Chemical carcinogenesis
林嬪嬪 研究員
國衛院 環職組
NHRI 學程, June, 12012
Outline
- Definition
- Multistage carcinogenesis
- Mechanisms of action of chemical carcinogenesis
- Test systems for carcinogenicity assessment
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Definition
Carcinogen: a physical or chemical agent that causes or
induces neoplasia.
Genotoxic: carcinogens that interact with DNA resulting in
mutation
Nongenotoxic: carcinogens that modify gene expression
but do not damage DNA
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Casarett & Doull’s Toxicology: the basic science of poisons. 7th ed.
Multistage carcinogenesis
1. Initiation (genotoxic)
Genetic changes (mutation and deletion)
2. Promotion (non-genotoxic)
Involves the selective clonal expansion of initiated
cells to produce a preneoplastic lesion; nongenotoxic
3. Progression (genotoxic)
Additional DNA damage (chromosomal aberration
and translocation)
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Anais da Academia Brasileira de Ciências (2007) 79(4): 593-616
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Initiation
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Nature Reviews Cancer 2005, 5, 113
Features of genotoxic and nongenotoxic carcinogens
Genotoxic carcinogens
Mutagenic
Can be complete carcinogen
Tumorigenicity is dose dependent
No theoretical threshold
Nongenotoxic carcinogens
Nonmutagenic
Threshold, reversible
Tumorigenicity is dose dependent
May function at tumor promotion stage
No direct DNA damage
Species, strain, tissue specificity
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Nature Reviews Cancer 2005, 5, 113
• Genotoxic carcinogens: initiated tumors by
producing DNA damage.
– Direct-acting carcinogens: do not require metabolic
activation or chemical modification to induce cancer. For
example, nitrogen mustard, some chemotherapeutic
chemicals.
– Indirect-acting genotoxic carcinogens: require metabolic
activation (from procarcinogen, proximate carcinogen, to
ultimate carcinogen). For example, benzo[a]pyrene,
alfatoxin B1, NNK.
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Summary of events involved in chemical carcinogenesis
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Metabolism is required for some carcinogens
(initiator) activation
Anais da Academia Brasileira de Ciências
(2007) 79(4): 593-616
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Phase I and phase II enzymes
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Oxidation catalyzed by cytochrome P450
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Metabolic activation of carcinogens
Procarcinogen
Proximate carcinogen
Ultimate carcinogen
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Nature Reviews Cancer 2005, 5, 113
Example of phase II reaction : glutathione
conjugation (by glutathione-s-transferase)
Acetyl
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Metabolic activation and detoxification (glucuronic acid
conjugation) of tobacco-specific nitrosamine: NNK
Detoxification
by conjugation
Activation by CYP2A
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Covalent binding between “ultimate
“ carcinogens and endogenous macromolecules
“Ultimate“ carcinogens: electrophiles
Endogenous macromolecules (DNA, RNA, protein and lipids):
nucleophiles

H

..

O H
..

Pauling Electronegativity Scale
.. 
F:
..
H
Li
Be
B
C
N
O
F
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Na
Mg
Al
Si
P
S
Cl
0.9
1.2
1.5
1.8
2.1
2.5
3.0
Electronegativity increases from left to right in the periodic table.
Electronegativity decreases going down a group.
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In general, soft electrophiles prefer to react with soft nucleophiles (low charge-toradius ratio in both). Whereas hard electrophiles react more readily with hard
electrophiles.
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Covalent binding with DNA: AFB1 and AAF
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Covalent binding with DNA: BaP
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One cell cycle is necessary to lock in
mutation: G to T transversion mutation
following BPDE-DNA adduct formation
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Structures of 8-oxoG-containing base pairs and of
several nucleosides of guanine oxidation products
Missmatched!
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Nature, 447, 941-950 (21 June 2007)
Metabolic activation of carcinogens and cancer risk
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Metabolic activation of benzo[a]pyrene
Activator: cytochrome p4501A1
(CYP1A1)
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CYP1A1 SNP
Allele
Low activity
High activity
Protein
Nucleotide changes
Trivial name Effect
CYP1A1*1A CYP1A1.1
None
Wild-type
CYP1A1*1B CYP1A1.1
-3219C>T
CYP1A1*1C CYP1A1.1
-3229G>A
CYP1A1*2A CYP1A1.1
3801T>C (MspI)
CYP1A1*2B CYP1A1.2
2455A>G; 3801T>C (MspI)
CYP1A1*2C CYP1A1.2
2455A>G
m2
CYP1A1*3
CYP1A1.1
3205T>C
m3
CYP1A1*4
CYP1A1.4
2453C>A
m4
CYP1A1*5
CYP1A1.5
2461C>A
R464S
CYP1A1*6
CYP1A1.6
1636G>T
M331I
2346_2347insT
frame shift
CYP1A1*7
m1
I462V
I462V
T461N
CYP1A1*8
CYP1A1.8
2414T>A
I448N
CYP1A1*9
CYP1A1.9
2461C>T
R464C
CYP1A1*10 CYP1A1.10 2500C>T
R477W
CYP1A1*11 CYP1A1.11 2546C>G
P492R
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Low ratio of NNAL-Gluc/free NNAL increases urothelial carcinoma risk
Individual susceptibility (metabolic index):
Total – free (NNAL-Gluc)/free NNAL
Detoxification index:
Total – free (NNAL-Gluc)
/free NNAL
Cancer risk
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Common forms of DNA damage
Abasic site
Strand breaks (single and double)
DNA cross-link
Hydoxylated (oxidized ) base
Bulky adducts
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DNA repair
Base excision repair
Nucleotide excision repair
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DNA substitution mutations are of two types
Transitions are interchanges of two-ring purines (A G) or of one-ring
pyrimidines (C T).
Transversions are interchanges of purine for pyrimidine bases.
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The presence of 8-oxoG (O) in DNA causes G-to-T
transversions
O: 8-oxoG
The actions of AP endonuclease,
deoxyribophosphate lyase, DNA
polymerase and DNA ligase
8-oxoG DNA glycosylase
DNA glycosylase MutY
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Nature, 447, 941-950 (21 June 2007)
Tumor promotion
AhR: aryl hydrocarbon
receptor
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Nature Reviews Cancer 2005, 5, 113
• Nongenotoxic carcinogens: not involve direct
binding, damage, or interaction of the chemical or its
metabolites with DNA.
Proposed mode of action:
Cytotoxicity,
Receptor mediated : CAR, PPARa, and aryl hydrocarbon
receptor (AhR),
Hormonal,
Epigenetic change,
Oxidative stress
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Pathogenesis of preneoplastic lesions leading to
hepatocellular carcinoma in animal models.
DENA: diethylnitrosamine (initiator)
Arch Toxicol (2008) 82:623–631
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• Receptor mediated : CAR, PPARa, and aryl
hydrocarbon receptor (AhR)
CAR, PXR, PPAR, LXR, and FXR belong to NR1 (a nuclear receptor
family): share a common heterodimerization partner, retinoid Xreceptor (RXR); Highly expressed in liver
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Ligand-mediated PPAR activation
Uncertainty:
1.Species difference in sensitivity to PPs exists, with humans appearing to be more
resistant to the toxic effects of these compounds.
2.The peroxisome proliferation and regulation of peroxisomal enzymes do not appear to
be as highly inducible in the human liver.
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3.Peroxisome proliferators are classified as rodent-specific hepatocarcinogens.
AhR
(Aryl hydrocarbon receptor; dioxin receptor)
- Ligand-activated transcription factor
- Transcriptionally regulate cytochrome P450 family 1
1. Location: chromosome 7
2. Protein Size: 105 KDa
3. mRNA size: 6.6Kb
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AhR Activation Mechanism
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From Wikipedia,
Hypothetical model of the role of AhR in tumor initiation,
promotion and progression.
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Carcinogenesis, 2010
CYP1A1 involves in metabolic activation of benzo[a]pyrene
Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl
hydrocarbon receptor (Shimizu et al., PNAS, 2000)
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Epithelial to mesenchymal transition (EMT)
One consequence is sequestration of b-catenin, thereby preventing its nuclear
translocation. Over-expression of master regulators of EMT, such as Snail and
Slug (and others) lead to down-regulation of E-cadherin, hence allowing nuclear
translocation of b-catenin. In association with transcription factors (TF) of the
TCF/LEF family, b-catenin induces transcription of proliferative and mesenchymal
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genes
Potential role of the AhR in EMT and tumor progression
NFAT: nuclear factor of activated T—cells
Autotaxin exhibit lysophospholipase D activity
LPA: lysophosphatidic acid
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Carcinogenesis, 2010
• Epigenetic changes
Numerous potential environmental
factors can lead to the formation of
CSCs from normal SCs and/or
progenitor cells.
Anais da Academia Brasileira de
Ciências (2007) 79(4): 593-616
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Toxicol Sci. 2011 March; 120(suppl_1): S192–S203.
• Hormonal
Estrogens
Synthesis, Distribution, Metabolism
ER Ligands
Metabolites
ER-mediated Signal
Non ER-mediated
Transduction
Signal transduction

Cell Differentiation 
Growth
(Spontaneous Mutations)
JNCI Monograph, 2000
Elimination /or
Protection
DNA Adducts
Ox. DNA Damage
Lipid Peroxidation
Redox Imbalance 
Altered Transcription (?)
Initiation and Progression of
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Cancer
Oxidative metabolism of estrogens
2-OHCE and 4-OHCE : 2-hydroxy and 4-hydroxy catechol estrogens;
COMT : catechol-O-methyltransferase;
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GSH: glutathione
JNCI Monograph, 2000
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JNCI Monograph, 2000
• Oxidative stress
Cancers 2010, 2(2), 338-363
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J Cancer Sci Ther 3: 070-075.
Selected mechanisms responsible for liver tumor
promotion in humans and animal model
Arch Toxicol (2008) 82:623–631
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- Test systems for carcinogenicity assessment
Method
Short term
Mutagenesis assays
Transformation in cell culture
Medium term
Qualitative and quantitative analysis of
preneoplasia
Long term
Chronic bioassay in animals
Time frame
Several weeks
1-3 months
2-8 months
18-24 months
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Mouse model for skin carcinogenesis and
genetic changes
Molecular and Biochemical Toxicology 4th ed. 2008
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The report identifies agents, substances, mixtures, and exposure
circumstances that are known or reasonably anticipated to cause
cancer in humans.
Known To Be Human Carcinogen:
There is sufficient evidence of carcinogenicity from studies in humans,* which indicates a causal relationship between
exposure to the agent, substance, or mixture, and human cancer.
(*This evidence can include traditional cancer epidemiology studies, data from clinical studies, and/or data derived from
the study of tissues or cells from humans exposed to the substance in question, which can be useful for evaluating
whether a relevant cancer mechanism is operating in humans.)
Reasonably Anticipated To Be Human Carcinogen:
There is limited evidence of carcinogenicity from studies in humans,* which indicates that causal interpretation is
credible,
but that alternative explanations, such as chance, bias, or confounding factors, could not adequately be excluded,
or
there is sufficient evidence of carcinogenicity from studies in experimental animals, which indicates there is an increased
incidence of malignant and/or a combination of malignant and benign tumors (1) in multiple species or at multiple tissue
sites, or (2) by multiple routes of exposure, or (3) to an unusual degree with regard to incidence, site, or type of tumor,
or age at onset,
or
there is less than sufficient evidence of carcinogenicity in humans
or laboratory animals; however, the agent, substance, or mixture belongs to a well-defined, structurally related class of
substances whose members are listed in a previous Report on Carcinogens as either known to be a human carcinogen or
reasonably anticipated to be a human carcinogen, or there is convincing relevant information that the agent acts through
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mechanisms indicating it would likely cause cancer in humans.
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Agents Classified by the IARC Monographs, Volumes 1–104
Group 1
Group 2A
Group 2B
Group 3
Group 4
Carcinogenic to humans
107 agents
Probably carcinogenic to humans
63
Possibly carcinogenic to humans
271
Not classifiable as to its carcinogenicity to humans
509
Probably not carcinogenic to humans
1
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Adapted from Table 4 in Cogliano et al. (2011) available at:
http://jnci.oxfordjournals.org/content/early/2011/12/11/jnci.djr483.short?rss=1
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