B.B.

Sangameswaran
Cancer Biology
IV/I sem
Unit III

Chemical Carcinogenesis

Objectives
Name the major classes of chemical carcinogens, with some
examples
Describe a PAH, what is a bay region, and why is it
important
Know the difference between a direct acting carcinogen and
an indirect acting carcinogen. Give some examples.
Understand the pathways of metabolic activation of chemical
carcinogens (Give some examples).
DNA adducts: what are the major forms, how are they
formed, what is the significance of DNA adducts, how are
DNA adducts measured, how do DNA adducts cause
mutations and tumor initiation
What are the stages of carcinogenesis in the multi-stage
model (mouse skin), and what are some key characteristics
of each?


Carcinogenesis
4Background
4Multistage carcinogenesis - mouse skin model
4Metabolic activation of chemical carcinogens
4DNA adducts, mutations, tumor initiation
4Biomarkers of carcinogen exposure
4Modifiers of chemical carcinogenesis
4Mutagenesis, models, and assays

The leading cause of breast cancer is the use of
antiperspirant.
Agent orange is a major cause of cancer in Vietnam
Veterans.
Heating plastic in the microwave releases toxins, like dioxin
and DEHA.
Regularly eating meat cooked on a charcoal grill wont
increase cancer risk.


Fiction: The leading cause of breast cancer is the use of
antiperspirant. There have been many extremely thorough
epidemiological studies of breast cancer risk factors published
in medical journals. There is no evidence that antiperspirants
cause breast cancer.
Darbre 2004: Paraben esters were detected in human breast
tumors but causality was not established.
The liver is the main route of elimination of toxins from the
body, not the underarm. Also, lymph nodes do not clear toxins
via perspiration.

Source: The American Cancer Society www.cancer.org
Antiperspirants and Breast Cancer
Source: The American Cancer Society www.cancer.org
Sufficient evidence of an association Soft-tissue sarcoma, Non-Hodgkin
lymphoma, Hodgkin disease, Chronic
lymphocytic leukemia (CLL)
Limited/suggestive evidence of an
association
Respiratory cancers (lung,
trachea/bronchus/larynx), prostate
cancer, multiple myeloma
Inadequate/insufficient evidence to
determine whether an association exists
Hepatobiliary cancers,
nasal/nasopharyngeal cancer, bone
cancer, breast cancer, female
reproductive cancers (cervical, uterine,
ovarian), urinary bladder cancer, renal
cancer, testicular cancer, leukemia (other
than CLL), skin cancers, acute
myelogenous leukemia in the children of
veterans
Limited/suggestive evidence of NO
association
Gastrointestinal cancers (stomach,
pancreas, colon, rectum), Brain tumors
Agent Orange and Cancer
Heating plastic in the microwave releases toxins, like dioxin
and DEHA. The US Food and Drug Administration (FDA) says DEHA
may leach into foods wrapped in plastics, especially fatty foods such as
meat and cheese. But the levels are very low. The levels of DEHA that
might be consumed as a result of plastic film use are well below levels
showing no toxic effect in animal studies. As for dioxin, the FDA says it
has seen no evidence that plastic containers or films contain dioxins and
knows of no reason why they would.

Source: The American Cancer Society www.cancer.org
Microwaving plastics
Myth: Regularly eating meat cooked on a charcoal grill wont
increase cancer risk. Grilled meats may increase cancer risk,
so it is recommended to enjoy barbecued meats in
moderation. Heterocyclic amines are formed during grilling,
broiling, or even searing meat in a very hot frying pan -- when
the very high temperatures break down the amino acid
creatinine. There is also some concern that fats from the meat
dripping onto coals create additional chemicals in smoke that
may land back on the meat. While heterocycylic amines cause
cancer in animals, it is uncertain whether the amounts
encountered on grilled meat actually increase cancer risk in
people.
Source: The American Cancer Society www.cancer.org
Barbecued meat and cancer
Background
4 Historical significance
4Factors contributing to the incidence of human
cancer
4Identification and classification of human
carcinogens
4Diverse classes of chemical carcinogens
4Life style and cancer
4Occupational exposure and cancer
Historical Significance
+1761 - John Hill Observed increased incidence of nasal cancer among
snuff users.
+1775 - Percival Pott Reported increased incidence of scrotal cancer in
chimney sweeps.
+1918 - Yamagiwa and Ichikawa Multiple topical applications of
coal tar to rabbit ears produced skin carcinomas.
+First demonstration that a chemical could produce cancer in animals.
+Confirmed Potts observations, providing a link between animal studies
and epidemiology studies.
+1930s - Kennaway Isolated benzo[a]pyrene from coal tar.
+1940s - James and Elizabeth Miller Relationship between metabolic
activation, DNA adduct formation, and tumorigenesis.

The University of Madison
Wisconsin Medical School,
McArdle Laboratory for Cancer
Research
Elizabeth C. Miller (1920-1987)
and James A. Miller (1915-
2000)
Factors Contributing to the Incidence of
Human Cancer
Diet (35%)
Alcohol (3%)
Industrial Products (1%)
Tobacco (30%)
Reproductive & Sexual behavior (7%)
Occupation (4%)
Infection (10%)
Food Additives (<1%)
Geophysical Factors (3%)
Pollution (2%)
Medicines (1%)
Adapted from Doll, R. and Peto, R.(1981) The Causes of Cancer. Oxford Medical Publications.
Unknown (?)
Types of Human Carcinogens
Physical Carcinogens
Biological Carcinogens
Chemical Carcinogens
(UV exposure, Ionizing radiation)
(Viruses - EBV, HBV, HIV, HPV;
Bacteria - H. pylori;
Parasites - liver flukes, Schistosoma)
(Organic, Inorganic, Fiber,Hormones)
Group 1 Human Carcinogens
Epidemiological
evidence
Sufficient
Animal data
Group 2A Probable Human Carcinogens
1
2
Limited
Inadequate/
No human data
Sufficient
Group 2B Possible Human Carcinogens No human data Limited
Group 3 Not classified as to human
carcinogenicity
Inadequate/
No human data
Inadequate/
No animal data
Group 4 Evidence of non-carcinogenicity
for humans
No evidence of carcinogenicity
in at least two adequate animal
tests in different species/in both
adequate epidemiologic and
animal studies
IARC Classification of the Evaluation of
Carcinogenicity for the Human
GROUP
Inorganic Carcinogens e.g., Chromium, Nickel
Organic Carcinogens
4 Polycyclic aromatic hydrocarbons (PAHs) e.g., Benzo[a]pyrene
4 N-Nitrosamines e.g., NNK
4 Aromatic amines e.g., 4-aminobiphenyl
4 Heterocyclic aromatic amines e.g., PhIP
4 Alkyl halides e.g., Vinyl halide, cyclophosphamide
4 Nitro aromatics and heterocyclics e.g., 2-nitrofluorene, nitrofurans
4 Azo dyes and diazo compounds e.g., 3-methyl-4-aminoazobenzene

4 Alkylating agents e.g., bis(chloromethyl)ether, b-propiolactone
4 Mycotoxins e.g., AFB
1
Diverse Classes of Chemical Carcinogens
Film or Fiber Carcinogens e.g., Asbestos
Hormones e.g., DES
Aromatic Amines
NH
2
2-Naphthylamine

NH
2
4-Aminobiphenyl

N
N
N=O
CH
3
O
N-Nitrosamines
N-Nitrosodiethylamine
4-(Methylnitrosamino)-1-
(3-pyridyl)-1-butanone (NNK)
Organic Chemical Carcinogens-1
Benzo[a]pyrene
B[a]P
1
2
3
4
5 6 7
8
9
10
11
12
Polycyclic Aromatic Hydrocarbons (PAHs)
Bay-region
7,12- dimethylbenz[a]anthracene
(DMBA)
CH
3
CH
3
Bay-region

N
N
N
NH
2
CH
3
2-amino-1-methyl-6-
phenyl-imidazo-[4,5-
b]pyridine
(PhIP)
Heterocyclic
Aromatic amines
Mycotoxins
Aflatoxin B1
O O
OCH
3
O
O O
Organic Chemical Carcinogens-2
O N N
CH
3
CH
3
Dimethylnitrosamine
H
2
N C OC
2
H
5
O
Ethyl carbamate
Alkylating Agents
Alkyl Halides
CH
2
CH Cl
Vinyl chloride
Reactive Oxygen Species (ROS)
O
2
-
, H
2
O
2
, OH

, ROO

E.g, Cigarette Smoke
bis-(Chloromethyl) ether
Direct-acting
Alkylating Agents
Methyl nitrosourea (MNU)
Methyl N-nitro-N-nitroso-
Guanidine (MNNG)
4Polycyclic aromatic hydrocarbons (e.g., B[a]P) 15
4Aza-arenes (e.g., DB[a,h]A) 3
4N-Nitrosamines (e.g., NNK) 7
4Aromatic amines (e.g., 4-ABP) 3
4Heterocyclic aromatic amines 8
4Aldehydes (e.g., acrolein) 2
4Miscellaneous organic compouds (e.g., 1,3-butadiene) 15
4Inorganic compounds (e.g., Ni, Cr) 7
Total 55
Hecht, S.S. (1999) JNCI, 91:1194-1210.
It contains > 3000 chemicals.
Cigarette Smoke : A Mixture of Chemicals
Environmental causes of cancer
Agent Neoplasm
Aflatoxin Hepatoma of liver
Alcohol Carcinoma of mouth,
pharynx, esophagus, and
larynx
Alkylating agents Bladder carcinoma and
leukemia
Aromatic amines Bladder carcinoma
Asbestos Mesothelioma of pleural
surfaces
Benzene Leukemia
Environmental causes of cancer, cont.
Agent Neoplasm
Cadmium Prostate carcinoma
Estrogens Carcinoma of endometrium and
vagina
Polycyclic aromatic
hydrocarbons
Carcinoma of skin and lung
Tobacco smoking Carcinoma of mouth, pharynx,
esophagus, larynx, lung, bladder,
and lip
UV light Squamous carcinoma of skin and
lip
Environmental causes of cancer, cont.
Agent Neoplasm
Vinyl chloride Angiosarcoma of liver
Hepatitis viruses Hepatoma of liver
Source: Doll and Peto, 1981
Does Ones Life Style Predispose
to Cancer?
Carcinogenic Factors Associated with Lifestyle
Chemical,physiological
condition/Natural Process
Associated Neoplasm
Tobacco smoking Mouth, pharynx, larynx, lung,
esophagus and bladder
Aflatoxins
Liver
Excess fat, protein, calories Breast, colon, endometrium
and gall bladder
Reproductive history
Late age at first pregnancy Breast
Zero or low parity Ovary
Adapted from Pitot (1986) Fundamentals of Oncology, 3rd Edn, New York: Marcel Decker;
Vainio et al (1991) Env. Hlth. Perspec. 96:5-9.
Dietary exposure
Life style
Evidence for
Carcinogenicity
Sufficient
Alcohol beverages Esophagus, liver,oropharynx
and larynx
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Chemical Carcinogens Implicated in Human Cancer



Occupation/Exposure
Cigarette smoke
Miners, insulation
material producers,
shipyard & dockyard
workers
Lung, trachea & bronchi,
pleura & peritoneum
Carcinogen Target organ
PAHs, aromatic amines,
nitrosamines, free
radicals
Lung, trachea,bronchi, larynx,
pharynx, mouth, esophagus,
bladder, prostate, cervix
Asbestos

Lung, trachea & bronchi
Coke/gas plant workers
and painters
B[a]P, b-naphthylamines,
coal carbonization products
Gasoline/rubber industry Benzene
Bone marrow
Dye manufacturing
4-aminobiphenyl,
auramine,2-naphthyl-
amine, benzidine, magenta
Bladder
Charbroiled meat/
Barbecued meat
Heterocyclic amines (PhIP) Colon
Carcinogenesis
4Background
4Multistage carcinogenesis - mouse skin model
4Metabolic activation of chemical carcinogens
4DNA adducts, mutations, tumor initiation
4Biomarkers of carcinogen exposure
4Modifiers of chemical carcinogenesis
4Mutagenesis, models, and assays
4DNA repair
Multistage Carcinogenesis and Human Cancer:
Relation of Mouse Skin Model
4Many concepts now currently applied to other tissues and model
systems, including cell culture models for multistage carcinogenesis
and transformation, were originally derived from the mouse skin
model.
4Epidemiological studies also suggest that human cancer is a
multistep process.
+For example, Vogelsteins model of colorectal cancer
Clonal origin of colorectal tumors
Hypomethylation - chromosomal deletions at 5q (activation of ras
gene), 17p (loss of p53 tumor suppressor gene) and 18q

Complete Carcinogenesis: A single large dose or more
commonly, multiple low-dose applications of a genotoxic
carcinogen (e.g., benzo[a]pyrene), is used to induce tumors on
the backs of susceptible strains of mice.

Two-stage Carcinogenesis: A subcarcinogenic dose of a
carcinogen (e.g., B[a]P or DMBA) [initiator] followed by multiple
applications of a non-carcinogenic agent [promotor] that
possesses the ability to induce epidermal hyperplasia (e.g., 12-
O-tetradecanoyl phorbol-13-acetate [TPA]) is used to induce
tumors.
Mouse Skin Model of Chemical Carcinogenesis
Mouse Skin Model of Chemical Carcinogenesis
GENETIC SUSCEPTIBILITY

1. Covalent binding
of carcinogen to
DNA, cell
replication, and
fixation of mutation.
2. Mutation
induction in critical
target genes of
stem cells, e.g. H-
ras
3. Phenotypically
normal epidermis
1. Expansion of
initiated stem cells
through epigenetic
mechanisms
2. Altered gene
expression/ enzyme
activities
3. Angiogenesis
1. Production and
maintenance of
chronic cell
proliferation
2. Development of
clonal outgrowths;
benign papillomas
3. Altered
differentiation
4. Diploid stem line
1. Additional genetic
events occurring
stochastically
2. Aneuploidy e.g.
nonrandom
trisomies of
chromosomes 6 & 7
3. LOH
4. Further alteration
in differentiation
5. Dysplasia
1. Invasion
2. Metastasis
3. Loss of tumor
suppressor activity
e.g. p53 mutation
4. Gene
amplification e.g.
mutated H-ras allele
Initiation
Promotion Progression
Characteristics of Tumor Initiators and
Promoters
*Initiators
* Are chemically reactive or
require metabolic
activation to chemically
reactive intermediates.
* React covalently with
cellular macromolecules
such as DNA, RNA,
protein.
* Produce an essentially
irreversible event after a
single application.
* Are mutagenic in
bacterical and mammalian
cells.
*Promoters
* Do not require metabolism,
i.e., are active in their
parent form.
* Require prolonged and
repeated exposure.
* Actions are essentially
reversible.
* Produce biochemical and
cellular responses typical
of gene derepression.
* Can be subdivided into at
least two distinct stages:
Stage I (conversion); and
Stage II (propagation).
BK5.IGF-1
Transgenic mice are
more susceptible to
spontaneous
papillomas and
TPA-skin tumor
promotion
Wilker et al., Mol. Carc. 25:122, 1999
Histological changes in dorsal skin of
BK5.IGF-1 mice
H&E staining of 8-week old untreated mice. Panel A, non-transgenic littermate,
Panel B, BK5.IGF-1 mouse; X150
Wilker et al., Mol. Carc. 25:122, 1999
BK5.IGF-1 mice are more sensitive
to TPA skin hyperplasia
H&E staining of mice treated with four topical treatments of 1.7 nmol TPA. Panel A,
non-transgenic littermate, Panel B, BK5.IGF-1 mouse; X150
Wilker et al., Mol. Carc. 25:122, 1999
Carcinogenesis
4Background
4Multistage carcinogenesis - mouse skin model
4Metabolic activation of chemical carcinogens
4DNA adducts, mutations, tumor initiation
4Biomarkers of carcinogen exposure
4Modifiers of chemical carcinogenesis
4Mutagenesis, models, and assays
4DNA repair
Millers Electrophilic Theory on
Chemical Carcinogenesis
4All chemical carcinogens that are not themselves chemically reactive
must be converted metabolically into a chemically reactive form.
4The active metabolite is an electrophilic reagent, which reacts with
nucleophilic groups in cellular macromolecules such as DNA to initiate
carcinogenesis
4Carcinogens which can bind directly to DNA without metabolic
activation are called direct carcinogens (e.g., MNU, MNNG) while
carcinogens which require metabolic activation are called indirect
carcinogens (e.g., B[a]P, DMBA)
Direct Acting Carcinogens
4 Includes active halogen compounds, alkyl imines, industrial
intermediates, alkylene epoxides, sulfate esters,
nitrosamides, and nitrosoureas.
4 Some of these are used as industrial intermediates and
others as cytostatic drugs.
4 Among the latter are the inorganic and organic platinum
amine chelates.
4 With these, the cis isomers are usually more active than the
trans compounds.
H
2
N
Cl
alkaline
or neutral
pH
CH
2
CH
2
CH
2
Cl
H
3
C
N
Carbonium ion
CH
2
CH
2
CH
2
CH
2
CH
2
Cl
Imonium ion
Carbonium ion
Reaction with another guanine
H
3
C
N
CH
2
CH
2
Cl
CH
2
CH
2
Cl
Nitrogen mustard
+
H
3
C
N
Imonium ion
+
Guanine in DNA
Guanine alkylated at the 7-nitrogen
CH
2
CH
3
CH
2
N CH
2
CH
2
Cl
Cross-linked guanines in different chains of DNA
R
N
N
N
N
NH
2
CH
2
CH
3
H
2
C CH
2
N CH
2
R
N
N
N
N
HN
N
O
N
N
R
H
2
N
HN
N
O
N
N
R
+
+
OH
+
H
2
N
OH
DIRECT ACTING CARCINOGENS
Overview of Metabolic Activation of Xenobiotics
Procarcinogen or
Parent compound
Proximate carcinogen
Ultimate carcinogen
Inactive metabolites
Detoxification
Phase I metabolism
Phase II metabolism
Covalent binding to DNA
Initiation of carcinogenesis
Phase I metabolism can either activate or detoxify. Phase II
metabolism makes the metabolite more polar so it can be
excreted, and usually detoxifies (with some exceptions).
Bay Region PAH
1
2
3
4
5
6 7 8
9
10
11
12
L Region
K Region
Bay Region
Bay-Region hypothesis
4 The bay-region hypothesis (Jerina et al.) suggests that bay-region
diol-epoxides of polycyclic aromatic hydrocarbons (PAHs) are
probable candidates as ultimate carcinogens because of their
propensity to form carbonium ions. Their propensities to form
carbonium ions is due to their unique electronic properties. (I.e.,
Dewar perturbational molecular orbital calculations predict ease
of ring opening and formation of carbonium ion on benzylic
carbon). This theory was proven by:
4 Correlating Dewar perturbational calculations with mutagenicity,
carcinogenicity of PAH.
4 Demonstrating that substitutions on the aromatic rings which
prevented bay region diol-epoxides blocked mutagenicity,
carcinogenicity.
Pathways of Carcinogen Metabolic Activation of
Chemical Carcinogens
^Cytochrome P450 enzymes
^Epoxide hydrolases
^Lipoxygenases
^Cyclooxgenases
^Myeloperoxidases
^Monoamine oxidases
^Glutathione S-transferase
^One-electron oxidation
^Lipid peroxidation
Phase I Metabolism
Hydrolysis Carboxylesterase Microsomes, cytosol
Peptidase Blood, lysosomes
Epoxide hydrolase Microsomes, cytosol
Reduction Azo- and nitro-reduction Microflora, micros. cyto.
Carbonyl reduction Cytosol
Disulfide reduction Cytosol
Sulfoxide reduction Cytosol
Quinone reduction Cytosol, microsomes
Reductive dehalogenation Microsomes
Oxidation Alcohol dehydrogenase Cytosol
Aldehyde dehydrogenase Mitochondria, cytosol
Aldehyde oxidase Cytosol
Xanthine oxidase Cytosol
Monoamine oxidase Mitochondria
Diamine oxidase Cytosol
Prostaglandin H synthase Microsomes
Flavin mono-oxygenase Microsomes
Cytochrome P450 Microsomes
P450 Isoform-specific activities
1A1 Induced by cigarette smoke. Activates PAH's.
1A2 Activates aromatic amines, heterocyclic amines, AFB1.
Also metabolizes ace taminophen.
1B1 Inducd by T CDD. Activates DMBA-3,4-diol.
2A6 Activates N-nitrosodimethylamine, N-
nitrosodiethylamine, AFB1, B[a]P, NNK.
2B6 Metabolizes B[a]P, AFB1, cyclophosphamide,
ifosphamide, NNK.
2C9 Metabolizes tolbutamide, hexobarbital, (S)-warfarin,
torsemide, tienilic acid, diclofenac, B[a]P.
2D6 NNK
2E1 Acetaminophen, acrylonitrile, benzene, carbon
tetrachloride, chloroform, dichloromethane, N-
nitrosodimethylamine, styrene, trichloroethylene, vinyl
chloride
3A4 Acetaminophen, AFB1, AFG1, 6-aminochrysene, B[a]P-
7,8-diol, cyclophosphamide, ifosphamide, 1-nitropyrene,
sterigmatocystin, senecionine
Phase II Metabolism
Glucuronide conjugation Microsomes
Sulfate conjugation Cytosol
Glutathione conjugation Cytosol, microsomes
Amino acid conjugation Mitochondria, microsomes
Acylation Mitochondria, cytosol
Methylation Cytosol
H
2
N

Direct epoxidation
N-hydroxylation
Aromatic
Benzidine (Pr)
N-hydroxy dacetyl
Benzidine (Px)
Procarcinogen (Pr); Proximate (Px); Ultimate (Ut)
O O OCH
3
O
O O
Aflatoxin B
1
(Pr)
Aflatoxin B
1
-2,3-epoxide (Ut)
O O
OCH
3
O
O
O
O
NH
2
N
H
C
O
H
3
C
N
O
C
CH
3
OH
N
H
C
O
H
3
C
N
+
N-Acetyl benzidine
Nitrenium ion (Ut)
H
P450 1A1
1B1, other
O
Two-step epoxidation

1
2
3
4
5 6
7
8
9
10
11
12
Benzo[a]pyrene
(B[a]P) (Pr)
OH
HO
() B[a]P-trans-7,8-diol (Px)
P450 1A1
1B1, other
O
OH
HO
(+) anti-B[a]P-trans-7,8-diol-
9,10-epoxide (Ut)

Epoxide hydrolase
Metabolic Activation of Benzo[a]pyrene (B[a]P)
Diastereomers

OH
O
HO

O
HO
OH
(+) syn BPDE (-) syn BPDE
Enantiomers
DE-1 DE-1
OH
HO
O

O
HO
OH
(+) anti BPDE (-) anti BPDE
Enantiomers
DE-2
DE-2
Metabolic Activation of NNK

OH
O
N
N
CH
3
N=O

N
O
O

CH
3
N NOH [ ]

CH
2
=O

O
N
N
CH
3
N=O

N
O
N=NOH
O
N
N
N=O
CH
2
OH
Nitrosamines
4-(Methylnitrosamino-)-
1-(3-pyridyl)-1-butanone
(NNK)
a-methylene hydroxylation
a-methyl hydroxylation
methanediazo-
hydroxide
+
+
4-(3-pyridyl)-4-oxobutane-
diazohydroxide
4-oxo-4-(3-pyridyl)-
butanal
DNA
DNA
CYP2A6, 3A4
CYP2A6, 2D6 ?

Aliphatic
O
CH
2
O
CH
2
CH CH
2
Safrole
1'-OH-Safrole (Px)
OH
O
CH
2
O
CH CH CH
2
Safrole 1'-O-ester (Ut)
O
O
CH
2
O
CH
CH CH
2
ester
O N N
CH
3
CH
3
Dimethylnitrosamine (Pr)
Hydroxymethyl, methyl
nitrosamine (Px)
O N N
CH
3
CH
2
OH
Methyl carbonium ion (Ut)
HO N N CH
3
HCOH
[
]
+
CH
3
+
+
N
2
+
H
2
O

Elimination (detoxification) reactions
Benzo[a]pyrene
4,5-oxide
O
Glutathione (GSH)
GSH-S-transferase
OH
SG
OH
OH
UDPGA
Glucuronosyl
transferase
O
glucuronide
Activation reactions
Ethyldibromide (Pr)
S-2-bromoethyl
glutathione (Px)
S-episulfonium ethyl
glutathione (Ut)
BrCH
2
CH
2
Br + GSH GSCH
2
CH
2
Br
H
2
C CH
2
S
+
G

Activation reactions
N
N-OH-2-Acetylaminofluorene (Px)
HO
C
O
CH
3
Sulfotransferase
Acetylation
UDPGA
Glucuronosyl
transferase
N-glucuronyl-S-AAF (Ut)
N C
O
CH
3
O
glucuronide
N
N-sulfate-2-AAF (Ut)
C
O
CH
3
O
S O O
OH
-
N
N-acetoxy-S-AAF (Ut)
C
O
CH
3
C H
3
C
O
O
Polymorphic Genes Studied in Relation to Population Cancers
Phase I Enzymes (Hydrolysis, Oxidation, Reduction)
CYP1A1 PAHs, TCDD & estrogens Lung, stomach, colon, breast
CYP1A2 Heterocyclic & aryl amines Bladder, colon
CYP2D6 Nicotine? (NNK?) Lung, bladder, breast
CYP2E1 N-nitrosamines, alcohol Lung, bladder, colon

Phase II Enzymes (Glutathione conjugations, glucuronidation, sulfation, acylation, amino acid
conjugation etc.)
GSTM1 Organic epoxides Lung, bladder, colon, stomach, breast, liver
GSTT1 Ethylene oxide, butadiene Lung ?
NAT1 Aromatic amines Gastric, bladder, colorectal
NAT2 Arylamines & heterocyclic Bladder, lung, colorectal, breast
arylamines

Gene Carcinogen or metabolite Hypothesized Cancer
Carcinogenesis
4Background
4Multistage carcinogenesis - mouse skin model
4Metabolic activation of chemical carcinogens
4DNA adducts, mutations, tumor initiation
4Biomarkers of carcinogen exposure
4Modifiers of chemical carcinogenesis
4Mutagenesis, models, and assays

DNA Adducts
What are the major forms of DNA damage ?
How are the DNA adducts formed ?
What is the significance of DNA adducts ?
How are the DNA adducts measured ?
How do DNA adducts cause mutations and
tumor initiation?
Major Forms of DNA Damage

+Endogenous - Normal and Oxidative Metabolism
+Exogenous - Chemical or Physical Carcinogens
Types of DNA Damage
4Bulky DNA adducts (e.g., BPDE-dGuo)
4Unstable DNA adducts (by one-electron oxidation; e.g., DMBA)
4Apurinic sites (depurination or depyrimidination)
4Deamination
4Oxidative damage (e.g., 8-oxodG, thymidine glycols etc.,)
4Alkylated DNA bases (ethylated or methylated bases)
4Strand breaks (single strand or double strand)
4Crosslinks (DNA-protein, intra strand, interstrand)
4Indigenous compouds (I-compounds)
4Exocyclic adducts (Malondialdehyde, 4-hydroxynonenal, acrolein,
crotonaldehyde)
Electromagnetic spectrum of sunlight that
reaches the earths surface
From: Ananthaswamy, H. 1997 Ultraviolet light as a carcinogen. In:
Comprehensive Toxicology V. 12
Structure of the major UV-induced
photoproducts in DNA
A, Two adjacebt thymine
molecules in normal DNA. B,
Thymine-thymine cyclobutane
dimer. C, Two adjacent thymine
and cytosine molecules. D, a
thymine-cytosine (6-4)
photoproduct.
From:
Ananthaswamy, H.
1997 Ultraviolet light
as a carcinogen. In:
Comprehensive
Toxicology V. 12
P450, EH
CH
3
CH
3
1
2
3
4
5
6
7
8
9
10
11
12
7,12-Dimethylbenz[a]anthracene
(DMBA)
1 e- [ox] of CH
3
?
(-)anti-DMBA-trans-dGuo/dAdo
CH
3
CH
3
OH
OH
OH
dN
CH
3
CH
3
OH
OH
OH
dN
(+)syn-DMBA-trans-dGuo/dAdo
CH
3
CH
2
HN
N
N
N
NH
2
CH
3
CH
2
H
2
N
HN
N
O
N
N
7-MBA-12-CH
2
-N7Gua
7-MBA-12-CH
2
-N7Ade
Unstable Adducts (depurination)
Stable DNA Adducts
CH
3
CH
3
DMBA-3,4-diol-1,2-epoxide
O
OH
OH
Formation of Stable and Unstable DNA Adducts by DMBA

HN
N
N
N
O
H
2
N
Sugar
1
2
3
4
5
6
7
8
9

N
N
N
N
NH
2
Sugar
2
1
3
4
5
6
7
8
9
Adenine Guanine
Purines Pyrimidines
PAHs (e.g.,B[a]P, DMBA)
Mycotoxins (e.g.,AFB
1
)

N
N
NH
2
O
1
6
5
4
3
2
Sugar

HN
N
CH
3
O
O
1
2
3
4
5
6
Sugar
Cytosine Thymine
Aromatic amines (e.g.,4-ABP)
N1
N3
N7
O
6

N3
N7
O
2

N3
O
4

O
2

N3
Alkylating agents (e.g., MNU, MNNG)
Sites of DNA Adduct Formation
DNA Adducts as Markers of Exposure
in Human Populations
4Early event in the overall process leading to carcinogenesis
4Measure of the internal dose or biologically effective dose
4Adducts in target tissues - correlated with cancer status
4Easily measured in surrogate tissues (e.g., lymphocytes)
4Correlated with genetic risk factors (i.e., DNA repair capacity and
polymorphisms in drug metabolizing enzymes).
4The nature of DNA adducts can give clues to the etiological agent.
4Mutagenicity of DNA adducts depends on (i) the type of DNA adduct
formed, (ii) extent to which it is repaired, (iii) its sequence context and
(iv) the polymerases involved.




Carcinogen-DNA Adducts in Animal Models: Rationale
for Study
Practical to use animal models
Dose-response of the carcinogen of interest can be studied
Levels of total and individual adducts can be measured
Specific metabolites involved in adduct formation can be examined
Possible to study DNA repair process in the target tissue
Specific mutations caused in target genes such as H-ras can be
studied and correlated with DNA adducts
Methods of DNA Adduct Analysis
^
32
P-Postlabeling
^High performance liquid chromatography (HPLC)
^HPLC with electro-chemical detection (HPLC-ECD)
^Immunohistochemistry (IHC)
^Enzyme-linked immunosorbent assay (ELISA)
^Radio-immuno assay (RIA)
^Gas Chromatography/Mass Spectrometry (GC/MS)
^Single cell gel electrophoresis (Comet assay)
^Fluorescence Spectroscopy
^Slot-blot analysis
Sensitivity of DNA Adduct Detection Methods


Method Limit of mg DNA Adduct : ntds
detection required
(fmol)

32
P-postlabeling 0.01 1-10 1:3 x 10
9

Immunoassay
RIA 40 ~10000 1: 10
8

ELISA
competitive 1 50 1: 6 x 10
8

noncompetitive 3 0.1 1: 10
7

USERIA 1 25 1: 10
7

Slot-Blot 1 1 1: 3 x 10
6

Fluorescence spectroscopy
Low temperature 3 1000 1: 3 x 10
8

Synchronous scanning 20 100 1: 5 x 10
6



Line narrowing 1 1000 1: 10
8

GC-MS 0.5 - -

Data adapted from Jeffery (1986) and Farmer et al. (1987)
32
P-Postalabeling Assay for DNA-Adduct Detection
T-G-A*-C
A-C-T-G*
Adducted DNA
Enzymatic
Hydrolysis
Micrococcal Nuclease + Spleen Phospho-
Diesterase
Np(m5Cp+Cp+Tp+Ap+Gp) + G*p+A*p
Adduct Enrichment
n-Butanol
Nuclease-P1
Butanol, G*p + A*p
Aqueous, Np
N + G*p + A*p G*p + A*p
32
P-Postlabeling
Quantitation by Scintillation Counting or Instant Imager
Analysis
TLC and Autoradiography
32
P-G*p +
32
P-A*p
--
--
--
--
--
--
--
--
-
-
-
Aromatic DNA Adduct Profiles as Analyzed by
32
P-Postlabeling
Assay in Lymphocyte DNA of Cigarette Smokers with and
without Lung Cancer
Current Smokers (Cases)
Current Smokers (Controls)
Never Smoker
Control
()BPDE-CT DNA
Marker
DRZ1
DRZ2
DRZ1
DRZ2
Vulimiri et al., Mol. Carcinogenesis, 27: 34-46, 2000.
Representative Autoradiograms Showing
32
P-labeled DMBA- and
TH-DMBA-DNA Adducts Formed in SENCAR Mouse Epidermis
Acetone control DMBA 10 nmol TH-DMBA 100 nmol
In Panel C: Solid circles: DNA adducts specific to TH-DMBA
Dotted circles: TH-DMBA-DNA adducts that co-migrated on TLC where DMBA-DNA adducts moved
Formation of DNA Adducts by DMBA and its Diol-epoxides in
SENCAR Mouse Epidermis and in vitro
Atomic force
microscopy of
BPDE-modified
linear DNA
containing
increasing
concentrations of
adducts.
From: Pietrasanta
et al., CRT13,
2000.
Mechanisms of DNA repair
Direct reversal of DNA damage
Alkyltransferases
Base excision repair
Glycosylase and AP endonuclease
Nucleotide excision repair
T-T, C-C, C-T repair
Bulky adduct repair
Double strand break repair
Homologous recombination (HR)
Nonhomologous DNA end joining (NHEJ)
Mismatch repair
Repair of deaminaition of 5-Me-cytosine
Repair of mismatches in DNA due to defective repair, etc.
DNA adducts induce signature mutations in hot
spots of oncogenes (e.g., H-ras) and tumor
suppressor genes (e.g., p53) in the DNA.
Mutations
Mutational Theory of Carcinogenesis
4 Agents that damage DNA are often carcinogenic.
Chemical carcinogens
UV light and ionizing radiation
4 Most carcinogenic agents are mutagens.
Ames assay (Salmonella tester strain)
Chinese hamster V79 cells
4 Individuals with DNA repair deficiencies have higher incidences of
cancer
Xeroderma pigmentosum (XP)
Ataxia telangiectasia (AT)
Fanconis anemia (FA)
Blooms syndrome (BS)

Carcinogen-DNA Adducts, Mutations and Tumor Initiation
Species Tumor Carcinogen Mutation Oncogene
Human Lung cancer B[a]P G --> T p53
NNK G --> A p53
Human Hepatocellular AFB G --> A p53
carcinoma
Human Skin cancer UV-B CC --> TT p53
C --> T p53
Rat Mammary carcinoma MNU G
35
--> A H-ras (12)
DMBA A
182
--> T H-ras (61)
Mouse Skin carcinoma DMBA A
182
--> T H-ras
MNNG G
35
--> A
DMBA
MNNG/MNU
MCA
DMBA
MNNG/MNU
TPA
UV
NORMAL
INITIATED
CELL
PAPILLOMA CARCINOMA
H-ras p53
DMBA MCA
A or G
G A or G G C C
T T T T T/C A T T
DMBA MNNG UV TPA
Carcinogen-specific mutations in ras and p53 genes
in skin tumors.
Codon 61 Codon 13
MNNG/MNU
G
A
Codon 12
Carcinogenic mutations in ras gene
Bertram (2001) The molecular biology of cancer. Molecular Aspects of Medicine, 21:167-223.
The mutagenic hotspots in ras located in codons 12, 13 and 61 are localized to regions of the protein
involved in interactions with GTP and the accessory protein p120.
Mutational hotspots in p53 are involved
in protein/DNA interactions.
Bertram (2001) The molecular biology ofcancer. Molecular Aspects of Medicine, 21:167-223.
175
248
249
273
281
X-ray cystallography of wild-type p53 (green) associated with DNA (purple)
reveals that these highly mutated regions (white) are intimately involved in
DNA binding.
Bertram (2001) The molecular biology of cancer. Molecular Aspects of Medicine, 21:167-223.
Binding of wild-type p53 to DNA as
deduced by X-ray Crystallography
Carcinogenesis
4Background
4Multistage carcinogenesis - mouse skin model
4Metabolic activation of chemical carcinogens
4DNA adducts, mutations, tumor initiation
4Biomarkers of carcinogen exposure
4Modifiers of chemical carcinogenesis
4Mutagenesis, models, and assays
4DNA repair
Biomarkers of Carcinogen Exposure
+ Measurements of DNA damage



Bulky DNA adducts (e.g., BPDE-dGuo), Oxidized bases (e.g., 8-oxodG);DNA breaks
+ Carcinogen metabolites or products of DNA damage
themselves in urine

(e.g., 1-nitropyrene; 8-oxodG; NNAL-Gluc; NNAL)
+ Surrogate markers of chemical modification to blood
proteins



Protein adducts (e.g. B[a]P-albumin, 4-ABP-hemoglobin adducts)
+ Mutations in marker genes



e.g., p53, ras
+ Differential expression of different enzymes
(e.g., CYP450, GST)
+ Cytogenetic markers



Sister Chromatid Exchange [SCE])
Chromosomal aberrations (e.g., micronuclei;
Carcinogenesis
4Background
4Multistage carcinogenesis - mouse skin model
4Metabolic activation of chemical carcinogens
4DNA adducts, mutations, tumor initiation
4Biomarkers of carcinogen exposure
4Modifiers of chemical carcinogenesis
4Mutagenesis, models, and assays

Modifiers of Chemical Carcinogenesis
=Exposure to other chemical carcinogens
=Capacity to metabolize carcinogens
=DNA repair capacity
=Age
=Sex
=Hormonal
=Immunological status
=Trauma
=Radiation
=Viral diseases
=Diet, nutrition, and lifestyle
=Genetic constitution
Carcinogenesis
4Background
4Multistage carcinogenesis - mouse skin model
4Metabolic activation of chemical carcinogens
4DNA adducts, mutations, tumor initiation
4Biomarkers of carcinogen exposure
4Modifiers of chemical carcinogenesis
4Mutagenesis, models, and assays

Carcinogenicity and Toxicity Testing
4 Short-Term Tests-Mutagenesis Assays
Salmonella Typhimurium S9
Mouse Lymphoma L5178 (MOLY assay)
Chinese hamster ovary (CHO) assay
Dominant lethal assay
LacZ- mouse
Sister chromatid exchange
Unscheduled DNA synthesis
4 Chronic Bioassays
2-year bioassay The gold standard
Uses both male and female F344 rats and B6C3F1 mice
10th Edition of the Report on Carcinogens

4 Newly listed as known human carcinogens
Steroidal estrogens
Broad Spectrum Ultraviolet Radiation
Wood dust
Nickel compounds
4 Upgraded from reasonably anticipated to known human carcinogen:
Beryllium and beryllium compounds


http://ntp-server.niehs.nih.gov
10th Edition of the Report on Carcinogens

4 Newly listed as reasonably anticipated to be human carcinogens:
IQ (2-amino-3-methylimidazo[4,5-f]quinoline
2,2-bis-(Bromomethyl)-1,3-propanediol (technical grade)
UVA, UVB, UVC Radiation
Chloramphenicol
2,3-Dibromo-1-propanol
Dyes metabolized to 3,3-dimethoxybenzidine
Methyleugenol
Metallic nickel
Styrene-7,8-oxide
Vinyl bromide
Vinyl fluoride





Further Reading
Vulimiri, S.V. and DiGiovanni, J. (1999) Carcinogenesis. In: Manual of
Clinical Oncology, 7th edition, Ed. By R.E. Pollock,Wiley-Liss, Inc., pp. 19-
43.
Ruddon, R.W. (1995) Cancer Biology, 3rd edition, Oxford University Press,
pp. 231-261.
Smart, R.C. (1994) Carcinogenesis. In: Introduction to Biochemical
Toxicology, 2nd edition, Ed. By E. Hodgson and P. E. Levi, Prentice Hall,
pp. 381-414.
Parkinson A. (1996) Biotransformation of xenobiotics. In: Casarett and
Doulls Toxicology: The Basic Science of Poisons, 5th edition, McGraw-Hill,
pp. 113-186.
Pitot H.C. III and Dragan, Y.P. (1996) Chemical carcinogenesis. In:
Casarett and Doulls Toxicology: The Basic Science of Poisons, 5th edition,
McGraw-Hill, pp. 201-267.
Taningher, M. et al., (1999) Drug metabolism polymorphisms as
modulators of cancer susceptibility. Mut. Res. 436, 227-261.

Further Reading
Pitot H.C. III and Dragan, Y.P. (2001) Chemical carcinogenesis. In:
Casarett and Doulls Toxicology: The Basic Science of Poisons, 6th edition,
McGraw-Hill, pp. 241-319.
Bowden and Fischer (1997) Comprehensive Toxicology Vol. 12: Chemical
carcinogens and anticarcinogens

Links:
www.toxicology.org
www.cancer.org
www.aacr.org
NTP home page: http://ntp-server.niehs.nih.gov