carcinogenesis handouts

8/8/2019 Carcinogenesis Handouts

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2008 Sinal - Intro Pharmacol II 1

Chemical Carcinogenesis

Dr. Christopher Sinal

Room 5E, Tupper Medical Building

[email protected]

Additional Resource: Luch, A. (2005) Nature and Nurture - Lessons from

Chemical Carcinogenesis.Nature Reviews in Cancer5:113-125

(www.nature.com)

Email for appointment

Handouts available online:

http://pharmacology.medicine.dal.ca/undergraduate/courses.cfm


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Cancer in a Pharmacology Course?

1. Chemical carcinogenesis involves the interaction of foreign compounds

(xenobiotics) with the body

2. Chemical carcinogens obey essentially the same pharmacokinetic

principles of absorption, distribution and elimination as therapeutic drugs

3. Chemical carcinogens are metabolized by the same enzymes as therapeutic

drugs

4. Conceptually, the biological effects of chemical carcinogens can be treated

the same as the unwanted side effects of therapeutic drugs

5. Cancer is a human disease treated with pharmacological agents


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What is Cancer?

a disease in which cells of the body divide and proliferate

in an uncontrolled manner

associated with a loss of normal cell cycle control

genetic mutation is a required event

Topics we will address:

chemical carcinogens

multi-stage model of carcinogenesis

metabolic activation of carcinogens metabolic deactivation of carcinogens

genetic changes associated with carcinogenesis

risk assessment


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A Brief History of Notable Discoveries in Chemical Carcinogenesis

1566

Paracelsus describes

wasting disease of

arsenic

miners in Austria

1500 1600 1700 1800 1900

1775Percival Pott links occupational

exposure to soot with

increased incidence of

scrotal cancer in English

chimney sweeps

1895

Ludwig Wilhelm Carl

Rehn reports increased

urinary bladder tumours

in aniline dye workers

in Germany

1700

Bernadini Ramazzini

publishes Diseases of Workers

a systematic analysis of

peculiar diseases associated

with different occupations


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Chemical Carcinogenesis - The Present

Approximately 200 chemical compounds or mixtures of

chemical compounds are known or are anticipated to be

human carcinogens

Mutation of approximately 100 genes has been described inresponse to exposure to chemical carcinogens

Exposure to chemical carcinogens is believed to be

responsible for a large proportion of cancer deaths in

Western industrialized societies Tobacco

Diet

Occupational/environmental exposure


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Chemical Carcinogens

Carcinogen - any substance or agent that significantly

increases tumor incidence

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


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Selected Examples of Known Human Chemical Carcinogens

BenzeneAll

Ethylene oxideLymphatics

Tobacco smoke

Aromatic amines (4-aminobiphenyl)Bladder

Vinyl chloride

Aflatoxin B1Liver

Coal soot

Cutting oilSkin

Smokeless tobacco

Tobacco smokeOral Cavity and Esophagous

Asbestos

Diesel exhaust

Tobacco smoke

Metals (As, Be, Cd, Cr, Ni)Lung

Chemical CarcinogenOrgan System


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Structures of Some Selected Chemical Carcinogens

O

O

OCH3

OO

NH2

O

O

R

R

R

R

R

R

R

R

R R

R

RR

RR

R R

R

N N OH3C

H3C

Cl S ClCl N Cl

Aflatoxins

Aflatoxin B1

Polycyclic Aromatic Hydrocarbons

Benzo[a]pyrene

Naphthylamine

Aromatic Amines

Dioxins Polychlorinated Biphenyls

Alkylating Agents

R = Cl, H R = Cl, H

Dimethylnitrosamine Sulphur Mustard Nitrogen Mustard


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Genotoxic and Non-Genotoxic Carcinogens

Genotoxic

DNA adducts Chromosome breakage Chromosome fusion Chromosome deletion

Genetic Damage

Non-Genotoxic

Inflammation Immunosuppression Oxygen radicals Receptor activation Epigentic silencing

Altered Signal Transduction

Genetic instabilityLoss of proliferation control

Resistance to apoptosis

Cancer

Metabolism

NH2


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Multi-Stage Model of Carcinogenesis


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Tumour Initiation

genetic damage by a genotoxic chemical carcinogen is a key event

chemical carcinogens form covalent addition products (adducts) with DNA

faulty repair of DNA damage leads to mutation

- aberrant levels of gene expression or protein products withaltered function

in general, a positive correlation exists between the number of detectable

carcinogen:DNA adducts and the development of tumours

genotoxic

chemical

carcinogen genetic

change

normal cell initiated cell


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Tumour Promotion

expansion of initiated cells to form a preneoplastic lesion

mutations in genes that control cell growth increases proliferation

(replication, growth) of initiated cell (clonal expansion)

non-genotoxic carcinogens can increase the proliferation of both normaland initiated cells

- but, initiated cells have an exaggerated growth response to these

agents

- inflammation, hormones (e.g. estrogen), growth factors, chemicals

clonal

expansion


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Malignant Conversion

transformation from primed preneoplastic state to

malignant state (conversion to tumour cell)

associated with additional genetic change and expansion

- affected by genotoxic and non-genotoxic chemicals low probability of malignant conversion is increased bycontinued exposure to chemical carcinogens

preneoplasticlesion malignanttumour

genetic

changeexpansion


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Tumour Progression

progression of a malignant tumour to a more aggressive state

associated with additional genetic change (carcinogen exposure,genetic instability) and expansion

invasion beyond the primary tumour (metastasis)

genotoxic and non-genotoxic agents can accelerate this process

genetic change

metastasis


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Summary - Multi-Stage Model of Carcinogenesis

multiple stages are involved in the progression from a

normal cell to metastatic cancer

conversion to a cancerous phenotype is initiated by geneticchange (genotoxic carcinogen)

chemical carcinogens (genotoxic and non-genotoxic) have

effects at all stages in the progression of carcinogenesis


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Metabolic Fate of Chemical Carcinogens

Tissue

Accumulation

Phase II Product

Phase I Product

Elimination

Elimination urinefeces

Carcinogen

environment


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The Liver and Carcinogen Metabolism

extensive capacity to metabolize a wide variety of chemicalstructures

metabolic capacity of various tissues: liver > lung > kidney

intestine adrenals > other tissues

liver metabolism is a primary defense against the

accumulation of toxic chemicals within the body

but, can also generate reactive carcinogens from non-reactive

precursors

drug, carcinogen and other xenobiotic metabolism is broadly

classified as Phase I or Phase II

conversion of lipophilic substances to more water-soluble

metabolites that are more readily eliminated in urine and bile


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Phase I Metabolism

predominant liver metabolism pathway

usually precedes Phase II reactions

most common outcome is deactivation (detoxification)

occasionally compounds are activated to metabolites

with greater biological activity or chemical reactivity(bioactivation)

Enzymes

microsomal cytochrome P450 (CYP)

epoxide hydrolase (EH) flavin-containing monooxygenase (FMO)

alcohol and aldehyde dehydrogenases

monoamine oxidases


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Phase I Bioactivation Reactions

HydroxylationCYP +H20

epoxide alcohol

O

H

OH

Amine Oxidation CYP

hydroxylamine

RHN C CH3

O

R N C CH3

OOH

Reductive

DehalogenationCYP

phosgene

C

Cl

Cl

Cl Cl C

Cl

Cl

Cl HCl+

Epoxide

HydrationEH

dihydrodiol

O

OH

OH


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Phase II Metabolism

usually preceded by Phase I reaction(s)

addition of polar moieties to exposed functional groups

(e.g. -OH, -COOH, -NH2)

produces more water-soluble metabolites

most common outcome is detoxification occasionally compounds are bioactivated by this route

Enzymes

UDP-glucuronyltransferases (UDP-GTs)

sulfotransferases (STs) glutathione S-transferases (GSTs)

N- and O- acetyltransferases (OATs and NATs)


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Direct and Indirect Chemical Carcinogens

the carcinogenic potential of a chemical is directly related to the ability to

covalently modify DNA

for many carcinogens, this process requires metabolism (indirect)

for others, metabolism is not required(direct)

Procarcinogen: parent compounds which are upstream of the ultimate

mutagen

Ultimate Carcinogen: the reactive metabolite which covalently modifies

DNA

Proximate Carcinogen: an intermediate metabolite between pro and

ultimate


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Metabolic Activation of DAB

diazo dye used in textile manufacture

known human hepatocarcinogen

one of the first characterized procarcinogens (1947)

1. CYP1A2; 2. Acetyltransferase

N

N

N

H3C CH3

N

N

N

H3C OH

N

N

N

H3C O

C

CH3

O

N

N

N

H3CDNA

Dimethylaminoazobenzene (DAB)"Procarcinogen"

Ultimate CarcinogenProximate Carcinogen DNA Adduct

1. 2.


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An important message regarding chemical

carcinogen metabolism.

bioactivation of procarcinogens by phase I or phase II

metabolism is a rare occurrence

most commonly, metabolism results in the successfulelimination of the procarcinogen without adverse effects


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Benzo[a]pyrene is eliminated through metabolism

benzo[a]pyrene"Procarcinogen"

O

OH

HO

OH

HO

O

OH

HO

HO

DNA

benzo[a]pyrene-7,8-diol 9,10-oxide

"Ultimate Carcinogen"

1 2 3

proximate

carcinogen

proximate

carcinogen


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Summary - Metabolism of Carcinogens

the liver is quantitatively the most important site for carcinogen

metabolism

direct chemical carcinogens do not require metabolic activation

indirect chemical carcinogens require metabolic activation to generate theultimate carcinogenic agent

phase I and phase II metabolism can result in bioactivation of chemical

carcinogens

most commonly, metabolism results in the detoxification and eliminationof chemical carcinogens


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Genetic Modification by Chemical Carcinogens

direct and indirect genotoxic chemical carcinogens covalentlymodify DNA (adduct formation)

almost all DNA damage is reversed by very effective and

efficient cellular DNA repair mechanisms

cells with unrepairable DNA damage usually undergo

programmed cell death (apoptosis)

errors in DNA repair lead to mutations (alteration of DNA

sequence) that can result in cancer initiation

mutations can be passed on to daughter cells by mitosis and

clonal expansion


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Spontaneous Decomposition of NMNU

H3C N C NH2

ON

O

H3C N

HC NH2

O

N

HO

CH3+

N H2ON

H2

O+

+ +

N-methylnitrosourea (NMNU)

methylcarbonium ion


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Metabolic Activation of Benzo[a]Pyrene

O

OH

HO

OH

HO

O

OH

HO

HO

DNA

benzo[a]pyrene"Procarcinogen"

benzo[a]pyrene-7,8-diol 9,10-oxide

"Ultimate Carcinogen"

1 2 3


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Carbonium ion-DNA Adducts

CH3+

NH

NNH

N

O

NH2

NH

NNH

N

OCH3

NH2

NH

NNH

N

O

NH2

CH3

+O6-methylguanosine

N7-methylguanosine

guanosine


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Implications of Faulty Repair of DNA-Carcinogen Adducts

DNA repair errors

DNA replication errors

altered cell

function and growth

mutations

aberrant gene regulation

incorrect gene products

tumour development

MUTATION-proto-oncogene

-tumour suppressor gene

REPAIR NO REPAIR

NO CANCER CANCER


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Proto-Oncogenes

Function: normal cellular genes that control cell:

- growth (proliferation)

- specialization (differentiation)

- death (apoptosis)

almost all proto-oncogenes encode signal transduction proteins activated proto-oncogene = oncogene

Activation:

Proto-oncogene Oncogene


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Mechanisms of Proto-Oncogene Activation

1. Overexpression of the gene leading to increased

concentration and biological activity of the protein product

- regulatory mutation

2. Expression of the gene at an inappropriate time or context

- regulatory mutation

3. Expression of the gene in an inappropriate cell type

- regulatory muation

4. Expression of an altered protein product

- structural mutation


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Ras Proto-Oncogenes

Ras Proteins

large family of membrane-bound guanine nucleotide binding proteins (G-proteins) GTP binding promotes formation of signal transduction complexes with other proteins

hydrolysis of GTP -> GDP terminates activity (transient activation)

exert a powerful proliferative influence in many cell types

Incidence of Ras Mutationsin Human Cancers

Pancreas 90%

Colon 50%

Thyroid 50%

Lung 30%

Myeloid Leukemia 30%Ovarian 15%

Bladder 6%

http://fig.cox.miami.edu/~cmallery/150/gene/c7.19.12a.Ras.jpg


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Ras and Human Cancers

activation is very common in human cancers in animal models of chemical carcinogenesis and in human cancers

arising from environmental exposure:

- most mutations occur within the 12th

or 61st codons

- amino acid substitution

Why the specificity of these sites?

1. Selectivity of ultimate carcinogen for these sites

- accessibility, nucleotide preference, physiochemiststry

2. Functional effect of mutations

- growth advantage to cells possessing mutation- reduced or absent GTPase activity (activation)


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Tumour Suppressor Genes

Function: normal cellular genes that :

- limit tissue growth

- contribute to the destruction of cells with damaged

genomes

almost all tumour suppressor genes encode signal transduction

proteins

Deactivation:

Tumour Suppressor Gene Loss of function


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Mechanisms of Tumour Suppressor Gene Deactivation

1. Expression of a protein with altered or absent function

- structural mutation

2. Absence of gene expression- regulatory mutation


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p53 Tumour Suppressor Gene

cell cycle control

DNA repair

differentiation

apoptosis

most well studied tumour

suppressor gene

fully functional p53 can

overcome the effects ofone or more activated

oncogenes

http://fig.cox.miami.edu/~cmallery/150/gene/c7.19.12b.p53.jpg


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p53 Mutations

point mutations resulting in amino acid substitutions or chain termination areextremely common in human cancers (50%)

mutational spectra exhibits hot spots

chemical carcinogens frequently cause mutations at codon 249 (AGG)

AGG(arginine)

AGT

ATG

O

(serine)

(methionine)

Protein with

reduced or absent

function


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Selectivity of p53 Mutations?

1. Unusual susceptibility of codon 249 to ultimate carcinogens

- accessibility, nucleotide preference, physiochemiststry- bulky PAHs form N7-deoxyguanosine adducts

2. Functional effect of mutations

- growth advantage to cells possessing mutation

- reduced or absent protein activity (deactivation)


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Summary - Genetic Modification by Chemical Carcinogens

genotoxic chemical carcinogens covalently modify DNA, a process

which can be reversed by cellular DNA repair

faulty repair of DNA adducts results in changes in the structure and/or

regulatory properties of affected genes and the corresponding protein

products

proto-oncogene activation and loss of tumour suppressor genes are

important outcomes of the genotoxic actions of carcinogens

the vast majority of genetic changes are efficiently repaired, have no

oncogenic consequence, or are lethal to the host cell


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Genetics and Cancer Susceptibility

interindividual variability in cancer susceptibility exists

arises from subtle differences in gene sequence between

individuals (polymorphisms)

can affect the level of expression of the gene or the

function of the protein product exhibits familial patterns of inheritance

Example: Polymorphisms of Carcinogen Metabolism


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Polymorphisms of Carcinogen Metabolism

Glutathione S-transferases- variant GST genes encoding proteins with reduced function have been

linked to increased susceptibility to PAH-induced (tobacco smoke) lung

cancer

UDP-glucuronyltransferases

- variant UDP-GTs genes encoding proteins with reduced function have beenlinked to increased susceptibility to various chemical induced cancers

N-acetyltransferases

- variant NAT1 and NAT2 genes with encoding proteins with reduced activity

have been linked to increased risk for aromatic amine induced bladder cancer

Phase I Bioactivation Enzymes

- no conclusive evidence exists linking polymorphisms of genes encoding Phase I

enzymes with human cancers, but

- evidence exists for interaction with other polymorphisms


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Risk

The probability of a particular adverse effect (e.g. cancer)after exposure to a chemical agent

Example:

The lifetime risk of cancer of is 2.5 x 10-4 from exposure to 1 part per million (ppm) of

chemical X present in the air, when breathed 24 hours a day for 70 years.

a probabilistic statement based upon short, high-dose exposure not derived from direct measurement

assumptions and uncertainties

Is equivalent to:

There is a risk of 1 additional cancer in a population of 1 x 106 individuals from

exposure to 1 part ppm of chemical X present in the air, when breathed 24 hours a

day for 70 years.


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Dose-Response Assessment

A quantitative description of the relationship between the

dose of the agent of interest and a detrimental effect

Description of potency (EC50) and efficacy (Emax)

Considers modifiers of response such as sex, age, species, route of exposure,exposure pattern

Data derived from animal studies with high-dose, short-term exposure

PotentialProblems:

1. Extrapolation from animals to humans

2. Extrapolation from acute to chronic exposure

3. Extrapolation from high dose to low dose


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Dose-Response Curves

ThresholdLinear

DoseDose Dose

Hormetic

+

Net

Effect

_

Damage > Repair

Damage > Repair

Damage = Repair Damage < Repair

Damage > Repair


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0.1 1 10 100

0

1

2

Annual Radiation Exposure (cSv)

Global

Average

Risk

An Example of Hormesis?

GlobalAverage

Exposure relative risk of leukemic

cancer is lower in certain

geographical areas with

higher than average

background radiationlevels

mechanism?

confounding factors?


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The Big Picture

cancer is a disease of uncontrolled cell growth characterized by defects in the

expression of proto-oncogenes and tumour suppressor genes cancer is a multistage disease which requires genetic change for transition from onestage to another

chemical carcinogens act by causing genetic mutations (genotoxic) and/or bystimulating the growth of cancerous cells (non-genotoxic)

metabolism of chemical carcinogens most commonly results in deactivation and

elimination - bioactivation is rare

genetic modification of proto-oncogenes and tumour suppressor genes by chemicalcarcinogens at regions that affect protein structure/function is a rare event

mutation is rare due to efficient and effective repair by DNA repair mechanisms

chemical carcinogenesis requires the occurrence of multiple rare events

the probability of cancer development from exposure to a chemical carcinogen isaffected by genetic polymorphisms, dose and time

the linear dose-response model is preferred for risk assessment

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