be mistaken for other
diseases. Often difficult to determine cause-and-effect

 asbestos related cancer appears 20-30 years post-exposure.

• acute exposure may result in acute or chronic effects.....

 acute exposure to asbestos may lead to cancer..

• chronic exposure may result in chronic or acute effects.....

 chronic lead exposure may lead to subacute or acute

symptoms.....

4. Accumulative Effects: occurs two ways...

• accumulation of toxin: exposure to heavy metals (lead, mercury) with long

half-lives result in toxic response due to metal accumulation....

• accumulation of effect: exposure to low level organophosphate pesticides

inhibit acetylcholine esterases to an extent where symptoms begin

5. Delayed Effects: effect may occur only after long exposure; agent cannot
be found in blood or tissues. Damage to system already done....radiation sickness

5
C. Dose and Effect: The dose-response curve,

dose (independent variable) on x-axis, and the effect (dependent variable) on y-

axis...

effect may be quantal (all-or-

none) death; or continuous
(graded), blood pressure,
respiration rate, urinary output,
hormone levels....

D. Evaluating Dose-response:

1. For precision, dose-response curve usually plotted as log dose vs. % response....

2. Both are from the same experimental data, but log-dose scale results in a more
linear data representation..

3. the linear portion of the curve (~16 - 84%) is used to calculate toxic potency.

4. One measure of short-term toxic potency is the LD50 -- the statistically-derived

single dose of a substance that can be expected to cause mortality in 50% of the
test animals...

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Here, LD50 = 100 mg/kg

This dose-response curve is roughly

linear between ~16 and 84%, representing
± 1 standard deviation (SD) of the mean.
This curve can be transformed to more
accurately describe the data...

⇐ Dose-response curve..

In the histogram, each bar represents the %

of affected at each dose minus those
affected at the immediately lower dose. In
the normal distribution, the mean ± 1 SD
represents 69.3% of the population, ± 2 SD
is 95.5% and ± 3 SD is 99.7% of the
population. Most respond to the mean dose.
Those responding to lower doses are
sensitive, while those responding to higher
doses are resistant.

Response is transformed to units of

deviation from the mean, called normal
equivalent deviations (NEDs). The NED for
50% response is 0; -1 for 15.9%, +1 for 84.1%
etc.

To avoid negative numbers, probability units

(“PROBITS”; = NED + 5) result in a more
linear curve, improving the accuracy of
species-to-human extrapolation, and the
eventual the risk calculation.

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 % response NED PROBIT
0.1 -3 2
2.3 -2 3
15.9 -1 4
50.0 0 5
84.1 +1 6
97.7 +2 7
99.9 +3 8

E. What can we learn from dose-response?

1. Predictability: slope of curve...

The slope of the dose-response curve gives an

indication of data variability. Compounds "A" and
"B" have the same LD50 value, although the slopes
are different. The slope of “B” is greater than
that for "A." For "A," more chemical is required
to cause a significant change in response
compared to “B.” A chemical showing a steep
dose-response is usually associated with less
variability and greater predictability.

2. Potency: position along the x-axis.....

Position along the x-axis
indicates potency. The curve
associated with the more potent
compound is to the left because
less is needed to produce an
equivalent response compared to
the curve on the right.

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Comparable toxic potencies:

Chemical Agent LD50 (mg/kg; rat, oral)

ethyl alcohol 10,000
table salt 4,000
sodium pentobarbital 150
strychnine 2
tetrodotoxin 0.10
Tetrachlordibenzodioxin ("dioxin") 0.001
botulinum toxin 0.00001

3. Margin of safety.....

Desirable and undesirable effects of drugs.

ED is effective dose, TD is toxic dose,
resulting in some sort of adverse or side
effect(s), while LD is lethal dose.

Therapeutic index = TD50/ ED50;

Margin of safety = TD1/ ED99.

4. Potency vs. Efficacy.....

Left: Compound "A" is more potent than
compound "B." Both "A" and "B" also reach
maximum efficacy.

Right: difference between potency and

efficacy . At low doses, "C" is more potent
than "D." But "D" has a greater maximal
efficacy than "C." Higher doses of "C" cause
no increase in effect.

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5. Chemical interactions:

a. Addition.....

The effects of two chemicals, when

given together are often additive -
the combined effect is predictable
based on their known toxicity. 1 + 1 = 2

The combined effect of two

b.Synergism.... compounds may be greater than the
predicted sum of their effects or
synergism. The pesticide synergist
piperonyl butoxide is added to some
insecticides to increase insecticidal
activity. 1+ 1 = 10

Effect of A is reduced by antagonist B.

1) functional antagonism: simple
counterbalancing of the toxic effect (
phenobarbital for strychnine poisoning);
c. Antagonism.... 2) chemical antagonism: antagonist
reacts with toxin (dimercaprol
chelates As);
3) receptor antagonism: antagonist
binds to same receptor as agonist,
(atropine with organophosphate
insecticides);
4) dispositional antagonism: alters
fate of toxin (cholestyramine binds to
toxic chemicals, preventing absorption).
1+1=0

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F. Target Organ Toxicity: many toxins do not produce general effects but are
specific to one or more tissues or organs......

• vinyl chloride: liver cancer

• asbestos: mesothelioma

• paraquat: pulmonary fibrosis

• cadmium: renal tubular damage

target organs are often not the site of the highest concentration of a chemical

• Lead concentrates in bone, but its effects are mainly seen in soft
tissues, such as liver, kidney and blood cells…

• DDT accumulates in adipose tissue (fat stores), but produces no

effects there...it is primarily a central nervous system toxin......

Reasons for Tissue-Specific Toxicity:

• Preferential accumulation : toxicant may accumulate in only certain

tissues, and cause toxicity....

Cd in kidney, paraquat in lung

• Selective metabolic activation: enzymes needed to convert a compound

to the active form may be present in highest quantities in a particular
organ…..

CCl4, aflatoxin B1, nitrosamines in liver

• Characteristics of tissue repair: some organs may be susceptible

because they lack sufficient repair capabilities....

nitrosamines in liver

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• Specific receptors and/or functions: toxicant may interact with
receptors in a given tissue....

curare: a receptor-specific neuromuscular blocker

• Physiologic sensitivity:

nitrite: oxidizes hemoglobin (methemoglobinemia)

cyanide: inhibits cytochrome oxidase (cellular anoxia)

barbiturates: interfere with medullary O2 and CO2

sensors....(asphyxia)

Study questions:

1. Describe how toxicology evolved into a modern science. Name some of the
persons who made significant contributions.
2. List and describe several sub-disciplines that are part of toxicology.
3. What is the single most important factor in chemical toxicity?
4. Define dose-response relationship. Sketch out a sample dose-response curve,
properly labeling the axes.
5. Define and compare acute, subacute, and chronic exposures and responses.
6. Does acute exposure necessarily result in acute response? Does a chronic
exposure necessarily result in a chronic response?
7. Define antagonism; what is the difference between functional, chemical,
receptor, dispositional antagonism?
8. Why do many chemicals affect certain target organs?
9. What is a probit transformation, and why is it used? Transform a log-dose
response curve to a log-dose probit curve.
10. How can we use the dose-response curve to describe potency, predictability
and various types of chemical interactions?
11. What is chemical synergism? Is it a common phenomenon? Give two examples.
12. What is a margin of safety? a therapeutic index?

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 III. DISPOSITION OF CHEMICALS IN THE BODY

Routes of absorption, distribution and excretion of toxicants in the body:

ingestion inhalation intravenous intraperitoneal

subcutaneous
intramuscular
Gastrointestinal Lung
tract
Portal
Liver blood dermal
Blood and Lymph
Extracellular
fluid Fat
Bile

Kidney Lung Secretory Organ

structures s

Bladde Alveoli Soft Bone

r tissues

Feces secretion
Urine Expired s
air

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A. ABSORPTION - The process by which toxicants cross body membranes and
enter the bloodstream

For a chemical to cause its effect, it must enter a cell. To enter a cell, it must
penetrate a biological membrane:

1. Biological membranes are primarily lipid – fatty acids. Non-polar, lipid soluble
compounds are therefore absorbed most efficiently.....

Biological membranes are composed

of a protein-lipid bilayer. The lipid
portion is primarily phospholipid,
which have ionic, polar head groups
oriented outward (the spheres),
and nonpolar lipid chains (tails)
oriented inward. The globular
structures represent integral
membrane proteins, which may be
transport, receptor or other types
of proteins....

Transport of drug through membranes occurs by one of two general processes:

passive diffusion and specialized transport

2. Passive diffusion (major route--most toxicants enter this way……)

hydrophilic compounds: through aqueous channels in membrane

hydrophobic compounds: (most toxicants and drugs) via lipid

portions of membrane

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Rate and extent of absorption of hydrophobic chemicals depends on three
factors:

a. Lipid solubility: measured by octanol:water partition coefficient (Kow, Pc)

Lipid solubility of a compound is commonly

measured by adding it to a mixture of water (polar,
hydrophilic phase) and octanol (non-polar,
hydrophobic phase) in a separatory funnel. The
amount of compound in each phase is then
measured. The relative amount of compound in
either phase gives the partition coefficient (Pc;
octanol/water: Kow) of the compound. The higher
the Pc, the more lipid soluble the compound.....

a. a compound with a large Kow is generally able to efficiently enter a

cell....

b. Kow increases with increasing chain length

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 b. Molecular Size: small particles absorbed more readily than larger

c. Degree Of Ionization: many drugs and toxic chemicals exist in either

ionized or non-ionized form…..

a. Nonionized: more lipid soluble; diffuse through membranes

b. Ionized: susceptible to ionic interactions with membranes--

absorption is hindered (less lipid soluble)

Degree of ionization dependent upon:

dissociation constant of the chemical (pK)

pH of the environment,

according to the Henderson-Hasselbalch relationship:

for weak acids: pKa - pH = log [nonioinized]

Acids: like R-COOH, etc...
[ionized]

for weak bases: pKb - pH = log [ionized]

Bases: like R-NH2
[nonionized]

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consider the ionization of two compounds:

benzoic acid (weak acid; pKa = 4)

aniline (weak base; pK = 5)

Percent of species in non-ionized form

pH benzoate aniline
1 99.9 0.01
2 99.0 0.10
3 90.0 1.00
4 50.0 10.0
5 10.0 50.0
6 1.00 90.0
7 0.10 99.0

What is the theoretical proportion of sodium phenobarbital (pKa = 7.2) present in its
most lipid soluble form in the stomach (pH 2) and in the urine (pH 6.5)?

[HA] [HA] 15.8x104

pKa −pH = log ;7.2 −2 = 5.2 = log = = 99.999%
[A−] [A−] 1

[HA] [HA] 5
pKa −pH = log ;7.2 −6.5 = 0.7 = log = = 80%
[A−] [A−] 1

17
€
3. Special transport

a. active transport:

• absorption against concentration gradient

• saturable
• selective
• requires energy

b. carrier-mediated

c. endocytosis: pinocytosis/phagocytosis

• requires energy

• peptides, antigen:antibody complexes

4. Absorption of chemicals by specific routes

a. percutaneous route

 skin layer is several cells thick and good barrier to absorption

A. transepidermal (most here)

B. transfollicular--via sweat glands and follicles (Pb)

 absorption of chemicals through the skin is promoted by:

• high lipid solubility and nonionization

• sweaty, hot skin

• wrapping of skin

• abrasion, injury, rash, presence of solvents

18
b. Inhalation -- rapid absorption

 Airway epithelium is 2-3 cells thick

 respiratory epithelium is 1-2 cells thick…

• principal route of absorption for gases, vapors, aerosols

c. Gastrointestinal route (G.I.)

• epithelial linings one cell thick

• intestines have extremely large surface area in many areas

 stomach not as good an absorptive surface and is continually

emptying)

• pH gradient: diffusion occurs where it is present in most lipid

soluble form:

weak acids diffuse best at lower pH (stomach--pH 2)

weak bases diffuse best at higher pH (intestines--pH 6)

• some compounds absorbed well, regardless of pH/pK

characteristics:

paraquat (has two positive charges), 2-PAM

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 • factors promoting absorption:

ulceration, GI irritation, fasting

• factors decreasing absorption:

 stomach contents

Chemical absorption and

subsequent “bioavailability” is
often decreased on a full
stomach…

Plasma
concentration
of chemical

time

20
B. DISTRIBUTION -- Movement of chemical throughout the body….

1. Factors affecting characteristics of distribution:

a. binding to charged particles:

the blood contains charged proteins to which chemicals will bind (eg.,
albumin, immune globulins, and transport proteins...)

chemical ⇔ bound chemical

(crosses membranes) (does not cross membranes)

b. fat storage

depends on lipophilicity of compound

fat person: 50% of body weight is fat

lean person: 10-20% of body weight is fat

c. apparent volume of distribution (Vd) = total amount of drug in body

drug blood concentration

theoretical volume of fluid into which the total drug administered would have
to be diluted to produce the concentration in plasma

Body water exists in three distinct compartments:

Compartment % of total L in 70kg [P] after 1g

person of chemical
Plasma water 4.5 3 333 mg/L
Total extracellular 20 14 71 mg/L
Total body water 55 38 26 mg/L

21
Vd of water soluble chemicals with a low Kow will be close to 3L or equal to that of
plasma water

Vd of lipid soluble chemicals with high Kow will be greater than 3L

examples:

Acetylsalicyclic acid Vd = 0.15 L

Chlorpromazine (thorazine) Vd = 21 L

C. EXCRETION AND ELIMINATION

toxicants are eliminated from the body by a variety of routes:

1. urinary excretion (by far the most important)

kidney receives 25 % of cardiac output, 20 % of which is filtered at

the glomerulus

2. fecal excretion

3. pulmonary tract most important for gases and volatile drugs: benzene,
anesthetics, CCl4, toluene, methanol

4. other minor: vomiting, milk, perspiration, tears

22
D. BIOTRANSFORMATION

Overview of chemical metabolism:

Absorbed chemicals are modified by the body to increase excretion and limit
toxicity

• benzene is metabolized by an initial (or "phase I”) reaction to phenol

• phenol is more hydrophilic is easily excreted.....

• phenol may be further metabolized (called a “phase II”reaction – in this case by a

sulfate molecule) to further increase hydrophilicity.....

23
 Cytochrome P450 (CYP). The most important enzyme in chemical metabolism –
normal function is steroidogenesis

the majority of the phase I reactions are catalyzed by a family of enzymes

called cytochrome P-450 (CYP) (P = pigment; 450 = absorption max when
bound to CO)

a. Characteristics of P450

• hemoprotein (contains iron atom in active site), > 6000 genes; >2200 in
animals

• highest in liver but also present in other tissues

• membrane bound, in the smooth endoplasmic reticulum (SER)

(microsomes)

• inserts a molecule of oxygen into substrate

• requires electrons to reduce Fe+3 to Fe+2

• product may either be excreted or further metabolized (see phase II)

Results usually a stable detoxified metabolite, sometimes a more toxic metabolite!

b. CYP nomenclature:

CYP1A2

Family: Subfamily:
> 40% amino acid > 55% amino acid
sequence homology sequence homology

Contribution of Human P450s in metabolism of all commercial drugs

24
Human P450 Substrate specificity:

Chemical CYP
Benzo(a)pyrene, other PAHs 1A1
Aflatoxin B1, heterocyclic amines 1A2
Tobacco-specific nitrosamine (NNK) 2D6
Benzene, styrene, TCE, vinyl chloride 2E1
Nitropyrene, 6-aminochrysene, aflatoxin 3A4
B1

Endogenous substrates for P450s –

cholesterol and bile acids (CYP7, 8),
steroids (CYP11, 19, 21),
prostaglandins (CYP5, 8)
Vitamins A and D (CYP24, 26, 27)
Other eicosanoids (CYP 2C, 2J, 4A, 4B)

25
Codeine and P450 polymorphisms

b. mechanism and sequence of P-450 catalysis:

26
PHASE I REACTIONS

a. Reactions catalyzed by Cytochrome P450:

1. aromatic and aliphatic hydroxylation:

27
2. Nitrogen hydroxylation:

3. Dealkylation and deamination:

28
4. Epoxidation:

5. De-sulfuration:

B. Reduction reactions. By oxidoreductases, P450, and by intestinal microflora.....

29
C. Hydrolysis reactions (add H2O to ester groups)

30
Epoxide hydrolase –important hydrolytic enzyme for environmental chemicals:

• in microsomes and soluble cellular fractions in liver, lung, kidney and

intestines

• catalyses the trans addition of H2O to epoxides to produce trans diols

• Deactivates many reactive "ultimate carcinogens," but is also involved in

the "activation" process of some carcinogens, such as benzo(a)pyrene...

31
  
Carcinogenic products
from P450 metabolism

The majority of P450

reactions result in less toxic,
more hydrophilic metabolites.

However, P450 also

“bioactivates” thousands of
environmental chemicals into
highly reactive, unstable
electrophilic (“electron loving”;
Lewis acids) intermediates
that react with critical cellular
nucleophiles (“nucleus loving”;
Lewis bases), such as DNA,
RNA, proteins, etc. resulting in
toxicity and cancer.

“Pro-carcinogens” which are

not carcinogenic per se but
become so after bioactivation,
include benzene, styrene,
nitrosamines, aflatoxin,  
benzo-a-pyrene, vinyl
chloride, and many others.

32
 PHASE II REACTIONS

• chemicals modified by phase I reactions can be further metabolized by a

variety of “phase II” reactions to form "conjugates...

• phase II reactions further increase hydrophilicity, promoting excretion.....

• products of phase II reactions (called "conjugates), are generally detoxified

compared to the product of phase I metabolism.

A. Sulfotransferase reactions:

• Enzyme: Sulfotransferase

• Coenzyme: Phoshoadenosine-
5'-Phosphosulfate
("PAPS")

B. Glucuronyl transferase
reactions:

• Enzyme: UDP-Glucuronyl Transferase

• Coenzyme: Uridine diphosphoglucuronic acid (UDPGA)

(during conjugation UDPGA is interconverted from α to β configuration)

33
C. Methyl transferase reactions:

• Enzyme: O-Methyltransferase

• Coenzyme: S-Adenosyl Methionine ("SAM")

D. Glutathione S-transferase reactions: important detoxifying pathway for

electrophiles

• Enzyme: glutathione S-transferase (GST)

• Coenzyme: glutathione (γ-glutamyl L-cysteinyl glycine; GSH)

glutathione is a tripeptide with a backwards or γ peptide bond; The SH group is

nucleophilic (Lewis base: electron rich), and reacts with electrophiles (Lewis acids:
electron poor)

Inactivates compounds that are metabolized to an electrophilic (electron poor) atom,

which are highly reactive and toxic...

34
GSH conjugates then cleaved to cysteine derivatives in the kidney....

GST detoxifies Aflatoxin B1

1. GSH/GST detoxifies dinitrochlorobenzene through a nucleophilic substitution

reaction with the electrophilic intermediate, leaving a detoxified, safely excreted
GSH conjugate

"SG" is usually the cysteine derivative of GSH; glutamate and glycine are
by-products.....

2. GSH/GST detoxifies bromobenzene, which is metabolized to a reactive,

carcinogenic electrophile by P450s. The detoxified GSH conjugate is safely
excreted

35
Enterohepatic circulation:

• Polar conjugates (products of phase I and II metabolism) formed in the

liver are secreted into the intestine via the bile duct and gall bladder.

• Conjugates are often hydrolyzed in the intestines by endogenous and

bacterial enzymes.

• Resultant free xenobiotics are reabsorbed and transported back to the

liver via the portal vein.

• A chemical may undergo several cycles of this process resulting in a

significant increase in retention time, possibly increasing its toxicity.

liver

stomach

Gall
bladder

Bile
duct

intestines Portal
vein

36
Study questions:

1. Describe the molecular structure and function of a biological membrane.

What is the significance of the cell membrane in toxicology?
2. How does lipid solubility affect absorption and distribution of a drug or
chemical?
3. How is lipid solubility measured?
4. Describe three factors that directly affect chemical absorption.
5. How does ionization affect chemical absorption and distribution. How is this
determined quantitatively?
6. Can a chemical still be toxic if it is not absorbed?
7. Name and describe three factors that affect chemical distribution.
8. What is volume of distribution and how is it calculated?
9. What is P450, and why is it so critical to toxicology?
10. What is the purpose of NADPH cytochrome P450 reductase?
11. Diagram the proper sequence of a P450-mediated metabolic cycle. Why is the
heme so important to the function of P450?
12. List and describe four types of phase II reactions, including the appropriate
enzymes and cofactors.
13. Does chemical metabolism always produce a detoxified product?
14. Explain the importance of GST in chemical detoxification. What kind of
substrates does GST interact with?
15. Explain how chemical metabolism may result in reduced or increased toxicity.
16. How are phase I or phase II enzymes induced by environmental chemicals or
food components?
17. Define enterohepatic cycling, and explain why it is important to chemical
disposition.
18. What kind of chemicals are most likely to be subject to enterohepatic
cycling?

37
 IV. TOXICITY TESTING AND RISK ASSESSMENT

Toxicity testing
Short-term genetic testing
Epidemiology
Low Dose estimation
Biological Monitoring
Risk Assessment and Risk Management
The Precautionary Principle

A. TOXICITY TESTING

Goal: to determine potential chemical toxicity to people

two underlying principles:

1. effects in laboratory animals are applicable to humans

how do we extrapolate from animal species to humans?

• by mg/kg body weight: on a body weight basis, humans are generally 10-
times more susceptible than animals

• by surface area: related to body weight2/3: determine surface area by

empirical formula:

m2= K•W2/3 /100,

K=species specific factor (rats=9; 70 kg human=10.6),
W = body weight in kg.

Equivalent dose levels in four species:

species weight dosage dose surface dosage

(g) (mg/kg) (mg/animal) area mg/cm2
cm2
mouse 20 100 2 46 0.043
rat 200 100 20 325 0.061
dog 12000 100 1200 5770 0.207
person 70000 100 7000 18000 0.388

38
2. Animal studies usually use high doses.

• a minimum of 30,000 animals would be required to detect a 0.01%

incidence of cancer (20,000 in 200,000,000)

• mathematical low-dose extrapolation methods are then employed to

estimate human risk associated with lower, “real world” chemical
exposures....

B. Types of Toxicity Tests:

1. Acute lethality: acute effects within 14 days (usually much sooner)

• a single dose

• lethality (different routes-usually oral)

• both male and female

• two species (usually rats and mice)

• acute oral LD50

• goals:

1. determine potency relative to other toxicants

2. identify target organs
3. determine if reversible

2. Subchronic testing: ~1-3 month duration

• daily dose

• two species (usually rat and dog)

• three dose levels

• blood chemistry, hematology, urinalysis

• complete organ pathogenesis

39
3. Chronic toxicity tests:

GOAL: to determine the accumulative toxicity and/or the carcinogenic potential

of a chemical or compound

• lifetime daily ingestion of toxicant in two species – rats (24 mo), mice (18
mo)

• use intended exposure route..... usually oral (but may be by inhalation...)

• dose selection: use maximum tolerated dose (that dose that slightly
suppresses body weight gain i.e., 10%) and fractions thereof

• pathology: all animals have gross necropsy after death by board-

certified pathologist

• complete gross and microscopic tissue examination (15-20 organs)

• complete hematology and blood chemistry workup

• special emphasis on target organs and animals that died prior to

termination of experiment

4. Other tests

• testing for carcinogens

• reproductive effects

• inhalation toxicology

• modern alternatives to animal testing - dermal toxicity using skin cells

40
5. Short-Term Genetic Toxicology Assays

goal: short-term, relatively inexpensive in vitro or in vivo testing to predict

possible carcinogenic effects of the test chemical

alternative: Investment of millions of $ in long-term animal cancer studies

there are two main types of carcinogens:

• genotoxic (interacts with DNA)

• epigenetic (not known to interact with DNA)

Because genetic assays detect Genotoxic Agents Only, some types of carcinogens
will not be detected.

IARC Classification of Human Carcinogens:

Group Evidence Examples

1.Confirmed carcinogen sufficient (human) arsenic, aflatoxin, benzene,
estrogens, vinyl chloride
2A. Probable limited human/sufficient benz(a)anthracene,
animal diethylnitrosamine, PCBs,
styrene oxide
2B. Possible limited-inadequate TCDD, styrene, urethane
human/sufficient animal
3. Not classifiable Sufficient animal, but strong 5-azacytidine, diazepam
evidience that cancer not
relevant to people
4. Probably not inadequate human and animal caprolactam

41
A. Salmonella / Mammalian Microsome (Ames) Assay: Most widely-used predictive
assay:

principle: bacteria is “test animal” whose DNA becomes mutated

when exposed to a mutagen. Because most mutagens are
also carcinogens, this assay is a powerful predictor of
chemical cancer risk.

endpoint: number of mutant colonies which are capable of growing on

plates of histidine-free medium; these represent the
-
number of cells that have mutated his his+ genotype.

Components Of Assay System:

a. Salmonella typhimurium (gram (-) enteric

bacteria) engineered with the following features
for increased sensitivity to mutagens:

• auxotrophic mutant--designated his-

unlike the "wild-type" bacteria, requires
histidine for growth

• “pkm 101”: a plasmid that codes for

"error-prone" repair

• “uvrβ” : UV light sensitive-DNA repair deficient

• “rfa” mutation: cell wall defect for increased permeability to test

compound

b. rat liver fraction: ("S9 mix" contains liver enzymes and cofactors)

1. "S9" liver homogenate (contains CYP)

2. glucose-6-phosphate, NADPH, MgCl2, KCl, NaHPO4, pH 7.4 buffer

c. plates of histidine-free minimal glucose agar

42
6. "Decision point" approach to results from Short and Long-term assays

A. structure of chemical

B. in vitro short-term tests

B.' decision point: tests under A and B

C. limited in vivo bioassays:

C.' decision point: tests under A, B and C.

D. chronic (lifetime) bioassay

E. final evaluation of all tests

43
B. Epidemiology -- early detection and prevention of risk and the study of the
distribution and determinants of disease..

….or toxicology studies where they let the subjects (people) out of their cages…

1. Three major types of epidemiology studies:

a. Case-control or “retrospective”—what are the cause(s) of the observed

disease?

b. Cohort or “prospective” “longitudinal” – what disease(s) will be caused by an

exposure?

c. Cross-sectional- “horizontal” – what is the exposure and outcome of a

population at a single time point? Or, “am I like my neighbors?”

44
 Three Types of Epidemiology Study Designs

Methodological Case-Control Cohort Cross-sectional

attributes
Initial Disease-non disease Exposure-non- Either
classification exposure

Time sequence Retrospective Prospective Present time

Sample Cases and controls Non-diseased Survivors
composition persons

Comparison Proportion of cases Proportion of Either

with exposure exposed with
disease
Rates Fractional (%) Incidence Prevalence
Risk Index Relative odds Relative risk— Prevalence
attributable risk
Advantages Inexpensive, small Lack of bias in Quick results
number of subjects, exposure; yields
rapid results, suitable incidence and risk
for rare diseases, no rates
attrition
Disadvantages Incomplete Large number of Cannot establish
information, biased subjects required; causation,
recall, problem in long follow-up, population of
selecting control and attrition, change survivors;
matching, yields only in time of criteria inadequate for
relative risk—cannot and methods, rare diseases
establish causation, costly, inadequate
population of for rare diseases
survivors

Another study design, not typically classified as “epidemiology” is the

Randomized, double-blind placebo-controlled clinical trial

Tobacco – lung cancer hypothesis – case study for acceptance of epidemiology as

science

45
2. A. Bradford Hill’s Criteria for Causation

a. Association is strong --- higher relative risks being more likely to indicate

cause…

b. Risk increases or decreases with exposure in a dose-responsive gradient

c. Consistent findings in several studies with different investigators,

different populations, and different designs

d. Exposure (cause) precedes the disease

e. It is biologically plausible that exposure could cause the disease;

f. The association is specific between exposure and a single disease

g. Epidemiologic findings fit coherently with information with other types of

research and other epidemiologic studies.

Reading assignments:

1. Case control study: Wynder and Graham Tobacco smoking as a possible

etiologic factor in bronchogenic carcinoma (JAMA 145:329 1950)--Landmark

study on the effects of smoking and health.

2. Chort study: Doll and Hill. The Mortaility of Doctors in Relation to their

Smoking Habits. (BMJ June 26, 1954).

46
 C. Low Dose Estimation

How do we extrapolate data from animal studies which typically use higher than
“real world” doses?

1. Reference dose (RfD), Acceptable Daily Intake (ADI), Benchmark Dose

NOAELs generated from these low-dose models often form the basis of maximum
exposure limits called reference doses (RfD) or concentrations (RfC), or Acceptible
Daily Intakes (ADI), depending on the regulatory agency:

(EPA, FDA) RfD = NOAEL / UF

(Reference dose is updated version of ADI…)

(WHO, FDA) ADI = NOAEL / UF

UF = uncertainty factor

47
These standards represent “safe” exposure limits that won’t result in an excess
incidence (usually > 1/106) of adverse health effects

Limitation of NOAELs:

1. arbitrary—by definition must be one of the experimental doses selected

2. once selected, rest of dose-response is ignored
3. experiments using fewer animals result in higher NOAEL values…
4. may not be available or reliable
5. depends on experimental design

Because of these limitations, the Benchmark Dose developed as an alternative

exposure standard (BMD = BMDL/UF)

Uncertainty factors (UF) take into account inherent uncertainty in interspecies (animal-
to-human) and intraspecies (human-to-human) extrapolations, as well as other
limitations:

BMD is modeled using the lower confidence limit for a dose at a specified
response level. Here, the BMDL of 8.3 µg/kg is determined from the 95%
confidence extrapolated from the dose-response curve fitted from
experimental data…

48
Uncertainty factors account for:

• Extrapolation: animal-to-human

• Extrapolation: human-to-human

• Experimental inadequacies:

o Less-than-lifetime exposure in animal studies

o Variations in NOAEL, LOAEL between studies

o Absence of NOAEL (extrapolation from LOAEL)

The majority of uncertainty comes from a combination of differences in

Toxicodynamics and Toxicokinetics inherent between and within species

10 10
interspecies

intraspecies

49
D. Risk and Hazard Assessment

The primary goal of toxicology is to assess risk posed by exposure to a toxic agent.

1. For risk assessment, a thorough knowledge of the terms hazard, safety, toxicity,
potency is essential:

• hazard: the probability that injury will result from a chemical under specific
conditions.

• safety: the practical certainty that injury will not result from use of a
substance

• toxicity: capacity of a compound to produce injury

• potency: the relative toxicity of a compound....

Risk is a function of hazard and exposure:

Risk = f(hazard, exposure)

Since every compound is toxic to some degree, and nothing is totally harmless, the
conditions of use must be defined for an accurate assessment of risk ......

Depending on conditions of use, a slightly potent compound may be

very hazardous, and .

an extremely potent chemical may be only moderately hazardous.....

50
2. Perception of risk. Illustrated by “risk-space” diagram:

3. Final management of risk

51
The Precautionary Principle: is a response to uncertainty, in the face of risks to
health or the environment. In general, it involves acting to avoid serious or
irreversible potential harm, despite lack of scientific certainty as to the
likelihood, magnitude, or causation of that harm. “Preponderance of evidence”
rather than scientific proof.

“Better Safe than Sorry”

4. Final outcome of risk assessment: exposure standards

a. Environmental Chemicals:

Reference Doses - environmental chemicals (EPA - Integrated Risk Information

System):

Chemical RfD (µg/kg/day)

PCBs 0.05
Mercury 0.1
Pentachlorophenol 30
Chloroform 1
Vinyl chloride 0.1 mg/m3 (RfC)
Arsenic 0.3
Bisphenol A 50
Nitrite 1.0 mg
acrylamide 0.2 mg

b. Chemicals in Foods:

Acceptable Daily Intakes- chemical residues in foods (FDA – CEDI/ADI Database):

Chemical ADI (mg/kg/day)

Ethylene Glycol 0.15
Mercury 0.4 (µg/kg/day)
Acrylic acid 0.135
1,4-butylene glycol 0.125
Castor oil, hydrogenated 1.0

52
c. Occupational exposures:

Threshold Limit Values (TLV-TWA)

Chemical TLV-TWA (ppm)

Arsine 0.05
Benzene 0.5
Chloroform 10
dinitrobenzene 0.15
nickel carbonyl 0.05

1. How are occupational exposure limits set?

OSHA sets only few industrial standards via in-house research and risk assessment,
called "Permissible Exposure Limits" (PELs)

• in practice PELS are derived from TLVs.

• TLVs are derived from volunteer committee assembled by the American

Conference of Government and Industrial Hygienists (ACGIH), called
the "TLV Committee."

• OSHA has adopted over 650 TLVs from this committee as PELs

• TLVs are generally lower than PELs for the same chemical

using accepted methods of risk assessment, committee creates non-binding,

"recommended" threshold limit values....

1. TLV-time weighted average (TLV-TWA)

The time-weighted average concentration for a normal 8 hr work day

and a 40 hr workweek, to which nearly all workers may be repeatedly
exposed without adverse effect....

53
 2. TLV-threshold limit value-short-term exposure limit (TLV-STEL)

The concentration to which workers can be exposed continuously for a

short period of time without suffering from a) irritation, b) chronic
or irreversible tissue damage or c), narcosis...

3. TLV-ceiling (TLV-C)

The concentration that should not be exceeded during any of the

working exposure...

E. BIOLOGICAL MONITORING

Evaluating dose through measuring internal concentration of chemical, metabolite or

a biochemical parameter related to dose (usually for chronically acting chemicals)......

1. what is measured?

• concentration of chemical in blood,

excreta or expired air....

• chemical species bound to a tissue

target (hemoglobin, albumin)

• biochemical alteration due to mechanism

of action (e.g., acetylcholinesterase in
plasma after organophosphate
exposure...)

biological monitoring is effective only when

the relationship between external
exposure, internal dose and adverse effects are known....

54
2. conditions for practical biological monitoring:

• test must be specific and sensitive

• biological samples easily taken

• parameter must be stable

• assay for parameter must be simple and rapid

• complementary to workplace monitoring

3. Selected Biomarkers: biological exposure index adopted by ACGIH

Chemical Biological Sample BEI

parameter type
Acetone acetone urine 50 mg/L
Benzene S-phenylmercapturic acid urine 25 µg/g crt*
1,3-butadiene 1,2-dihydroxy-4- urine 25 mg/l
(N acetylcysteinyl) butane
Nitrobenzene p-nitrophenol urine 5 mg/g crt
Nitrobenzene Methemoglobin blood 1.5% of Hb
Parathion p-nitrophenol urine 0.5 mg/g crt
Trichloro- trichloroacetic urine 100 mg/g
ethylene acid crt
Aniline methemoglobin blood 1.5%
Toluene Hippuric acid urine 1.6 g/g crt
Toluene Toluene blood 0.5 mg/L
Toluene o-cresol urine 0.5 mg/L
xylenes Methylhippuric acid urine 1.5 g/g crt
*creatinine

55
 WHO guidelines on urine specimens:

Creatinine concentration: > 0.3 g/L and < 3.0 g/L;

Or Specific gravity: > 1.010 and < 1.030

Biomarkers are based on either:

• the chemical itself

• a metabolite of the chemical

• a biochemical endpoint of toxic effect

F. REGULATORY TOXICOLOGY

1. why is regulation needed?

• scientists develop information

• information used to set “safe” exposure standards

workplace, environment, consumer goods, etc...

• therefore, legislative control is necessary...

• Rules are then formulated by various government agencies, who then

have enforcement responsibilities....

56
2. regulations are modified as:

• more scientific data becomes available

• increased sensitivity of analytical procedures...

• public pressure to increase or decrease legislation....

• regulatory climate of prevailing administration

burden of proof of safety or risk....varies depending on law:

• FDA: on the manufacturer or supplier to prove safety..

• OSHA: on regulatory agency, to prove that substance is

hazardous, before exposures can be restricted.....

3. Government agencies involved in regulating environmental and industrial

toxicants....

a. Federal Food, Drug And Cosmetic Act (1938)

b. Occupational Safety And Health Administration (OSHA; 1970)

• worker protection act

• worker “right-to-know”

• hazard communication standards, labeling of chemicals

• under Department of Labor

• established NIOSH

• MSDS: materials safety data sheets

57
c. Environmental Protection Agency

most comprehensive agency for control of toxic substances..

• Pesticides (Federal Insecticide, Fungicide And Rodenticide Act;

"FIFRA"

• Toxic Substances (Toxic Substances Control Act; "TOSCA")

• Hazardous Wastes (Resource Conservation And Recovery Act;

"RCRA")

• Toxic Water Pollutants (Federal Water Pollution Control Act)

• Toxic Air Pollutants (Clean Air Act)

• Toxic Water Pollutants (Clean Water Act)

• Pesticide Residues in Foods (Food Quality Protection Act “FQPA”)

EPA Information Databases: National Center for Environmental Publications and

Infomation (NCEPI)

a. Toxic Release

Inventory:

(http://www.ep

a.gov/tri/)

58
2006 EPA TRI Data ; TRI Releases by State 2006

TRI Releasing Facilities in the USA (2006):

A separate category of toxic chemicals reported in the TRI:

PBT (Persistent, Bioaccumulative, and Toxic) chemicals

16 PBT chemicals and 4 PBT chemical compound categories subject to reporting

PBT Chemical List

Study questions:

1. Explain how experimental data from animal studies are extrapolated to people.
2. What is the advantage of using an animal’s surface area as a dose criterion?
3. Why are high doses of chemicals used in animal studies?
4. Does a short-term acute animal study accurately predict all types of toxicity?
5. What is a carcinogen? Are all carcinogens “created equal”?
6. How are carcinogens ranked in the IARC classification scheme?
7. Explain how you would conduct an Ames assay, including all assay components.
8. Describe the process of risk assessment and risk management.
9. Identify and describe the three major types of epidemiology studies, and their
advantages and disadvantages.
10. What are the Hill’s Criteria for Causation?
11. Explain how data from animal studies are extrapolated to determine risk to “real
world” doses of chemicals people are typically exposed to.
12. What is a reference dose, and how is it calculated?
13. When is benchmark dose used to determine risk?
14. What is the difference between a reference dose, an acceptable daily intake and
a threshold limit value?
15. What are some factors that determine the uncertainty factor?
16. Define the “Precautionary Principle,” and explain its impact environmental
toxicology.
17. What is a biomarker? Give four examples of chemical biomarkers and explain how
they arise in the human body.
18. What is the TRI?
19. What is a PBT chemical, and why is it important?

59
 V. CHEMICALS IN THE ENVIRONMENT

Topics:

• Chemodynamics—adsorption, evaporation

• Half-life of chemicals in the environment

• Biomagnification

A. CHEMODYNAMICS

Chemical movement and fate are dependent upon:

water and soil characteristics (water and soil are the ultimate sinks for all
chemicals)

physical-chemical characteristics of the chemical

60
1. ADSORPTION to soils: tendency of chemical to distribute among two phases, from
solution to soils...

chemical (adsorbate)
soil (adsorbent) water (solvent)

in aqueous systems, many chemicals adsorb to soil particles or to soil particles

suspended in water...

• has an effect on distribution of compounds in the environment

• if compound is strongly adsorbed to soil, it won't be leached easily....

characteristics of soil important in adsorption:

1. surface area

2. presence of charged sites

3. hydrogen bonding sites

4. hydrophobic areas

A. Major soil fractions:

1. Mineral fraction

A. Clay: crystal structures of silica and alumina

Characteristics of clay:

1. Hydrophilic surface and water-holding capacity

2. Has cation exchange capability due to hydroxyl groups (pH

dependent)

3. large surface area

4. hydrogen bonding

61
Two types:
Kaolinite: 2-layered alumino-silicate bilayer

Montmorillonite: 3-layered... silica-alumina-silica trilayer

Building blocks for clay minerals:

-2
Silicon-Oxygen tetrahedron - Si2O5

-3
Aluminum Octahedron: Al(OH)6

62
 B. Sand: not important in toxicant movement….

2. Organic fraction: dark brown soil components formed through microbial

breakdown of plant and animal tissues.

Major components Humin, Humic Acid, and Fulvic Acid

1. High cation exchange capability (higher than clay)

2. Contain hydrophobic domains that bind toxicants directly....

3. Hydrogen bonding

63
B. Chemical bonds resulting in adsorption (in clay or organic matter)

1. Vanderwalls forces – weak interactions between molecules

2. Hydrophobic bonding:

Hydrophobic regions attract lipid soluble toxicants via

hydrophobic interactions.....

3. Hydrogen bonding: when H is bonded to F, O, N, Cl

C. Properties of adsorbate (chemical) affecting binding to soil:

1. pK of adsorbate /pH of environment (soil pH usually 4.5-8.5)

• Bases (cationic) bind to soil best: bind to cation exchange sites in clay

• Acids (anionic) bind poorly: anionic sites in clay repel acids

• Neutral compounds bind more easily than anionic species: aren’t repelled by

clay

Acidic chemical (benzoic acid)....

when pH < pK when pH > pK

• benzoic acid is neutral when soil pH is less than pK of chemical

• benzoic acid is anionic when soil pH is greater than the pK of chemical

64
Basic chemical (aniline)....

is either cationic (when pH < pK) or neutral (when pH > pK)

cationic species is more tightly bound than neutral species because it can bind with
cation exchange sites......

2. Solubility and partition coefficient

Adsorption (measured by Kad) to soil directly proportional to:

• low water solubility

• high partition coefficient

65
2. CHEMICAL EVAPORATION

Chemical properties that are important in

evaporation:

1 ATM = 760 mmHg = 760 Torr

(from Torricelli -- student of Galileo)

a) Vapor pressure:

pressure of a gas in equilibrium with liquid or a solid at a given

temperature.....

examples of vapor pressure (in Torr: higher number --more evaporation)

At 25 °C At 30 °C
Lindane 9.4 x 10-6 1.28 x 10-4
DDT 1.5 x 10-7 7.26 x 10-7

vapor pressure is directly proportional to temperature:

66
b). Vapor density do= PM/RT

Related to vapor pressure by the Ideal Gas Law: PV = nRT

where: P = vapor pressure

R = 0.082 L atm/mole/°K
V = 1L
T = temperature in °K (°C + 273)
n = number of moles
M= molarity

vapor pressures and densities of three pesticides at 30 °c :

molecular weight Vp (torr) Vapor Density

(g/l)
Lindane 291 1.28 x 10-4 1.97 x 10-6
Dieldrin 399 1.0 x 10-5 2.1 x 10 -7
DDT 354 7.26 x 10-7 1.36 x 10-8

A. Evaporation of chemicals from soil

Besides vapor pressure and vapor density, other factors affecting chemical
evaporation from soil:

1. concentration of chemical

• vapor density (i.e. evaporation) increases as concentration of chemical

increases...

• maximum vapor density results from saturation of soil binding sites...

67
2. temperature

• as temperature increases, vapor density increases

3. Soil moisture

as soil becomes wet, compounds are displaced from binding sites and
evaporation increases....

4 . Soil type

chemical evaporation decreases with increasing soil organic matter (organic

matter has hydrophobic binding sites...)

68
B. Evaporation from Water

1. Henry's Law constant chemical evaporation is proportional to

vapor pressure and inversely proportional to
solubility

H =Henry's law gas constant

H = Pv/S S = water solubility of chemical
Pv= vapor pressure of the chemical

2. Ionization of compound:

• evaporation rate is inversely proportional to ionization

• ionized chemicals hydrogen-bond with water, decreasing evaporation

69
B. Half-life of chemicals in biological systems:

1. Kinetics of compound following single exposure

Can derive t0.5 and

elimination rate constant
from this graph......
Log C in
body

For example, DDE t0.5 = 200

days....

time

2. Kinetics of chemical following multiple exposure:

The curve levels off because

intake and excretion of
chemical reach an equilibrium
Log C in after a certain time...
body
The time-to-reach plateau is a
function of the half-life of the
chemical.....

time

3. concentration of chemicals reaches a predictable plateau under the following

conditions:

• first-order elimination of chemical from body

• zero-order (constant) absorption of chemical

70
4. When a chemical is constantly absorbed (zero-order) and eliminated (first
order), its kinetics in the body looks like this:

x = amount of chemical in the body

Xmax at any time;

Xmax= maximum amount of chemical

in body
X
k1 = zero-order intake constant.
Units: amount /time

k2 = first-order elimination rate

time constant- fraction of the total
chemical present in the body
excreted per unit time. Units:
proportion of total chemical in
body, or fraction of X/time

5. Chemical half-life (t0.5)

From the derivative dx/dt = k1-k2x, this equation defines the amount of chemical
at time t, with input (intake) and output (excretion) rates known. e = base of natural
logs = 2.178

at Xmax (plateau).....

at half-plateau, i.e., when t=t½, e-k2t = 0.5, then

then:

71
What is the maximum body burden of lead following a daily exposure (k1 ) to 2

mg/day when the excretion (k2) is 10-2/day (or 0.01/day):

-1
Xmax = 2mg day-1 /0.01 day = 200 mg

what is the time required to reach plateau?

t0.5 = 0.693/k2 = 0.693/0.01 =69.3 days X 7 = 485 days

why multiply by 7?

By convention, seven half-lives are required to eliminate nearly all the chemical
or accumulate chemical to Xmax in the body:

Number of half- % eliminated or

lives accumulated
1 50
2 75
3 87.5
4 93.75
5 96.88
6 98.2
7 99.2

How long does it take to reach plateau if the excretion rate doubles (i.e., now
0.02 day-1 ) at the same input rate?

0.693/0.02 = 34 days x 7 = 242 days

if the input rate is three times original, (i.e., to 6 mg/day), what is the effect on
time-to-plateau and Xmax?

Xmax = 6 mg day-1/0.01 day-1 = 600 mg , X max triples

72
t0.5 = 0.693/0.01 = 69 x 7 = 485 days; tripling input has no effect on
time to-plateau

When input is increased

three-fold, the time-to-
plateau remains
unchanged, but the body
burden at plateau (i.e.,
Xmax) is now three-fold
higher...

When the excretion rate

of a chemical is increased
two-fold, both Xmax and
t0.5 to plateau are
decreased.

73
5. An illustration of this principle:

• Open a bank account with a plan to deposit $1 per day while withdrawing half of
your total balance each day

therefore:

• rate of deposit (i.e., input; k1) = $1 day-1;

• rate of withdrawal (i.e., excretion; k2) = 0.5 day-1
• account activity for the first ten days:

Day Deposit Total Withdrawal Maximum

(k1) (k2) daily
balance
($)
1 $1 $1 .5 1
2 $1 $1 + .5 = $1.5 .75 1.5
3 $1 $1 + .75 = $1.75 .875 $1.75
4 $1 $1 + .875 = $1.875 .9375 $1.875
5 $1 $1 + .9275 = $1.9375 .9688 $1.9375
6 $1 $1 + .9688 = $1.9688 .9844 $1.9688
7 $1 $1 + .9844 = $1.9844 .9922 $1.9844
8 $1 $1 + .9922 = $1.9922 .9961 $1.9922
9 $1 $1 + .9961 = $1.9961 .9981 $1.9961
10 $1 $1 + .9981 = $1.9981 .9990 $1.9981

1. What is the maximum balance ($ max) you can achieve in your account?

$max= k1/k2 = $1/0.5 day-1 = $2

2. When will you reach the maximum balance

($max)?

time to $ max=0.693/0.5 day-1 = 1.39 days x 7

= 9.7 days

74
3. How long to deplete your account if you stop depositing?

T0.5 = 0.693/0.5 day-1 = 1.39 x 7 = 9.7 days

C. BIOMAGNIFICATION

where concentration of chemical increases with organisms higher in the "food chain"

1. factors influencing bioaccumulation....

a. Persistence: long environmental t0.5

CHEMICAL t½ in soil (months)

Chlorinated hydrocarbons(e.g., dieldrin, aldrin) 24
Triazines 18
benzoic acid and amide 12
chlorphenoxy herbicides (e.g., 2,4-D) 6
carbamate insecticides (e.g., diazinon) 3
organophosphates (e.g., parathion, malathion) 2

b. Surface area of soil particles

increased surface area of soil particles allow for a large chemical

concentration in the sediment via binding.....

PARTICLE SURFACE
AREA (m2/g)
kaolinite clay 7-30
vermiculite 600-800
organic 500-800
matter

75
c. Kow of chemical

• bioaccumulation is proportional to lipid solubility...

2. Requirements for biomagnification:

a. Food chain dynamics - animals eating plants and animals

b. low elimination rate of chemical

Illustration of food chain dynamics....

76
 Results of study on concentration of PCBs in marine animals:

SPECIES PCB CONCENTRATION (µg/g fat)

Zooplankton 18 (3-35)
Herring 14 (0.5-180)
Salmon 13 (6-25)
Seal 112 (20-970)

Model systems to study bioconcentration and environmental chemodynamics:

1. "Farm Pond" system (Robert Metcalf, University of Illinois)

In most experiments, a 14C-labeled compound is added to a food

source, and then followed through the food chain.....

day added to Farm Pond removed for analysis

0 sand and water; equilibrate
2 plant 50 sorghum seeds; 10 snails, 30
water fleas, algae, plankton
20 treat sorghum leaves with 14C labeled
compound
22 10 caterpillar larvae
24 300 mosquito larvae
30 3 mosquito fish 50 mosquito larvae
32 samples of organisms

77
Chemical Kow and bioconcentration are closely correlated....

"EM" (ecological magnification) = concentration in organism/concentration in

water

78
2. Artificial streams and ponds - Lotic Mesocosm:

INERIS Verneiul-en-Hallate: outdoor lab, canal, canal1

Study questions:

1. Name four factors influencing chemical adsorption to soil. Name four factors
influencing chemical evaporation from water.
2. Why does a weak acid like the pesticide 2,4-D bind poorly to clay?
3. Calculate the theoretical proportion of the herbicide amitrole (pK = 3.0) that
would bind to kaolinite clay at pH 6.
4. Describe how the following chemical characteristics affects evaporation from
soil: vapor pressure, temperature, soil moisture and soil type.
5. Why aren’t chemicals with a low Kow biomagnified significantly?
6. How does a chemical’s H value affect its evaporation from water?
7. How is EM calculated, and how does it relate to chemical Kow ?
8. What is the Farm Pond, and how is it used to predict biomagnification?
9. What is the theoretical maximum body burden of lead assuming a constant
daily exposure of 0.02 mg, and a first order excretion rate of 10-4/day.
10. You are attempting to clean-up 5800 gallons of aniline on US Hwy 89, 5 miles
east of Logan. The majority of the spill is on the road shoulder adjacent to
the Logan River near the intake for the city’s water supply. Aniline (pK= 5) has
an appreciable vapor pressure. The soil on the spill site is a sand/clay mixture
(pH 6.5) but little organic matter; easterly (i.e., towards town) winds, warmer
temperatures and probable rain showers are forecast. In detail, a) what are
your immediate steps to reduce harm to people?; b) assess the
chemodynamics that will determine the movement of aniline from the
immediate site (consider all routes); c) detail the best course of action you
would take to contain the spill, to minimize the spread of contamination to the
local environment as well as to the inhabitants of Logan.

79
VI. Classes of Toxic Chemicals and Agents
2007 Comprehensive Environmental Response, Compensation, and Liability Act
(CERCLA or “Superfund”) Priority List Hazardous Substances

Criteria for list:

1. Frequency of occurrence

2. Potential toxicity and toxic potency

3. Chance of human exposure

A. METALS

Heavy metal: a metal of high specific gravity

some are toxic whereas others are required for nutrition:

• metals required in substantial amounts: Na+

• metals required in smaller amounts, and with limited tolerance:

Cu,+2 Zn,+2 Fe+2

• metals that are toxic at low concentrations:

Pb+2, Hg,+2 As+3

metal toxicity involves reactions with ligands that are essential for the normal
function of the system

80
Ligand: an anion and/or a molecule that contributes electrons to a metal.... bonds
range from strongly covalent to ionic...

ligand interaction with metals may be

• monodentate : form one bond with the metal ion, or:

• bidentate : can occupy more than one coordination position

the ligand binding ability of metals forms basis for some metal antidotes

British Anti-Lewisite (2,3 dimercaptopropanol; BAL) developed as antidote

to Lewisite, used in WWII.

BAL increases urinary excretion, reduces toxicity..

other antidotes for metal toxicity:

• EDTA (organic chelator)

• DTPA (diethylene triamine pentaacetic acid) similar to EDTA but

is more potent and can be used to treat iron poisoning...

• Sulfhydryl resins: (non-absorbable from GI tract)

• has been proposed to treat methyl mercury poisoning

• traps mercury in intestine, thereby reducing enterohepatic

cycling...

81
1. ARSENIC (As) (not officially a metal, but a “metalloid”)

A. Sources: 90% produced used as wood preservative (chromated copper

arsenate); pesticides (old: "Paris green" new: DSMA, MSMA for
cotton); animal feed, shellfish, ceramic glazes, tropical medicines, metal
ores (universal contaminant); fossil fuels, smelting operations (as arsine
gas...), mine leachates; naturally present in soil; volcanoes; drinking
water ; well water; no longer produced in USA

B. Disposition:
Arsenic (III) is the primary toxic form; Arsenic (V) [arsenate] is the most
prevalent
a. well absorbed through GI tract, lungs, skin,
b. accumulates in liver, kidneys, nails, bone, hair and skin
C. Toxicity:
acute toxicity (oral) : 120 mg causes 50-75% mortality in people
• burning and dryness of mouth, GI disturbances (diarrhea), projectile
vomiting
• capillary damage
• muscle spasms, coma due to extreme electrolyte loss

Chronic toxicity:
• difficult to distinguish from flu
• malaise, fatigue, GI disturbances, diarrhea or constipation, capillary
damage, renal damage (albuminuria), neuropathy, pale deposition bands on

fingernails, personality changes, anemia, skin lesions;

• Cancer: skin, liver, lung, kidney, bladder.

82
 ARSINE GAS (AsH3) – gas formed in metal manufacture; used in semiconductor
industry
• non-irritating, odor threshold 10x TLV( 0.05 ppm)
• most potent hemolyzer known (lyses red blood cells)
• between 10-60 ppm: symptoms after 30 min
• 250 ppm lethal in 30 min
• industrial exposure can cause skin and lung cancer

Arsenic – the King of Poisons, the Poison of Kings – Case studies: arsenic hot spots

2. BERYLLIUM (Be)

A. Sources: coal and combustion (steel and electric power industries), ceramic
industries, beryllium alloy manufacture, cigarette smoke. TLV-TWA= 2 µg/m3
of workroom air for an 8-hour work shift.

B.Disposition:

• inhalation primary route; residual has long t½ in lung

• distributed to all tissues, skeleton primary sink.

C. Toxicity:
• contact dermatitis most common toxicological effect—skin hypersensitivity
reaction

• acute pneumonitis—inflammatory reaction involving entire respiratory tract

immediately after inhalation; can be fatal; recovery complete after weeks or
months

83
 • chronic granulomatous disease “berylliosis;” “chronic beryllium disease (CBD):
 cyanosis, shortness of breath, mottled lung on X-ray, alveolar
granulomas

• probable human lung carcinogen (class 2A IARC)

3. CADMIUM (Cd)

A. Sources: electroplating, pigment for paints and plastics, cathode material for Ni-
Cad batteries, by-product of zinc and lead mining and smelting, "organ meats"
--liver and kidneys

B. Disposition:

• oral absorption ~ 6-10%

• highest Cd in kidney: binds to metallothionein, a metal binding protein in
kidneys--
• urine is primary route of excretion, although slow
• babies have little Cd: 30 mg body burden by age 50

C. Toxicity: chronic inhalation causes proteinuria and emphysema; teratogenic in

humans

Renal structure, Nephron and Renal Physiology and Mechanism of Renal Toxicity by
Metals

84
 Case study: environmental disease caused by Cd pollution in Japan:

"Itai-Itai" (イタイイタイ病 or “Ouch-Ouch”) disease.....

• Cd pollution in Jinzu River contaminated rice fields--caused kidney damage; Ca+2
loss leading to osteoporosis and deformity

4. CHROMIUM (Cr)

A. Sources: plating, pigments, tanning salts, wood preservatives, fly ash; an essential
element-glucose and lipid metabolism – cofactor for insulin action

B. Disposition: poorly absorbed from GI tract (ca. 1%)

excreted primarily in urine
found in kidneys

C. Toxicity: Cr+6 (chromate) is more toxic, the carcinogenic and mutagenic form of
chromium but less prevalent than Cr+3

• ascorbate (vitamin C) is an antidote for chromate GI toxicity:

Cr+6 + ascorbate → Cr+3

Clinical observations from occupational exposure to Cr:

• dermatitis (allergic reaction),
• ulcers (non allergic reaction),

• nasal septum perforation,

• asthma,

• lung cancer.....

85
5. IRON (Fe)

A. Sources: diet and vitamins--body burden ca. 4 mg (67% as Hb, 27% bound to
ferritin)

B. Disposition: absorption 2-15 % -- Fe+2 >> Fe+3 homeostatically controlled: little

excreted

C. Toxicity:

Acute (major): accidental ingestion by children (ferrous sulfate) is common--2000

cases/year
• GI tract irritation and bleeding, vomiting, pneumonitis, convulsions, coma, death

Chronic: iron oxide inhalation: causes siderosis silicosis-like disease

Iron is prime example of toxicants that acts through a Free-radical mechanism

86
 Free radical
mechanisms are
common in
toxicology

Iron, other transition

metals (Cu+1, Ni+2, Cr+5,
Mn+2), and agents like
paraquat, benzene,
phenols, cigarette
smoke, drugs (primaquin)
air pollutants are toxic
through the generation of
reactive free radicals,
which have unpaired
electrons in their bonding
orbitals.

Oxygen-based radicals
like hydroxyl (OH.) are
common in toxicology.
Radicals react with
unsaturated (double bond)
fatty acid residues in cell
membranes resulting
hundreds of toxic
products, many of them
reactive toward protein
and DNA. 8-OH-dG is one
common mutagenic DNA
base.

Malondialdehyde (MDA),
an abundant product of
lipid peroxidation, reacts
with DNA to form
mutagenic adducts.

87
6. LEAD (Pb)

• greatest incidence of poisoning than any other metal

• oldest metal poison known--most widely studied

Sources: paint, gasoline (tetraethyl--largest environmental source), plumbing,

solder, food packaging, moonshine whiskey, imported toys; CPSC regulation: 0.06 % in
coating

Handheld XRF: Testing for lead by X-ray Fluorescence

Common problem for workers in these occupations:

lead smelters, storage battery workers, painters and paint mfg, brick makers, cable
makers and splicers, galvanizers, miners, plumbers and pipe fitters, welders

Disposition: normal blood Pb: 17 µg/dl (17 µg/100 ml); > 30 µg indicative of exposure

• oral absorption ~5-10%; poor dermal absorption

• inhalation best when particle size < 0.5 µm;
• accumulates in body;
• slowly excreted in feces and urine--enhanced by mobilization of bone and tissue
reserves by dietary imbalance, acidosis, chelating agents
• Distributes along with Ca+2 : accumulates in bone, kidney, liver, teeth, nerve and

muscle

88
Toxicity:
• few acute problems, primarily occupational hazard
• GI symptoms from oral exposure--"Painter’s colic" (persistent constipation)
• CNS toxicity: encephalopathy, headache, tremor, blindness
• hematologic toxicity: anemia due to decreased heme synthesis
• renal toxicity;
• possible human teratogen
• gum line (purplish line on gum margin) characteristic... “Burton’s Line”

7. MANGANESE (Mn)

Sources: steel alloys, dry-cell batteries, electrical coils, glass, dyes, welding rods.

Disposition:
• an essential element; homeostatically regulated absorption
• not systemically toxic; high oral doses can cause GI irritation

Toxicity:
most human effects from inhalation of manganese dioxide dust during mining or
manufacturing, resulting in acute or chronic effects:

ACUTE: "Managanese pneumonitis"

CHRONIC (more serious) : "Manganism" irritability, walking and speech
disturbances, encephalopathy, Parkinson-like syndrome; slow recovery can use L-

dopa to treat symptoms

89
8. MERCURY (Hg)

HISTORY: Hg - Hydrargyum (Greek) “liquid silver”

Sources: natural degassing of earth's crust (predominant), catalysts, medicinals,

pesticides, by-product of chlorine, alkali and paper pulping (phenyl Hg)
industries, semiconductors; smelting; mercuric nitrate used in wool felt
process; greatest occupational Hg exposure is from Hg vapor

volatility + biotransformation makes Hg unique among toxic metals

Disposition and Toxicity– depends on form:

• has high affinity for sulfhydryl groups ("mercaptan")

Hg+2 (inorganic) ………….Hgo (elemental)……………….MeHg+ (organic)

higher lipid solubility

90
91
Hgo (elemental)—major risk is breathing vapors
• vapor absorbed via lung,
• if ingested, < 0.01% absorbed via GI
• can be oxidized to Hg+2 in vivo
• Hg+2 accumulates in kidneys after Hgo exposure
• more lipid soluble than inorganic forms:
• crosses blood-brain barrier: CNS toxicity -psychic, emotional symptoms
Hg+2 (mercuric)
• ~ 15% absorbed via GI tract causing GI disturbances
• selectively accumulated in kidney causing renal toxicity
• pink extremities, photophobia

MeHg+: MOST TOXIC AND LIPID SOLUBLE FORM

• crosses membranes (placental barrier) rapidly
• often formed from the bacterial alkylation of Hg
• common contaminant of fish
• readily absorbed (skin or GI);
• excretion: 90% via feces, 10% via urine
• accumulates in red cells, brain, kidneys, hair, teeth:

Symptoms of Mercury Toxicity:

• sensory disturbances in tongue, lips and limbs
• ataxia, "tunnel vision," deafness, seizures

• mental impairment, cerebral palsy-like symptoms

Treatment:
• chelation therapy to enhance excretion, thiol resins to prevent absorption
• hemodialysis with sulfhydryl compounds like L-cysteine;

92
Human Poisoning Case Studies:

DANBURY (CT) HAT COMPANY

• “Hat city” “Mad as a Hatter”
• mercuric nitrate used in felting process in hat making (banned in US 1941)
• “Danbury Shakes” neurologic symptoms, hallucinations
• High Hg contamination in Still River, lakes, ponds

STACO THERMOMETER CO. Poultney, VT 1970s-1984

workers experienced common series of health problems:
• headaches, bleeding or sore gums, upset GI, coordination problems
• Hg detected in air of worker’s homes, clothing, furniture, and in bodies of
workers and their families…
IRAQ 1971-1972

• from contaminated seed grain....

• 6,530 hospitalized, 500 deaths;
• people were exposed to between 4.8-14.6 mg/kg of Hg from bread....

JAPAN 1958- Minamata Bay:

• 2,265 victims officially recognized (incl. 1,784 mortalities); > 10,000
compensated
• Fish (dietary staple) contained 10 mg Hg/kg
• ~ 64 infants developed cerebral palsy-like signs, disfiguring birth
defects

Documented by photojournalist W. Eugene Smith

93
9. NICKEL (Ni)

Sources: nickel refining, stainless steel, coins, jewelry

Disposition: astringent properties limit GI absorption

Toxicity: (may be an essential trace element)

• dermal exposure causes contact dermatitis--"Nickel itch"
• direct contact (e.g. with jewelry) sensitization reaction

Ni(CO)4 --(nickel carbonyl) formed during Ni refining (reaction with CO) is most

important toxic form

Acute Toxicity:

• Ni at 30 ppm for 30 min can be lethal to humans

• initially causes headache and vomiting; 12-36 hours later: dyspnea, cyanosis,
fever, CNS toxicity, pneumonitis

can be effectively treated with broad-spectrum chelating agent ditiocarb

(sodium diethyldithiocarbamate)

Chronic Toxicity: respiratory tract carcinogen

• nickel refinery workers show a 150x increase in lung cancer Class 2B

Carcinogen

94
10. THALLIUM (Tl)

Sources: pesticide bait (roaches, rats), fireworks, "color logs," optics, costume
jewelry, catalysts, alloys, semi-conductors, pigments, herbal supplements

Disposition: absorbed through the skin, lungs and GI tract; follows potassium
distribution (similar ionic radius to K)

Toxicity:
• has been used in murders (tasteless, works slowly)
• onset is 2-4 days
• Treatment: a) Prussian Blue – acts as ion exchange agent, sequesters Th,
increases excretion; b) dialysis

Symptoms of toxicity:
• extreme GI irritation; vomiting, cramps, etc
• myelin sheath toxicity causing: ascending paralysis, psychic disturbances,
"wrist-drop," abnormal reflexes
• acute alopecia (10-14 days after exposure)
• residues in hair, nails and bones

95
B. Air Pollutants, Gases, Vapors, Particulates, Fibers.

-- Types And Sources

96
National Ambient Air Quality Standards (NAAQS) for the six “Criteria
Pollutants:”

http://www.epa.gov/air/criteria.html

Pollutant Primary Stds. Averaging Times Secondary Stds.

Carbon Monoxide 9 ppm 8-hour(1) None
3
(10 mg/m )
35 ppm 1-hour(1) None
3
(40 mg/m )
Lead 1.5 µg/m3 Quarterly Average Same as Primary
Nitrogen Dioxide 0.053 ppm Annual (Arithmetic Mean) Same as Primary
(100 µg/m3)
Particulate Matter (PM10) Revoked(2) Annual(2) (Arith. Mean) Revoked(2)
150 µg/m3 24-hour(3) Same as Primary
3 (4)
Particulate Matter (PM2.5) 15.0 µg/m Annual (Arith. Mean) Same as Primary
3 (5)
35 µg/m 24-hour Same as Primary
Ozone 0.08 ppm 8-hour(6) Same as Primary
(7)
0.12 ppm 1-hour Same as Primary
(Applies only in limited areas)
Sulfur Oxides 0.03 ppm Annual (Arith. Mean) -------
(1)
0.14 ppm 24-hour -------
------- 3-hour(1) 0.5 ppm
(1300 µg/m3)

(1)
Not to be exceeded more than once per year.
(2)
Due to a lack of evidence linking health problems to long-term exposure to coarse particle pollution,
the agency revoked the annual PM10 standard in 2006 (effective December 17, 2006).
(3)
Not to be exceeded more than once per year on average over 3 years.
(4)
To attain this standard, the 3-year average of the weighted annual mean PM2.5 concentrations from
single or multiple community-oriented monitors must not exceed 15.0 µg/m3.
(5)
To attain this standard, the 3-year average of the 98th percentile of 24-hour concentrations at each
population-oriented monitor within an area must not exceed 35 µg/m3 (effective December 17, 2006).
(6)
To attain this standard, the 3-year average of the fourth-highest daily maximum 8-hour average
ozone concentrations measured at each monitor within an area over each year must not exceed 0.08
ppm.
(7)
(a) The standard is attained when the expected number of days per calendar year with maximum
hourly average concentrations above 0.12 ppm is < 1, as determined by appendix H.
(b) As of June 15, 2005 EPA revoked the 1-hour ozone standard in all areas except the fourteen 8-hour
ozone nonattainment Early Action Compact (EAC) Areas.

97
 Pulmonary anatomy and function: Overall organization....

• lung bi-laterally organized 5 lobes..right: 3 lobes;

left : 2

• air enters nasal turbinate, then trachea (main

conducting airway)

• at carina, air divides to main bronchi

• after several generations, air reaches terminal

bronchioles then alveoli

O2 - CO2 exchange occurs at the

alveoli, which are heavily vascularized
for close association between air and
red blood cells.

Major Cell types of the pulmonary epithelium:

Cell type Location Key features Function

Ciliated cell Airways Apical cilia Defensive:
clearance of foreign
matter
Mucous or “Goblet” Airways Secretory; microvilli Secrete mucus
Non-ciliated Airways Contains majority of Secretory,
epithelial or “Clara” P450 metabolic
Type I or squamous Alveoli Small, flat, covers Structure and
alveolar epithelial 97% of respiratory maintenance of
cell surface; Most alveoli; ingestion of
susceptible to foreign matter
toxicants
Type II or granular Alveoli Large; Secretory; Secrete surfactin;
epithelial cell cuboidal with repair: “stem cell”
central nucleus of pulmonary
epithelium
Alveolar macrophage Alveoli Mobile, phagocytic Defensive:
phagocytic

98
 The Inflammatory Response

Obstructive lung diseases (COPD, asbestosis, silicosis, etc.) and some lung cancers
have similar inflammatory pathology:

99
Monitoring health affects: Pulmonary function tested by spirometry:

PARAMETERS DEFINITION
respiratory rate frequency of breathing per minute
tidal volume volume of air exchanged per normal
breath
total lung capacity air volume in entire lung
vital capacity air volume exhaled by maximum
expiration following maximum
inspiration
compliance index of flexibility of the lung;
decreased compliance indicates
increased stiffness
Blood gases: PO2 PCO2 efficiency of gas exchange and index
of total alveolar ventilation...

100
 inhalation of particles an occupational problem....
• grain dusts in agricultural workers
• silica and coal dust in miners
• metal dusts in foundry and smelting workers

Deposition pattern depends on particle size:

Nasal, pharyngeal, laryngeal

0.8

Alveolar
Regional
deposition

0.4

Tracheobronchial
0.2

0.0001 0.001 0.01 0.1 1 10 100

Particle diameter ( m)

101
1. Aldehydes

many aldehydes are formed in urban air--categorized as Volatile Organic Chemicals

(VOCs)

• reaction products from photo-oxidation of hydrocarbons

• incomplete combustion....
car and diesel exhaust,
forest fires
wood-burning stoves

the concentrations of aldehydes are:

• clean air: 0.0005 - 0.002 ppm

• ambient urban air 0.004 - 0.05 ppm
• indoors: < 0.8 ppm where formaldehyde-emitting materials are present--
plywood, particle board, furniture, upholstery, carpet

two aldehydes: formaldehyde (50% of total) and acrolein (5%)

these materials significantly contribute to the odor and eye irriation

caused by photochemical smog.

102
2. Acrolein (CH2==CHCOH)

Properties:
• unsaturated aldehyde, more irritating than formaldehyde
• major contributor to the irritating quality of cigarette smoke and smog
• combustion product of fuels and cellulose materials
• TLV-STEL=0.1 ppm

Toxicology:

• < 1 ppm: irritation of eyes and mucous membranes in sinus and respiratory tract.
• effects in exposed animals:
a. cilatoxic—decreased mucus clearance
b. increased tidal volume, respiratory frequency
c. vascular and myocardial toxicity

Mechanisms:

• reflex cholinergic stimulation causing bronchoconstriction and increased airway

resistance; treatable with epinephrine

103
3. Formaldehyde (COH2)

Sources:
leather, wood and paper, resins, plastic and textile processes, funeral homes, pathology
and biology laboratories, particle board, carpet, upholstery materials, insulation (urea-
formaldehyde foam), disinfectants, cosmetics, deodorants, dyes, vehicle exhausts, tobacco
smoke, gas, oil, coal, wood, and rubbish incineration; photochemical smog; a normal
metabolite in cells

Properties:
• colorless gas; has a pungent odor extremely irritating to mucous membranes of eyes,
nose and throat.
• polymerizes readily-- sold and transported in solution or as polymer
• highly water-soluble; predominant as aqueous solution containing stabilizers (methanol
or methyl/ethyl cellulose). most common form is formalin (37% formaldehyde, 6 -
15% methanol).

TLV-STEL-C: 0.3 ppm;

0.5-1 ppm: odor detectable
2-3 ppm: mild irritation
4-5 ppm: intolerable irritation

Toxicology :
Acute:

• irritation of mucous membranes, upper respiratory tract and eyes.

• thickening mucous layer, decreased ventilation, bronchoconstriction
• sensitization reactions on skin and lungs from either direct or by inhalation

104
 Chronic:
• Cancer: animal studies indicate 6 to 15 ppm exposure for 2 yr induces nasal
squamous-cell carcinomas
• Probable human carcinogen
• chronic irritation of lung, possible contributor to "sick building syndrome”
(multiple, flu-like effects)

4. Hydrogen sulfide (H2S)

Sources: petroleum refining (by-product) decaying organic matter, pulp mills, volcanic
steam vents

Exposure limits:
1. TLV-TWA=10 ppm; STEL=15 ppm; 0.01%--irritation; 0.1% --collapse and death;

Toxicology:

1. poor “warning properties” due to receptor saturation

2. acute exposures cause (in order of increasing severity):

headache, mucous membrane irritation, insomnia, loss of appetite,

collapse, convulsions, asphyxia, death

Mechanism: binds to heme proteins--produces sulfmethemoglobin causing asphyxiation

105
5. Nitrogen Dioxide (NO2)

Sources: automobile exhaust, silage (farmer's lung), cigarette smoke

Atmospheric formation of NO2:

N2 + O2 2NO (inside engine)
2NO + O2 2NO2 (in atmosphere)

exposure limits: TLV-TWA= 3 ppm TLV-STEL/C=5 ppm

Toxicology: deep lung irritant:

• decreased lung compliance, increased lung resistance

• increased susceptibility to pulmonary infections
• pulmonary fibrosis
• free-radical induced lipid peroxidation

6. Sulfur dioxide (SO2)

Sources: coal burning (coal contains < 6% S); steel smelters, other sulfur oxides (SOx)

from combination of emissions, fog and cool temperatures

Exposure limits: TLV-TWA= 2 ppm

106
Toxicology:
• soluble—mainly affects upper airways
• extreme irritant
• increased mucous secretion, loss of ciliated cells;
• bronchial constriction and increased pulmonary resistance;
• increased susceptibility to respiratory diseases

7. Ozone (O3): Major pollutant in metropolitan areas

Sources and formation: photolysis of NO2 (from auto emissions), electrical arcs

Theorized chemistry of ozone formation related to NO2:

NO2 + hν NO + O (NO2 an efficient absorber of uv light)

O + O2 O3

NO + O3 NO2 + O2

Hydrocarbons + NO + O2 NO2 + PAN (peroxyacetyl

nitrate—another atmospheric oxidant)

Formation of O3 favored by:

• Air temperature > 32 °C
• Low winds
• Intense radiation
• Low precipitation

• NO2, hydrocarbons (methane, benzene, formaldehyde)

• Atmospheric inversions

107
Exposure limits: TLV-TWA: 0.05 ppm (heavy work); 0.08 ppm (moderate work); 0.1 (light
work)

Toxicology: a strong oxidizing agent and deep lung irritant affecting terminal bronchioles
and alveoli

Acute Toxicity: from animal studies:

• irritation of mucous membranes, bronchoconstriction
• pulmonary edema, lethal 4.5-5.0 ppm/ 3 hr

Chronic Toxicity: (0.25- 1.0 ppm)

• bronchitis, fibrosis
• cell death, decreased pulmonary function, susceptibility to infection
• Jerrett NEJM (2009): associated with death from respiratory causes:
Death risk for each 10 ppb increase = 1.040

Mechanisms of Toxicity:

• induction of lipid peroxidation (free-radical process)

Reduction of surfactin production, altered cell membranes

108
8. Carbon Monoxide (CO)

Sources: automobile exhaust (20L CO min-1 hp-1)--Los Angeles air: 30-75 ppm; coal
burning, gasification, etc. (20-30% CO);

TLV-TWA: 25 ppm

Toxicology: Very efficiently binds to hemoglobin—prevents O2 binding causing

asphyxiation

Haldane equation describes the competition between O2 and CO for Hb

[COHb]= M [P CO] M= 210 (at pH 7.4 in blood)

[HbO2] [P O2]

At 50% CO; 50% O2 saturation:

i.e., 50 = 210 x (air is 20% O2) x= 0.1 %

50 20%

• 0.1% CO will produce 50% COHb

• CO has 200-fold greater affinity for Hb than O2

• Reduces O2 carrying capacity of Hb

109
 CO-HB in Signs and symptoms
Blood (%)
<2 No significant effects
2.5-4.0 Decreased short-term maximal exercise duration
in young healthy men
2.0 - 20.0 Decreased visual perception, hearing, motor and
sensorimotor performance, vigilance and other
measures of neurobehavioural performance
4.0-33.0 Decreased maximal oxygen consumption with
short-term strenuous exercise in young healthy
men
20-30 Throbbing headache
30-50 Shortness of breath, dizziness, nausea, weakness,
collapse, coma
> 50 Convulsions, unconsciousness, respiratory arrest,
death
Health Protection Agency

110
10. Urban Particulate Matter - PM2.5 , PM10

Atmospheric particulate matter (PM) with a median aerodynamic diameter < 10 µm

(larger particles quickly settle out of the atmosphere)

• “course particulates” >2.5 µm

• “fine particulates” < 2.5µm (more dangerous)

Sources: refineries, pulp mills, coal burning, wood burning, volcanism, transportation;

dust, asbestos fibers, metals, (~ 40% industry, ~15% vehicles)

Properties: Diverse chemical makeup, include: SOx, NOx, organic compounds. Cache

Valley PM2.5 contains high ammonium nitrate.

Environmental effects and Toxicology:

• reduces visibility: absorbs UV light, and fine particles are the same

wavelength of visible light

• fine PM inhaled and deposited into deep lung

• causes pulmonary diseases: increased respiratory infections, asthma,

bronchitis, pneumonia and emphysema

• premature deaths associated with high urban PM

111
Epidemiology of PM:

Pope et al.,J.A.M.A. 287:1132 (2002)

• PM2.5 is risk factor for cardiopulmonary mortality and lung cancer.

• each 10 µg/m3 elevation in PM2.5 associated with

 4% increase in all-cause mortality

 6% increase in cardiopulmonary mortality

 8% increase in lung cancer mortality

Cache Valley has some of the highest urban PM2.5 measured in USA!

Two weeks during January – February 2004: highest PM2.5 -- 135 µg/m3 (EPA

monitor)

Conditions promoting high PM2.5 in Cache Valley:

1. heavy vehicle use; cold starts  incomplete combustion;

2. atmospheric invervsions: cold stagnant air, low mixing traps and

concentrates pollution;

3. deep mountain basin, contained; no fresh air source,

4. daily release of 5.3 tons NH4+ vapor from 75,000 cows

5. NO3 from vehicle exhaust (from VOC + NOx emissions)

112
 6. formation of ammonium nitrate particles in atmosphere driven by

temperature and relative humidity

NH4NO3 formation
promoted by
temp RH

K
HNO3 + NH3 NH4NO3 (s,
(gas) (gas)
NO aq)
2 OH
N 

O O
3

hydrocarbon NH
s 3
ureas
e

ure
a

Possible solutions to Cache Valley PM2.5

• vehicle emissions inspection to reduce VOC and NOx input

• increased use of public transportation
• reduce cow population
• reduce protein in cow diet to reduce excess urea excretion

 2% reduction in protein will result in 50% reduction in NH3 without

affecting performance of cow

Health Effects Study of Cache Valley PM2.5

113
11. Asbestos

Properties:
• a large group of naturally-occurring hydrated silicates that crystallize in a

fibrous pattern....

• legal definition of an asbestos fiber: ≥ 3:1 aspect ratio

• asbestos is not dangerous until mined and processed, then becomes friable .

• aerodynamic behavior of fibers is determined by diameter and not length;

• fibers ≥ 1 µm diameter do not penetrate the deep lung....

• high tensile strength; resists heat and chemicals

Sources/Uses: (p. 17 Virta USGS 1255-KK)

• construction materials: pipes, roofing, shingles, concrete

• friction materials: brake linings, clutch pads

• other: joints, gaskets, asphalt, sealants

Use pattern 1900 -2000

Types of Asbestos: amphiboles and serpentine

a) amphibole

• crocidolite Na2 (Fe+3)2 (Fe+2)3SiO22(OH)2

• amosite (Fe,Mg)7 Si8O22(OH)2

• anthophyllite (Mg,Fe)7 Si8O22(OH)2

114
 characteristics:

• sharp, rod-like needles, chemically diverse

• less important in industry

• commonly contaminates talc and vermiculite

• penetrates the lung readily, has long t0.5....

b) Serpentine

• "curly"

• lodges in airway bifurcation

• less potent than amphibole: more soluble and cleared from lung faster

Chrysotile Mg6Si4O10(OH)8

• most common form of serpentine

• accounts for > 90% of the world's asbestos

115
Asbestos Toxicology: Occupational diseases (all chronic)

Asbestosis

• pulmonary interstitial fibrosis: excessive deposition of collagen in lung

• progressive lung stiffening; impaired gas exchange

Lung cancer

• tumors of airways or alveoli

• occurs 20 years or more after exposure

• much more common in smokers--7-30x increase in risk

• incidence higher in those exposed to amphibole (miners, shipbuilders)

compared to those exposed to chrysotile (auto-repair/brake

specialists)

Diffuse malignant mesothelioma

• extremely rare but fatal tumor of the peritoneal lining

• lag time from exposure to disease ≥ 30 years

• ≥ 18% of the mortality in crocidolite workers

also seen in household members and those living in close proximity to

mines

116
 High risk occupations to asbestos diseases:

• miners, shipyard workers, service station workers, demolition workers

• spouses/families of the above

Initial clinical signs:

• shortness of breath

• "asbestos bodies" microscopic protein-coated fibers in lungs from lung

biopsy

• positive chest x-ray....small irregular opacity

“Amphibole hyphothesis”

• recognizes significant differences toxic potency between the main forms of

asbestos

• risk of cancer and other diseases thought to be more closely associated with
amphibole asbestos,

• Remedial efforts therefore focused on amphibole types

Asbestos Law: regulated by variety of US Federal agencies:

• Consumer Products Safety Commission (CPSC)

o Asbestos in consumer products like paper and millboard, cement sheet,
dry-mix asbestos furnace or boiler cement, wood stove door gaskets,
hair dryers, laboratory gloves and pads, stove mats and iron rests,
central heating duct connectors..

• Occupational Safety and Health Administration (OSHA –agency of CDC)

o Asbestos exposures in the workplace

117
 • Agency for Toxic Substances and Disease Registry (ATSDR—agency of
PHS)
o waste sites, unplanned releases, and other sources

• National Institute of Standards and Technology (NIST)

o Accreditation of asbestos laboratories under the National Voluntary
Laboratory Accreditation Program (NVLAP)

• Mine Safety and Health Administration (MSHA)

o Asbestos related to mining activity

EPA-AHERA (Asbestos Hazard Emergency Response Act) requires building

inspections in public and private schools for asbestos-containing building materials

• inspections every 3 years

• asbestos management plan..

• yearly notification to parent, teacher, and employee organizations of the asbestos

management plan and any asbestos abatement actions taken or planned..

• designate contact person to ensure responsibilities local education agency are

implemented

• perform periodic surveillance of known or suspected asbestos containing building

material

• Provide custodial staff with asbestos awareness training

118
12. Nanoparticles and Nanotoxicology

nanoparticles are particles with diameter < 100µm

1. Examples of nanoparticles:

• Carbon nanotubes – cylindrical carbon tubes used in nanotechnology,

electronics, optics, materials science, architecture.

• Fullerene – ball shaped clusters of carbon, boron or other materials, used in

medicine, electronics, lubricants…
• Metal oxides: titanium dioxide - color agent in foods; zinc oxide - sunscreens
etc.
• Quantum dots – 5 – 50 nm semiconductors used in electronics, LEDs, solar
cells, medical imaging, scientific reagents…

U of U Nanoinstitute

2. Nanotoxicology – emerging field, safety issues unresolved

• nanoparticles are absorbed and widely distributed throughout the body

following inhalation, dermal and gastrointestinal absorption

• Zn oxide nanoparticles from sunsreeens are absorbed and distributed–

Australian study

• nanoparticles are ingulfed by pulmonary cells, macrophages, other cells

• nanoparticles induce cellular mutations, oxygen free radicals, inflammation –

potential disease risk – cardiovascular, cancer…

119
C. Industrial solvents, vapors, and other chemicals

1. Benzene

Sources:

• historically: inks, rubber, lacquers, paint removers……

toluene safer substitute in these products

• raw materials for organic chemicals, plastics, gasoline (1-3% in U.S.; ≤

30% in other countries)

• yearly U.S. consumption: 11 x 109 gallons

Occupational exposure: ~ 2 x 106 WORKERS in petroleum refining, coke and

coal,tire manufacture, truck transport

Exposure standards::

odor threshold: 12 ppm

TLV: 0.5 ppm; STEL: 2.5 ppm; REL: 0.1 ppm

Biomarker: urinary phenol normal value: < 20 mg/g creatinine; tentative max
permissible: 45 mg/g

Toxicology:

• confirmed A1 human carcinogen

• High Vp (75 mm Hg), most important as an inhaled toxicant

120
Acute Toxicity:

• narcotic effects

• inhalation of 20 ppt (2%) for 2 min is fatal

• produces cardiac arrhythmia: sensitizes cardiac muscle to catecholamine

hormones

Chronic toxicity: most critical to occupational risk: toxic to blood and blood-
forming cells
Causes appearance of immature, maturation arrested blood cells or
“Shift-to-the-Left”

• blood cell anemia: ↓ in blood cells

• myelogenous leukemia: ↑ in immature white blood cells

• aplastic anemia: ↓ in all cell types

Metabolism: benzene is metabolized by CYP2E1 to myelotoxic metabolites

121
 PERIPHERAL
BLOOD
Erythrocytes Lymphocyte
Reticulo (RBCs) s
-cyte

Basophilic
Erythroblasts

Orthochromatic Lymphoblast Plasmacyte

Erythroblasts

STEM CELL
Erythro- POOL
cytes Juvenile
Megakaryocyte

Proerythroblast
Polychromatic Megakaryoblast
Erythroblasts

Myeloblast

Granular Platelets
Promyelocytes
Megakaryocyte
Granulocytic
Myelocytes

Granulocytic
Metamyelocyte Spent
BONE
Megakaryocyte MARROW

Granulocytes Polymorphonuclear Basophil Eosinophil Monocytes

leukocytes (PMNs)

Adapted from Erslev and Gabuzda, Pathophysiology of Blood. W.B Saunders. 1975

122
2. Methyl tert-Butyl Ether (MTBE)

Sources/Uses:

• Fuel additive (1990 Clean Air Act Amendment:

gasoline must be 2-2.7% O2); anti-knock agent

• Primary gasoline oxygenator; 1999 US production: 255,000 Bbl/day

• no US standards in drinking water; California MCL (maximum contaminant

level) : 0.013 mg/L

Environmental Fate:

• high Vp (250 mm), H2O soluble (4.8g/100g)--leaches into groundwater

• slow biodegradation, but little ecological bioaccumulation

Human Exposures:

• occupational: service station attendants, mechanics, truck drivers

• general: fuel vapors, un-combusted fuel in exhaust, drinking water from

leaking storage tanks, sewers, runoff…

123
Toxicology of MTBE:

1. Acute:

• low animal and human toxicity (no effect: 5-50 ppm 2 hr exposures)

2. Chronic:

• high concentrations: rodent kidney and liver carcinogen

• human health risk unknown

Metabolized in people and animals to TBA and formaldehyde:

3. Styrene and ABS

Sources, uses: starting material for variety of end-products:

• polystyrene

loose-fill packaging, disposable dinnerware, appliances, eye

glasses, picnic coolers, magnetic tape....

• acrylonitrile-butadiene-styrene (ABS)

piping, automotive components (instrument panels,

consoles, trim) telephones, consoles, luggage, shower stalls...

124
 Exposure standards (styrene monomer): 30,000 workers in 1000 plants; >
300,000 exposed from styrene-containing products....

• TLV-TWA: 20 ppm; STEL: 40 ppm; NIOSH REL: 1 ppm

• biomarker: urinary mandelic acid (permissible value= 1 g/g

creatinine)

Toxicology: High Vp ( 2.34 mm Hg) –most important as inhaled chemical

• primarily CNS effects (~ 100 ppm): headaches, nausea, memory losses,

impaired balance; liver toxicity

• suspected human teratogen and carcinogen

• metabolized to styrene oxide in vitro

4. 1,3-butadiene (BD)

Properties: colorless gas; liquid < -4 °C ; high Vp: = 910 mm Hg

Uses: polymer component in synthetic rubber; 1997 production 1.7 x 106 tons (top
20 of synthetic chemicals)

Occupational Exposure: TLV-TWA: 2 ppm;

125
Toxicology:

• Class A2 carcinogen

• Skin, mucous membrane irritant

• Narcotic in high concentrations

• Lung carcinogen in animals

• Increased cases of leukemia in exposed workers

Metabolism: activated by CYP to an active, carcinogenic dual epoxide intermediate.

5. Vinyl Chloride

monomer used in the manufacture of polyvinyl chloride based plastics

CH2==CH--Cl -CH2--CHCl—CH2--CH--Cl

vinyl chloride polyvinyl chloride (PVC)

Sources: plastics, water pipe, food wrap, phonograph records, aerosol propellants;

126
popularity due to low cost, resistance to corrosion, can be molded to any shape

Exposure to VC:

• consumer exposure from new homes, new cars, food packaging (peanut oil:

3 ppm)

• Current TLV: 5 ppm

• Biomarker: thiodiglycolic acid > 2 mg/g creatinine

Toxicology:

• confirmed (A1) human liver and brain carcinogen

one of first identified human carcinogens (B.F. Goodrich plant)

50% ↑ incidence of total cancers in VC industry

320% ↑ in brain; 1000% ↑ in liver

signs of occupational exposure:

• scleroderma abnormal skin thickening and pigmentation

• capillary breakage: bruises on hands, etc.

• liver fibrosis, cirrhosis

Mechanism of action:

Probably mediated by an epoxide intermediate

127
6. Cyanide
Sources: fumigating products, gold and silver extraction, metal polishes, rat and
pest poisons, foods. Natural cyanogenic glycosides in foods

Disposition: cyanide gas well absorbed by all routes, but poisoning most
often following inhalation.....
Toxicology:
• exceptionally toxic, and rapidly acting...
• brain primary target organ
• characteristic bitter, almond-like odor and taste...
Symptoms:
• rapid onset if inhaled (1-2 seconds..); symptoms following ingestion of

CN- salts appear in 1 minute....

• nausea, salivation, convulsions
• paralysis, asphyxia, coma, death

Mechanism: blocks respiration by binds to heme moiety of cytochrome

oxidase in the mitochondrial respiratory chain

Treatment: two-fold therapy

1. sodium nitrite (NaNO2; produces methemoglobinemia---Fe+3) dissociates
CN- from cytochromes

2. sodium thiosulfate (Na2S2O3) : provides substrate for endogenous enzyme

rhodanese (highest in liver) catalyzes CN- conversion to thiocyanate (SCN-;
much less toxic)

S2O3= + CN- --------> SCN- + SO3=

128
7. Methylisocyanate CH3N=C=O

Source: reagent for manufacture of carbamate insecticides.......

Properties:
• boiling point= 39°C, vapor pressure= 348 mm Hg
• poor "warning properties"
a. human thresholds for odor detection (> 2 ppm) and mucous
membrane irritation (> 0.4 ppm) are greater than the TLV (0.02
ppm)

Toxicology:
• short half-life in body:
• < 2 min contact irritant to mucous membranes and respiratory tract
• initial injury confined to areas of direct contact (eyes, lungs)
• lasting lung damage may result from exposure--pulmonary congestion, cell
death

129
Acute effects:
• severe irritation of eyes, nose, throat, cough, choking sensation

• ataxia, vomiting, diarrhea, colicky abdominal pains,

• corneal ulceration, pulmonary edema, death within minutes

Chronic effects:

• pulmonary function: ↓ vital capacity, resting ventilation, O2 uptake

• pulmonary edema, obstruction, fibrosis

• pulmonary hypertension

• birth defects in children of exposed mothers

chemical involved in the Worst Modern Industrial Disaster:

• Union Carbide plant in Bhopal India December 2/3,

1984....
● Bhopal

• 30-40 tons released during a 2-3 hr period

• killed > 8000; seriously injured > 200,000

130
D. Aromatic and Low Molecular Weight Halogenated Hydrocarbons

• Aromatic Halogenated Hydrocarbons

PCBs, chlorophenols, tetrachlordibenzodioxin (TCDD)

• Low-Molecular Weight Halogenated Hydrocarbons

trihalomethanes, trichlorethylene

1. Aromatic Halogenated Hydrocarbons

A. Polychlorinated Biphenyls – “PCBs”

mixtures biphenyl isomers with varying

degrees of chlorination (between 12 and 68%
chlorine).... known under the tradename
"AROCLOR" (AROCLOR 1254® is most
common isomer - 54% chlorine)

Sources: cooling and insulating fluids for transformers and capacitors; hydraulics,
plasticizers, waxes, carbonless paper; banned in US 1977....

Toxicologic properties: PBT Chemical

• Persistent - resistant to biodegradation.... resistance proportional to % Cl

• Bioaccumulates in environment - highly lipophilic

131
 • Toxic

• residues worldwide: atmosphere, oceans, ponds, lakes, sediments, fish,

animals, human adipose and blood, breast milk

Toxicology: (well known and studied)

• thymic atrophy, wasting, immune system and reproductive toxicity

GE: Largest Superfund site in USA: 1947-1977 1.3 million pounds dumped into
Hudson River

Hudson Falls Plant; Map; EPA Hudson River PCB cleanup

B. Triclosan

Wide spectrum antibacterial, antifungal agent

• 0.1 % in deodorant, toothpaste, dishsoap, shave

cream, utensils, childrens toys…

• Low acute and chronic toxicity;

• moderate environmental accumulation

• Forms dioxin when exposed to sunlight

• Structurally similar to thyroid hormone; Endocrine disruptor in frogs

132
C. Chlorophenols

Sources, uses:

wood preservatives (railroad ties, telephone poles, fence

posts....), pesticides…

Toxicology:

• evaporates from treated surfaces

• acute toxicity (145 mg/kg rat, oral)

• immunotoxin

• liver and kidney damage, blood and reproductive disorders

D. 2,3,7,8-tetrachlorodibenzo-p-dioxin ("dioxin;TCDD)

133
Sources of TCDD:

Source g of TEQ per yeara

Municipal waste incineration 1,100
Backyard trash burning 1,000b
Inadvertent landfill fires 1,000b
Secondary copper smelting 541
Medical waste incineration 447
Forest, brush and straw fires 208
Cement kilns 171
Iron ore sintering 100b
a
Grams of toxic equivalency (TEQ) are mid-level estimates of grams of emissions of
dioxins and dioxin-like compounds. In computing these grams, each component of the
dioxin mixture is weighted with a factor that is a measure of its toxicity relative to
the toxicity of 2,3,7,8-TCDD.
b
Order-of-magnitude estimate.

Historical sources: a by-product of the manufacture of herbicide 2,4,5-T;

hexachlorobenzene, and chlorinated benzenes; "Agent Orange"......

Toxicological characteristics: one of the most toxic chemicals known in animals

• extremely toxic, highly lipid soluble

• stable in the environment

• soil contaminant (Times Beach, MO); tightly bound to soils, thus, not a

significant ground-or surface-water problem....

toxic responses in animals following exposure in the ppb range.....

• degeneration of liver and thymus

• porphyria (Greek: “purple pigment” liver disorder-- aberrant porphyrin

metabolism); King George III (1760-1820)

134
  purple stools, urine, abdominal pain, vomiting, neuropathy, seizures,

hallucinations, depression, anxiety, paranoia

• immunotoxicity

• most potent P-450 inducer known

• cancer

Possible effects in humans:

• chloracne is most common human response – curious case of Viktor Yuschenko

(blood and tissue levels 1000x normal)

• humans not as susceptible as many animals

• early animal studies over-estimated human health risk

• affinity of TCDD for human Ah receptor is less than that in animals

Mechanism toxicity: cytosolic receptor binding

• TCDD binds to receptor; TCDD-receptor complex travels to structural gene

in nucleus

135
 • gene repressor is lifted off by TCDD-receptor complex
• results in P-450 induction, TCDD metabolism, widespread toxicity.....

Incidents of accidental human exposure:

Seveso Italy (1976): Reactor explosion at Icmesa Chemical Co.; Chloracne major

human toxicity following large release of TCDD . Serum TCDD among highest

recorded.

• Increased cases of endometriosis

Times Beach, MO (1971): waste oil containing TCDD sprayed on road for dust

control, caused death of horses; flood waters of Meramec River spread

contamination throughout town.

• some in vitro immunological effects: otherwise, no detectable toxic

responses.

• Town evacuated, homes demolished, cleanup completed 1997.

2. Low-Molecular-Weight Halogenated Hydrocarbons

A. Trihalomethanes

136
 Sources::

• chlorination of naturally-occurring humic substances during

water purification;

• also in pre-chlorinated water (pre-filtering raw water before

chlorination decreases halomethanes concentrations in finished
water)

Toxicity: a possible risk of increased rectal, colon and intestinal cancer

B. 1,1,2-Trichloroethylene (TCE)

Sources:

• commonly used solvent, especially in degreasing metal parts and

semiconductors; dry cleaning of fabrics...
• most common contaminant of raw and treated drinking water in the
U.S.

Environmental Chemodynamics:

• not tightly bound to soil; highly volatile;

• readily leached from soil into ground and surface water....

Toxicology:

• animal lung and liver carcinogen; possible human carcinogen

• may be converted in environment to vinyl chloride

• decreased body weights in exposed animals

• effects in humans unclear...

137
C. Polycyclic Aromatic Hydrocarbons and Aromatic Amines

A. Polycyclic aromatic hydrocarbons

Sources: naturally formed: creosote, burned carbonaceous materials, exhaust,

burnt food, cooked meat...

Chemistry and Structure-Activity: based on the fused carbon anthracene skeleton

(non-carcinogenic)

“Bay Region” is an important structure that determines carcinogenic activity...

138
other PAHs (all are carcinogenic)

Substituents near Bay Region increase carcinogenicity:

139
Toxicology and Metabolism:

• lung, liver, skin carcinogens

• induce cytochrome P-450

PAHs are activated by a multi-step process to the ultimate carcinogen:

for Benzo[a]pyrene, the BP-7,8-diol-9,10-epoxide is the ultimate carcinogenic

form....binds to DNA

Elevated cancer risk associated with consumption of cooked meat:

Cancer hazard ratio: 1.22 (males); 1.20 (females)

140
D. Xenoestrogens (or “Endocrine Disruptors”): a foreign (Gr. xeno)
compound with estrogen-like properties

Theory: exposure to xenoestrogens may contribute to wide-ranging toxic effects

like birth defects and breast cancer

• xenoestrogens induce cell division and promote cancer by increasing total

lifetime exposure to biologically active forms of estrogen (principally estradiol)….

• xenoestogens interact with cellular estrogen receptors; estrogen-receptor

complex binds to nuclear DNA to activate cell division in estrogen-responsive
tissues…

• xenoestrogens may promote cancer by enhancing production of “bad” estrogen

metabolite the 16-α-hydroxyestrone (breast cancer tissue has elevated levels
of 16α)

• 16-α-hydroxyestrone increases binding of the estrogen receptor with growth-

promoting genes….

• 2-hydroxyestrone may be protective

141
Proven Xenoestrogens

Compound Use Details

Chlorinated aromatics
Atrazine herbicide widely used today
Chlordane insecticide (termite, ant) widely used before
banned in 1988
DDT insecticide widely used before
banned in 1972
Endosulfan insecticide widely used
Kepone bait in ant and roach traps banned in 1977
Methoxychlor insecticide close relative of DDT
some PCBs component of electrical banned but still in older
insulation transformers
Plastics
Bisphenol A breakdown product of leaches into fluids when
polycarbonate hot
nonylphenol softener of plastics leaches into fluids at
room temperature

Bisphenol A – prototype xenoestrogen

• Developed as possible synthetic estrogen

• 2.8 x 106 tons globally in 2002
• Improve strength and clarity of polycarbonate plastics –
• ADI 50µg/kg/day
• BPA migration from plastics - widespread exposure, and
universal residue in people
• In 95% of urine samples tested by CDC
• Numerous studies show adverse reproductive and physiological effects in
rodents at doses lower than ADI

142
Link between xenoestrogens and reproductive effects in wildlife?

• Elevated vitellogenin (a female-specific protein), in male fish near municipal

sewer outlets

• Alligators developed altered hormone levels, small reproductive organs and other
anomalies following a Kelthane spill in 1980, a pesticide with DDT as contaminant

• deformed shells in oysters harvested from Kepone-contaminated waters

• elevated rates of embryo death, deformities and abnormal nesting behavior in

fish-eating birds living in the Great Lakes regions contaminated by chlorinated
organics

Xenoestrogens act as “tumor promoters,” causing, among other things, cell

division in estrogen-sensitive tissues.

Xenoestrogens and other tumor promoters are carcinogenic, but they act in the
“promotion” stage in carcinogenesis.

143
E. Pesticides

• insecticides
• herbicides
• rodenticides
• fungicides

I. INSECTICIDES

A. Dichlorophenylethanes: DDT, methoxychlor, dicofol

First “miracle” pesticide. Aedes aegypti principal target

144
1. DDT

• extremely stable in the environment

• rapidly absorbed, slowly metabolized, enterohepatic cycling

• stored in adipose tissue—primary human exposure from "prime" red meat

• DDE is principal metabolite (major form of DDT in vivo);...still toxic and

very lipophilic....

145
 TOXICOLOGY OF DDT (in animals):

ACUTE (CNS toxicant): CHRONIC (hepatotoxicant)

tremors, ataxia
↓ seizure threshold
convulsions
respiratory failure
Synergizes other hepato-toxicants.....
6 ppm DDT in body fat increases
hepatic toxicity of CCl4 40-100x

2. METHOXYCHLOR

• DDT replacement, degrades rapidly in environment

• rapidly metabolized (short t½ )

• acute symptoms similar to DDT

• no biomagnification

3. DICOFOL (Kelthane® )

• DDT alternative ~ trace DDT contamination

(by law, < 0.1%)

• Agaricide (spider mites) on fruit, vegetables, tree nuts, ornamentals, hops,

cotton

• Not effective against insects

• Low acute toxicity against mammals

• Soil residues decrease rapidly, but traces remain for > 1 year

146
B. CHLORINATED CYCLODIENES

Endrin, Aldrin, Dieldrin, Chlordane, Toxaphene

*all uses canceled except chlordane

for fire ant control in power substations

Toxicology:

• as a class, more acutely toxic than DDT

• biotransformed to epoxides

Toxic effects of chlorinated cyclodienes in humans differ depending on exposure

characteristics:

ACUTE CHRONIC
dizziness, headache psychological disorders
nausea, vomiting intermittent twitching
clonic (thrashing) and tonic (tetani) convulsions
convulsions
effects eventually subside due to Hepatoxic, carcinogenic
redistribution into fat

147
148
C. LINDANE

(mis-named benezenehexachloride or "BHC")

TOXICOLOGY:

• restricted use: pet collars, dog shampoos, non-food animals, home

ornamentals, wood treatment, lice control in people..

• not well absorbed through skin

• stored in body fat

primary chlorinated hydrocarbon in human adipose!!

TOXICITY (in animals):

ACUTE CHRONIC
CNS toxicant--more violent blood disorders -- leukemia
convulsions than DDT
Hepatotoxic, carcinogenic

D. ORGANOPHOSPHATES:

1. Types

A. Nerve Gases - pyrophosphates

• developed in 1940s in Germany

• no metabolism required

• extremely acutely toxic!!!!

149
 B. Insecticides

• Parathion, Malathion, Diazinon

• require bioactivation

Estimated lethal doses to 70 kg human:

Organophosphate lethal dose

Sarin 0.0085 g
Parathion 0.1
Malathion 60.0
EPN 0.3
TEPP 0.05
OMPA 0.2
Phosdrin 0.15

2. Characteristics:

• well absorbed by all routes (except malathion)

• short t½ ; rapidly degrades in environment

• little groundwater contamination and bioaccumulation

• requires repeated application

• many require metabolic activation: from phosphorthioate (P=S) to phoshooxon

(P=O) functional group

150
 3. Toxicology:

• inhibits acetylcholinesterase (AChE)

• Acetylcholine accumulates at cholinergic receptors in CNS and PNS

4. Symptoms of acute exposure: (begin almost immediately; are consistent with

excess ACh)

a. weakness, pale skin

b. difficulty breathing; ↑ bronchial secretions

c. salivation, sweating

d. bradycardia

e. tremors, twitching

f. miosis (constricted pupils)

g. cyanosis, apnea

h. convulsions, death

151
 Autonomic nervous system Central N. S.
Parasympathetic Sympathetic Somatic

All Nerves Cholinergic

Preganglionic
Nerves:

ACh ACh ACh ACh ACh

Ganglion:
EPI Skeletal
Muscle
Cholinergic Adrenergic
nerves Nerves

Postganglionic Via the

Nerves: blood
ACh ACh NE

+ Exocrine Glands + Sweat glands + Heart Smooth muscle of:

+ Smooth Muscle of G.I. - Blood vessels + Spleen - G.I. tract
tract (some) + Blood vessels - Lungs
- Heart Rate (most)

*Classification of nerves by transmitter or mediator released and location of cell body Adapted from Selkurt
Physiology
and synapse; + = stimulatory - = inhibitory. Ach = acetylcholine; NE = norepinephrine; Epi
Little, Brown and Co. 1976.
= epinephrine

152
4. Mechanism of Toxicity: irreversible covalent phosphorylation of the serine
active site of the acetylcholine esterase (AChE) enzyme.....

153
5. Treatment of Organophosphate Poisoning: exposure to OPs is life threatening,
but specific dual antidote therapy is effective

first, decontaminate with warm alkaline soapy water, avoid self-contamination---

use gloves, administer CPR, then:

A. atropine sulfate 2-4 mg

B. 2-PAM (2-pyridine aldoxime methiodide) 1g

• accelerates dephosphorylation of the enzyme

• enhances hydrolysis of phosphate group from enzyme.

• must be given shortly after atropine - 2-PAM ineffective against "aged"

enzyme.

• recovery in days or weeks, depending on severity..

Atropine is an alkaloid from Solanaceous plants like Atropa belladonna (deadly

nightshade) and Datura stramonium (Jamestown weed). It occupies the same
muscarinic postsynaptic receptors of the parasympathetic nervous system as Ach,
which is why it is an Ach antagonist. It is extremely toxic, causing increased heart
rate, dilated pupils, dry mouth among other symptoms. Scopolamine, a related
alkaloid, is used for motion sickness..

154
MALATHION

• less hazardous

• acute toxicity (oral and dermal) much less than parathion

• widely used

• phosphate-enzyme ester is rapidly hydrolyzed..

AZINPHOS-METHYL (GUTHION)

• a popular alternative to parathion

• more toxic than parathion

rat oral LD50= 10 mg/kg vs 50 mg/kg (parathion)

rat dermal LD50= 200 mg/kg vs 491 mg/kg (parathion)

TRI-O-TOLYL-PHOSPHATE (TOTP)

• Plasticizer used in lacquer, shellac products

• causes delayed, long-term neurotoxicity

“OPIDN”

• muscle flaccidity of arms and legs causing

"shuffling gait"

• In early 1930s, 30,000 – 100,000 affected

during prohibition from contaminated or
adulterated Jamaican ginger extract (“Jake”)

• Read history of Jake Leg; Listen to Jake Leg Blues

155
E. Carbamates

• include Baygon, Carbaryl and Temik

• related to OPs in mechanism of action

• generally have lower dermal toxicity

156
 Carbamate toxicology:

mechanism of action: carbamylation of acetylcholinesterase

• direct-acting: do not require metabolism

• carbamylated cholinesterase spontaneously hydrolyzes

• less inherent hazard than OPs

• do not use 2-PAM to treat---use atropine alone

• symptoms similar to OPs, but shorter-lived....

Diflubenzuron (Dimilin)

• Chitin inibitor – used locally for control of

Mormon Crickets

• No observed long-term effects, non-

mutagenic; LD50 in mice/rats > 4600 mg/kg

• Possible estrogenic effects

157
F. BOTANICAL INSECTICIDES

Pyrethrins

• insecticides derived from flowers of Chrysanthemum cinerariaefolium and

coccineum

• ,a mixture of six compounds (pyrethrins, jasmolins, cinerins) collectively

termed “pyrethrins” or “pyrethrum”

• flowers are imported from Kenya, Rwanda, Tanzania and Ecuador (0.9-
1.3% pyrethrins)

• because of limited supply of flowers, most in use today are synthetic

derivatives (“pyrethroids”) of natural pyrethrum

Mechanism of action:

• block Ca+2, Mg+2+ ATPase—CNS toxins

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II. HERBICIDES

A. Chlorophenoxy herbicides

1. Characteristics:

• acids pKa = 3

• water soluble

• absorbed well orally, but not dermally

• rapidly excreted unmetabolized: people t0.5 = 48 hr

2. Toxicology:

• mainly nervous system effects

• toxicity of 2,4,5-T due to contamination of TCDD (0.02 ppm) -- 20 µg of

TCDD in usual application of 1 kg 2,4,5-T/acre

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B. Bipyridyl herbicides

1. Uses:

• defoliant—used in cotton harvesting; banned in many countries

• marijuana eradication (1970s-1980s)

• pasture renovation, weed control

2. Characteristics:

• highly polar (charged), poorly absorbed through skin and G.I. tract

• paraquat used in murders, suicides

• diquat much less toxic than paraquat

3. Toxicology of paraquat

• acute toxicity: 150 mg/kg oral rat LD50 (40-200 range)

• regardless of exposure route, concentrates in lung; specific pulmonary

toxicant

• high hazard rating

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 4. Pathogenesis of paraquat exposure: (regardless of route of exposure)

• lethargy, apnea, tachycardia

• labored breathing

• lung scarring; emphysema-like conditions

• convulsions

• slow, painful death

• symptoms begin in a few days

C. TRIAZINES

1. Uses:

• popular for pre-and post-emergence weed control for various agriculture

products--asparagus, corn, pineapple

• forestry---noxious weeds (in Northern Utah: Dyer's woad)

2. Toxicology

• low acute toxicity in rats 3000mg/kg (oral)

• chronic exposure causes thyroid toxicity

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III. RODENTICIDES

A. STRYCHNINE

natural alkaloid from plant Strychnos nux-vomica

• most violent poison known

• 10-100 mg can kill a large man

1. Historical uses:

• throat spray, aphrodisiac, anaesthetic

• once popular for rodent and pest control (U.S.); bear poison (Japan)....

2. Use today:

• in-ground control of gophers

2. Symptoms of poisoning:

• intermittent, severe convulsions lasting ½ -2 min in 15 min intervals.

• opisthotonos violent convulsion that tears major muscle groups...

• victim hypersensitive to noise and light---sets off more convulsions.....

3. Treatment

• short-acting barbiturate (hexobarbital) immediately after convulsion

• activated charcoal 15g orally: binds strychnine 1:1

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 • potassium permanganate (KMnO4) 1:5000-10,000 dilution to oxidize
strychnine

• has short t0.5 ....degraded rapidly; survival likely if alive after 2 hr

B. WARFARIN (COUMADIN)

• dispersed in grain-based baits

• vitamin K antagonist ---an anticoagulant

• induces internal hemorrhaging

• acts 8-12 hrs after ingestion

• human poisonings not a problem......multiple dosing necessary for

toxicity

• Wisconsin Alumni Research Fund--natural contaminant of sweet clover

Study questions

1. What was the first antidote specific for metals? How does it work?
2. What is the criteria used by the EPA for their CERCLA list of priority
pollutants?
3. Why does arsenic top the EPA’s CERCLA list of priority pollutants?
4. What is the mechanism of action of iron toxicity?
5. Arsenic contamination in drinking water is particularly high in Eureka and
Park City, UT. What is its likely source?
6. Lead is one of the first metals discovered to be poisonous. Describe how
lead is toxic to the CNS and to the blood system.
7. Identify real-life episodes involving poisoning by Cd and Hg
8. Is all mercury alike toxicologically?
9. High concentrations of Hg have been found in Utah trout. What is its
likely source, and why is it a health concern?

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10. re some properties of Th that have made it a popular poison for murders?
11. Describe the mechanism by which CO is toxic, and the physiological
sensitivity that makes it so.
12. How is pulmonary function measured? How would they change after
exposure to acrolein, H2S, and ozone?
13. What is PM2.5, and how does it impact human health?
14. How is Cache Valley PM2.5 formed, and what are some possible solutions?
15. Describe how benzene acts both acutely and chronically. Identify some
clinical signs of chronic benzene toxicity.
16. Both CN and ethyl parathion have two-stage antidote therapies. Compare
and contrast.
17. Are all forms of asbestos of equal toxic potency? Explain.
18. Describe the mechanism of action of TCDD toxicity. How is it similar to
some xenoestrogens?
19. Describe the multi-stage model of carcinogenesis.
20. Identify two instances of human poisoning with TCDD.
21. Why is TCE a commonly-found contaminant in drinking water in urban
areas?
22. What was the first chemical carcinogen identified? How made the
discovery?
23. What is a Bay-Region, and why is it important?
24. Describe the bioactivation of benzo-α-pyrene.
25. What are some of the major reasons that the sale and use of DDT was
banned in this country.
26. What are some of the similarities and differences between nerve gases
and organophosphate insecticides?
27. Describe the antidotes used for organophosphate poisoning and their
mechanism of action.
28. Define and give three examples of a xenoestrogen. Why are
xenoestrogens important toxicologically?

164