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CHEMICAL CARCINOGENESIS
INDRANIL CHATTOPADHYAY
Cancer is a group of diseases characterized by uncontrolled growth and
spread of abnormal cells […] that can result in death” (American Cancer
Society, 2006)
Genetic disease characterized by either acquired or inherited mutations.
Accumulation of non- lethal successive mutations or loss of function in the
genes that control the growth of cells
Any agent with the ability to initiate the formation of cancer.
Due to the ability to damage the genome or to the disruption of cellular
metabolic processes.
Cause damage after repeated or long-duration exposure.
Not immediate apparent harmful effects, with cancer developing only after a long latency
period.
Similarity to Drugs and Other Toxins:
• Exhibit clear dose-response relationships
• Undergo biotransformation (activation, deactivation)
• Response varies with species, sex, age
• Interact with co-administered substances (enhancement
or inhibition)
Difference from Drugs and Other Toxins:
• Biologic effect is persistent, cumulative and delayed
Proximate or direct-acting: act locally without metabolic change,
they are already electrophilic.
Examples:
Nitrogen mustard
Nitrosomethylurea
Benzyl chloride
Indirect acting : are metabolically activated into electrophilic
species (procarcinogen to carcinogen).
Examples:
Polycyclic aromatic hydrocarbons (PAH)
Produced by incomplete combustion of organic materials
Present in chimney soot, charcoal-grilled meats, auto exhaust,
cigarette
Which classes of chemicals tend to be carcinogens?
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Epoxides:
Ethylene oxide
Propylene oxide
Organohalogen comp.:
Vinyl
chloride
Carbon
tetrachloride
Chloroform
Hexachlorobenzene
Trichloroethylene
Hydrazines:
Hydrazine (and salts) 1,2Dimethylhydrazine
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N-Nitroso
compounds:
N-Nitrosodimethylamine
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Aromatic
Amines:
Benzidine
Aniline oAnisidine
Toluidine
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o-
Aromatic
hydrocarbons:
Benzene
Benz[a]anthracene
Benzo[a]pyrene
How do carcinogens enter the body?
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Skin absorption. Many solvents and other
chemicals go directly through the skin.
Ingestion. Swallowing of a carcinogen.
Inhalation.
Breathing gases, fumes and
vapors is the most common form of exposure.
What organs to carcinogens attack?
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Lungs
Liver
Kidney
Reproductive system
Skin
Many other organs and tissues
What factors influence the development of cancer?
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Dose--amount and length of exposure. The lower the dose the
least likely you are to develop cancer or related diseases.
Environmental or “lifestyle” factors.
 Cigarette smoking (co-carcinogen)
 Alcohol consumption (co-carcinogen)
 Diet--high fat consumption, natural antioxidants
 Geographic location--industrial areas, UV light
 Therapeutic drugs--some are known carcinogens
 Inherited conditions
Exposure of humans to chemical agents and the identification of the cancer-causing
molecular species.
Loeb L A , Harris C C Cancer Res 2008;68:6863-6872
Examples of Carcinogens Identified in the Normal Diet
Food
Compound
Black pepper
piperine
C o m m o n Agaritine
Celery
Furocoumarins,
mushroom
Rhubarb
Anthraquinones
psoralens
Cocoa powder
Theobromine
M u s t a r d , A
l
l
y
l
Alfalfa
sprouds
Canavanine
horseradish
isothiocyanate
Burnt material
Large number
Coffee
Caffeic coffee
An overview of primary examples of events that have generated important insights into
carcinogenesis.
Loeb L A , Harris C C Cancer Res 2008;68:6863-6872
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A substance that is not itself a carcinogen can sometimes be turned into
carcinogen.
Pro-carcinogens are activation dependent.
These compounds require cellular enzymatic metabolism into an ultimate
carcinogen in order to exert their carcinogenic action.
The resulting chemicals can be just as carcinogenic as direct carcinogens.
• Metabolism represents the organism's attempts to detoxify exogenous
chemicals to water soluble conjugates which can be excreted.
• But through the detoxifying process a chemical may be activated to an
ultimate carcinogenic form.
• These pathways may be modulated by drugs, age, nutrition, hormones or
genetics.
Metabolic Activation of Chemical compounds and Genotoxic and NonGenotoxic effects of Carcinogens
Metabolic Activation of Chemical Carcinogens
Phase I Metabolism usually precedes Phase II reactions with the most
common outcome is deactivation (detoxification).
Occasionally compounds are activated to metabolites with greater biological
activity or chemical reactivity (bioactivation).
•Oxidases(e.g. cytochromeP450)-catalyze the oxidation of chemicals to produce
unstable epoxide intermediates or stable hydroxylated compounds for
conjugation by Phase II enzymes.
•Loss of chemical leaving groups generates electrophiles which have the
potential to form covalent DNA adducts.
• microsomal cytochrome P450 (CYP)• epoxide hydrolase (EH)
• flavin-containing monooxygenase (FMO)
Metabolism of aromatic hydrocarbons
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First chemically identified carcinogens
The polycyclic aromatic hydrocarbons are chemically inert and require
metabolism to exert their biologic effects.
• This is a multi-step process:
Initial epoxidation (cytochrome P-450, CYP1A1 an inducible isoform)
Hydration of the epoxide (epoxide hydrolase)
Subsequent epoxidation across the remaining olefinic bond (principally
CYP3A4).
• The result is the formation of the ultimate carcinogenic metabolite a
diol-epoxide
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Phase II Metabolism produces more water-soluble metabolites
Most common outcome is detoxification
Occasionally compounds are bioactivated by this route as well.
Transferases(e.g. glutathione-S-transferase)-catalyze the conjugation of
water-soluble molecules onto oxidase-generated Hydroxyl groups of
potentially reactive chemicals (detoxification)
Conjugated products are unstable and may spontaneously break Down
into DNA-reactive electrophiles
UDP-glucuronyltransferases (UDP-GTs)
Phase II enzymes
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act on oxidized substrates and also contribute to xenobiotic
metabolism.
methyltransferases,
acetyltransferases, glutathione transferases,
Uridine 5'-diphosphoglucuronosyl transferases,
sulfotransferases,
nicotinamide-adenine dinucleotide (NAD)- and nicotinamideadenine dinucleotide phosphate (NADP)-dependent alcohol,
aldehyde and steroid dehydrogenases,
quinone reductases,
NADPH diaphorase,
azo reductases,
aldoketoreductases,
transaminases,
esterases,
hydrolases.
Induction of Metabolizing Enzymes
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May be a reason for tissue-, and/or species-selectivity of carcinogens
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Metabolites may be ligands for receptors
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Production of reactive oxygen species
Nebert & Dalton Nat Rev Cancer 2006
Action of carcinogen
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Change in DNA structure
– the formation of bulky aromatic-type adducts,
• exocyclic (N2) amino group of deoxyguanosine,
deoxyadenosine, C8-, N2-, and sometimes O6-positions of
deoxyguanosine as well as deoxyadenosine.
– alkylation (generally small adducts),
• generally attack the following nucleophilic centers: adenine (N1,
N3, and N7), cytosine (N3), guanine (N2, O6, and N7), and
thymine (O2, N3, and O4).
– oxidation,
• form thymine glycol or 8-hydroxydeoxyguanosine adducts
– dimerization,
– deamination
• deamination of methylated cytosine
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Epigenetic changes
– alteration in DNA methylation
EPIGENETICS
Epigenetic alterations – changes induced in cells that alter the
expression of the information on transcriptional, translational, or posttranslational levels without changes in DNA sequence
Methylation of
DNA
DNMT1
DNMT3a
DNMT3b
Modifications of
histones
Me
P
U
SAM SAH
A
A - acetylation
Me- methylation
P- phosphorylation
U - ubiquitination
RNA-mediated
modifications
• RNA-directed DNA
methylation
• RNA-mediated chromatin
remodeling
• RNAi, siRNA, miRNA …
Classification of Carcinogens According to the Mode
of Action
GENOTOXIC:
§ DNA-reactive or DNA-reactive metabolites
§ Direct interaction to alter chromosomal
number/integrity
§ May be mutagenic or cytotoxic
§ Usually cause mutations in simple systems
DNA Adduct
Mutation
Cancer
• Electrophilic species react with electronrich sites on DNA bases to form covalent
adducts
• Each class of agents reacts at selective
positions on purine and pyrimidine targets
Mechanism of Carcinogenesis:
Genotoxic Carcinogens
1. Carcinogen activation
Chemical
C
“inactivated“
carcinogen
4
P
Y
5
0s
"Activated“
carcinogen
2. DNA binding
4. Gene mutation
3. Cell proliferation
(fix mutation)
DNA Repair
APOPTOSIS
Schematic diagram showing the mechanism through which exposure to polycyclic
aromatic hydrocarbons is thought to cause cancer
Rundle, Mutat Res 600(1-2):23-36 (2006)
Williams J.A., Carcinogenesis 22:209-14 (2001)
Overview of Genotoxic and Non-Genotoxic Effects of Carcinogens
Mutations Result from Incomplete DNA Repair
Loeb L A , Harris C C Cancer Res 2008;68:6863-6872
Classification of Carcinogens According to the
Mode of Action
GENOTOXIC
NON-GENOTOXIC
Phenotypic characteristics of cancer cells:
• Immortalization
• Transformation
• Loss of contact growth inhibition
• Autonomy of proliferation
• Avoidance of apoptosis
• Aberrant differentiation
• Induction of angiogenesis
Four steps of Carcinogenesis
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Tumor Initiation
Tumor Promotion
Malignant Conversion
Tumor progression
Multi-stage chemical carcinogenesis
Cellular and Molecular Mechanisms in Multistage
Carcinogenesis: INITIATION
Initiating event involves cellular genome – MUTATIONS
Target genes:
- oncogenes/tumor suppressor genes
- signal transduction
- cell cycle/apoptosis regulators
“Simple”
genetic
changes
Chemical Exposure (air, water, food, etc.)
Internal Exposure
Metabolic Activation
Macromolecular Binding
DNA
RNA
Detoxication
Protein
(Biomarker)
Biologically Effective Dose
X
Efficiency of Mispairing
X
Cell Proliferation
Initiation
Clonal selection and expression
of initiated cells
Mutator
phenotype cells
Cancer cells
Normal cells
Endogenous
ACQUISITION OF ADDITIONAL RANDOM
MUTATIONS
Environmental
Normal cells
Endogenous
Environmental
GENETIC AND EPIGENETIC MODELS OF THE CANCER INITIATION
ALTERATIONS IN CELLULAR
EPIGENOME
Epigenetically
reprogrammed cells
Mutator
phenotype cells
Cancer cells
Cellular and Molecular Mechanisms in Multistage
Carcinogenesis: PROMOTION
Reversible enhancement/repression of gene expression:
- increased cell proliferation
- inhibition of apoptosis
No direct structural alteration in DNA by agent or its metabolites
Tumor Promotion
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Promoters - irritants or substances that produce cell activation and proliferation.
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Non-mutagenic, not carcinogenic alone, and often (but not always) able to mediate their
biologic effects without metabolic activation.
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To reduce the latency period for tumor formation after exposure of a tissue to a tumor initiator
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To increase the number of tumors formed in that tissue.
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To induce tumor formation in conjunction with a dose of an initiator that is too low to be
carcinogenic alone.
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Act like growth factors on cells that have already acquired one or more carcinogenic
mutations.
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Tumor promotion causes cells to proliferate but not to terminally differentiate, resulting in
proliferation of preneoplastic cells and the formation of benign lesions such as papillomas,
nodules or polyps.
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Increased rate of division increases the likelihood that they will acquire more mutations.
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Promotion may be reversible and most of these pre-neoplastic lesions (e.g. polyps,
papillomas) may regress if promoting agent is withdrawn.
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Threshold levels of promoter may be necessary to impart growth stimulus to initiated cells.
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exogenous (e.g. chemicals) or endogenous (e.g. hormonal status, chronic inflammatory
states).
Tumor-promoting chemicals, complex mixtures of chemicals,
or other agents
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dioxin,
benzoyl peroxide,
macrocyclic lactones,
bromomethylbenzanthracene,
anthralin,
phenol,
saccharin,
tryptophan,
dichlorodiphenyltrichloroethane (DDT),
phenobarbital,
cigarette-smoke condensate,
polychlorinated biphenyls (PCBs),
teleocidins,
cyclamates,
estrogens and other hormones,
bile acids,
ultraviolet light, wounding, abrasion, other chronic irritations (i.e., saline
lavage).
Tumor Promotion and Tumor Initiation
1. Initiation alone is not sufficient
for tumor formation.
2 and 3 An initiated cell is altered
making it more likely to give rise
to a tumor if exposed to another
agent called promoter.
4. Tumors do not result when the
promoting agent is applied before
the initiating agent.
5. Promoters can induce tumors in
initiated cells, but they are
nontumourigenic by themselves
(group5).
6. This indicates that unlike
initiating agents, promoting agents
do not affect DNA directly and are
reversible .
Cellular and Molecular Mechanisms in Multistage
Carcinogenesis: PROGRESSION
• Irreversible enhancement/repression of gene expression
• Complex genetic alterations (chromosomal translocations,
deletions, gene amplifications, recombinations, etc.)
• Selection of neoplastic cells for optimal growth genotype/
phenotype in response to the cellular environment
“Complex”
genetic
changes
Malignant Conversion
• Carcinogenesis requires the malignant conversion of
hyperplastic cells from a benign to malignant state
• Malignant conversion is the transformation of a
preneoplastic cell into one that expresses the malignant
phenotype.
• This process requires further genetic changes.
• Increased substantially by the exposure of preneoplastic
cells to DNA-damaging agents
Tumor Progression
• Expression of the malignant phenotype and the
tendency of already malignant cells to acquire more
aggressive characteristics with time.
• Propensity for genomic instability and uncontrolled
growth.
• Invasion and Metastasis are manifestations of further
genetic and epigenetic changes.
• Activation of proto-oncogenes and the functional loss
of tumor-suppressor genes
Used a strain of Salmonella
typhimurium, which requires
histidine to grow.
The reversion of a single gene will
allow the cells to grow without
histidine.
Highly accurate.
Now widely used in chemical
industries, such as pharmaceutical,
cosmetic, and agriculture .
Most chemicals found to be
mutagenic are not carcinogenic in
animal models or humans!
Carcinogenicity of non-genotoxic
chemicals (e.g. promotors) cannot
be assessed with the Ame’s assays.
Plate bacteria, add test chemical, there is
a little bit of histidine there, so cells
divide a few times, and if the chemical is
a mutagen, reversion is common; then
the mutant culture takes off, even if it
runs out of histidine.
Multistage carcinogenesis
in mouse skin
Conceptual Model for Understanding Mechanisms of Tobacco Carcinogenesis
DNA repair mediated by
p53 family target genes.
A modified molecular epidemiologic approach for validating causal relationships between
carcinogen exposure and cancer risk.
Loeb L A , Harris C C Cancer Res 2008;68:6863-6872
Recent Advances in Biochemistry and Molecular biology in Characterizing
chemical carcinogenesis
Method improvement
Experimental induction of cancers
DNA adduct quantification in humans
Genome sequencing
Epigenetics
Polymorphism genetics
Mutagenesis
Cataloguing oncogenes and tumor suppressor
genes
Clonal (driver) versus non-clonal mutations
Exogenous versus endogenous mutagenesis
Mechanisms of DNA adduction
Site-specific versus non-site-specific adduction
Biological effects of free radicals
Biochemistry and molecular biology
Metabolism of xenobiotic
Low-dose versus high-dose carcinogenesis
Cataloguing environmental carcinogens
Bioaccumulation of exogenous carcinogens in
the adipose tissue