Chemical Chemical

Carcinogenesis:Carcinogenesis:GENOTOXIC and NON-GENOTOXICGENOTOXIC and NON-GENOTOXIC

carcinogenscarcinogens

Initiating

Event

Cell Proliferation

(clonal expansion)

Progression

Cell Proliferation

Cell Proliferation

Malignancy

Second Mutating

Event

Third Mutating

Event

Initiation

Promotion

Stages of CarcinogenesisStages of Carcinogenesis

Initiating

Event

Cell Proliferation

(clonal expansion)

Progression

Cell Proliferation

Cell Proliferation

Malignancy

Second Mutating

Event

Third Mutating

Event

Initiation

Promotion

Stages of CarcinogenesisStages of Carcinogenesis

Classification of Carcinogens Classification of Carcinogens According to the Mode of ActionAccording to the Mode of Action

GENOTOXIC NON-GENOTOXIC NON-GENOTOXICGENOTOXIC

Classification of Carcinogens Classification of Carcinogens According to the Mode of ActionAccording to the Mode of Action

GENOTOXICGENOTOXIC:: DNA-reactive or DNA-reactive metabolitesDNA-reactive or DNA-reactive metabolites Direct interaction to alter chromosomal Direct interaction to alter chromosomal

number/integritynumber/integrity May be mutagenic or cytotoxicMay be mutagenic or cytotoxic Usually cause mutations in simple systemsUsually cause mutations in simple systems

DNA AdductDNA Adduct MutationMutation CancerCancer

Mechanism of Carcinogenesis:Mechanism of Carcinogenesis:GenotoxicGenotoxic Carcinogens Carcinogens

1. Carcinogen activation1. Carcinogen activation 2. DNA binding2. DNA binding 4. Gene4. Gene mutationmutation

Chemical "Activated“carcinogen

3. Cell proliferation3. Cell proliferation(fix mutation)(fix mutation)

“inactivated“carcinogen

CYP450s

DNA Repair APOPTOSIS

Interaction of the exo-epoxide of Interaction of the exo-epoxide of aflatoxin Baflatoxin B11 with DNA with DNA

Smela et al., Carcinogenesis 22:535-45 (2001)Smela et al., Carcinogenesis 22:535-45 (2001)

Classification of Carcinogens Classification of Carcinogens According to the Mode of ActionAccording to the Mode of Action

NON-GENOTOXICNON-GENOTOXIC:: Do not directly cause DNA mutation Mechanism of action is not completely

understood Difficult to detect - requires rodent carcinogen

bioassay

?? MutationMutation CancerCancer

Non-Genotoxic Carcinogens

1)1) Mitogens: Mitogens: • stimulation of proliferationstimulation of proliferation

• mutations may occur secondarily to cell proliferationmutations may occur secondarily to cell proliferation

• may cause preferential growth of preneoplastic cellsmay cause preferential growth of preneoplastic cells

2) 2) Cytotoxicants: Cytotoxicants: • cytolethalcytolethal• induce regenerative growthinduce regenerative growth• mutations may occur secondarily to cell proliferationmutations may occur secondarily to cell proliferation

Tissue Changes with Mitogenic Tissue Changes with Mitogenic and Cytotoxic Agentsand Cytotoxic Agents

Proliferation

Cell Death Proliferation

Cytotoxic Agent

MitogenicAgent

Tissue

Mechanism of Carcinogenesis:Non-Genotoxic carcinogens

Cell proliferation (to fix “spontaneous” mutation)Cell proliferation (to fix “spontaneous” mutation)

APOPTOSIS

CANCER

X

Mechanisms of Non-Genotoxic Mechanisms of Non-Genotoxic CarcinogenesisCarcinogenesis

(what’s in a “black box” ?)(what’s in a “black box” ?)

Increased cell proliferationIncreased cell proliferation

Decreased apoptosisDecreased apoptosis

Changes in gene expression Changes in gene expression

Induction of metabolizing enzymesInduction of metabolizing enzymes

Activation of receptors (signaling)Activation of receptors (signaling)

Oxidative stressOxidative stress

??????

Decreases time available for DNA repairDecreases time available for DNA repair Converts repairable DNA damage into non-repairable Converts repairable DNA damage into non-repairable

mutationsmutations Necessary for chromosomal aberrations, insertions, Necessary for chromosomal aberrations, insertions,

deletions and gene amplificationdeletions and gene amplification Clonally expands existing cell populationsClonally expands existing cell populations

Cell Replication is Essential for Cell Replication is Essential for Multistage CarcinogenesisMultistage Carcinogenesis

Mitogenic Cytokines andMitogenic Cytokines andInduction of Cell Proliferation Induction of Cell Proliferation

Complete Mitogens:Complete Mitogens:

EEpidermal pidermal GGrowth rowth FFactor, actor, TTumor umor NNecrosis ecrosis FFactor actor , ,

HHepatocyte epatocyte GGrowth rowth FFactor, etc.actor, etc.

Co-Mitogens:Co-Mitogens:

Insulin, glucagon, norepinephrin, estrogensInsulin, glucagon, norepinephrin, estrogens

Growth Inhibitors:Growth Inhibitors:

TTransforming ransforming GGrowth rowth FFactor actor , , IInternterLLeukin 1eukin 1

Reasons That Not All Agents That Increase Reasons That Not All Agents That Increase Cell Proliferation are CarcinogensCell Proliferation are Carcinogens

Quality of the dataQuality of the data Temporal association of the increase in cell Temporal association of the increase in cell

proliferationproliferation Selective cytotoxicity for initiated cellsSelective cytotoxicity for initiated cells Terminal differentiation of proliferating cellsTerminal differentiation of proliferating cells

Mutagenesis Mutagenesis Carcinogenesis Carcinogenesis

Cell Proliferation Cell Proliferation Carcinogenesis Carcinogenesis

Toxicity Toxicity Cell Proliferation Cell Proliferation

ApoptosisApoptosis

Programmed Cell Death (Apoptosis): Active, orderly and cell-Programmed Cell Death (Apoptosis): Active, orderly and cell-type-specific death distinguishable from necrotic cell death type-specific death distinguishable from necrotic cell death (passive process):(passive process):

Induced in normal and cancer cells Induced in normal and cancer cells Non-random eventNon-random event Result of activation of a cascade of biochemical, gene Result of activation of a cascade of biochemical, gene

expression and morphological eventsexpression and morphological events tissue and cell specifictissue and cell specific Growth factors and mitogens inhibit apoptosisGrowth factors and mitogens inhibit apoptosis

Alteration of Gene ExpressionAlteration of Gene Expression

Nuclear (hormone-like) receptors Nuclear (hormone-like) receptors

Kinase cascadesKinase cascades

Calcium-, nitric oxide-mediated signalingCalcium-, nitric oxide-mediated signaling

Transcription factorsTranscription factors

Gene methylation status (hypo -> enhanced gene Gene methylation status (hypo -> enhanced gene

expression; hyper -> gene silencing)expression; hyper -> gene silencing)

Induction of Metabolizing EnzymesInduction of Metabolizing Enzymes

May be a reason for tissue-, and/or species-May be a reason for tissue-, and/or species-

selectivity of carcinogensselectivity of carcinogens

Metabolites may be ligands for receptorsMetabolites may be ligands for receptors

Production of reactive oxygen speciesProduction of reactive oxygen species

Nature 407, 920 - 923 (2000) ©Nature 407, 920 - 923 (2000) ©

  The nuclear receptor CAR mediates specific The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolismxenobiotic induction of drug metabolism

ATCGGTTA……

XB

CAR

CYP 2b10

Oxidative StressOxidative Stress

Indirect DNA damageIndirect DNA damage

Induction of cell proliferation/apoptosis signaling Induction of cell proliferation/apoptosis signaling

cascadescascades

Peroxisome ProliferatorsPeroxisome Proliferators

A wide range of classes of chemicals: A wide range of classes of chemicals: lipid lipid lowering drugs, plasticizers, lowering drugs, plasticizers,

food flavors, industrial solvents, herbicides food flavors, industrial solvents, herbicides Cause marked increases in size and number of Cause marked increases in size and number of

peroxisomes peroxisomes Potent rodent liver carcinogensPotent rodent liver carcinogens Human exposure is from therapeutic, Human exposure is from therapeutic,

environmental, industrial and other sourcesenvironmental, industrial and other sources No clear epidemiological evidence for or against No clear epidemiological evidence for or against

carcinogenicity in humans carcinogenicity in humans

PEROXISOMEPEROXISOME

Fatty Acid

Acyl-CoA

Enoyl-CoA

Hydroxyacyl-CoA

Ketoacyl-CoA

Acetyl-CoA Acyl-CoA shortened by two carbons

Fatty acyl-CoA synthetase

Acyl-CoA oxidase

Enoyl-CoA hydrolase

Hydroxyacyl-CoAdehydrogenase

Thiolase

H2O2

• -oxidation of fatty acids• bile acid synthesis• purine and polyamine

catabolism• amino acid catabolism• oxygen metabolism

Peroxisome proliferation

• Liver growth– hypertrophy– hyperplasia

• Induction of liver enzymes– peroxisomal enzymes (peroxisome proliferation)– P450 - the CYP4 genes

• Proliferation of the endoplasmic reticulum and peroxisomes

• Hypolipidaemia

Adipocyte DifferentiationGlucose HomeostasisMacrophage Function

Peroxisome ProliferationLipid Homeostasis

Liver Carcinogenesis

Lipid HomeostasisSkin proliferation

Peroxisome Proliferator - Activated Peroxisome Proliferator - Activated ReceptorsReceptors

FA

FABP

DietFat Stores

FA StorageFA Metabolism

FA

PPRE

FA

PP

PPRE

??PPRE

LTP

G

PPAR agonist-induced hepatocarcinogenesis mode of action

Peters& Gonzalez, J. Mol. Med., 2005

Klaunig et al., Crit. Rev. Tox., 2003

Klaunig et al., Crit. Rev. Tox., 2003

Peters et al., Carcinogenesis, 1997

PPARPPAR (+/+) (+/+)

+ WY-14,643 (11 months)+ WY-14,643 (11 months)PPARPPAR (-/-) (-/-)

+ WY-14,643 (11 months)+ WY-14,643 (11 months)

Peroxisome Proliferators: Species Differences

• Mouse and rat: highly responsive• Marmoset: does not respond• Guinea Pig: no peroxisome proliferation, but have hypolipidaemia• Humans: believed to be unresponsive, but have hypolipidaemia

• PPAR exists in mouse, rat, guinea pig and human

• In humans: Lower hepatic levels of PPARa Lower ligand binding activityDifferent structure (polymorphisms)Different PP Response Elements in DNAPresence of competing proteins for PPREExpression of dominant-negative form of PPAR

So, we have a chemical that So, we have a chemical that is a non-genotoxic RODENT is a non-genotoxic RODENT

carcinogen!carcinogen!

If we would regulate this chemical, If we would regulate this chemical,

would it help to improve the quality would it help to improve the quality

of HUMAN life?of HUMAN life?

  Proportion Percentage

Chemicals tested in both rats and mice 350/590 59%

Naturally occurring chemicals 79/139 57%

Synthetic chemicals 271/451 60%

Chemicals tested in rats and/or mice    

Chem. in Carcinogen. Potency Database 702/1348 52%

Natural pesticides 37/71 52%

Mold toxins 14/23 61%

Chemicals in roasted coffee 21/30 70%

Innes negative chemicals retested 17/34 50%

Physician’s desk reference PDR    

Drugs with reported cancer tests 117/241 49%

FDA database of drug submissions 125/282 44%

Proportion of chemicals evaluated as Proportion of chemicals evaluated as carcinogeniccarcinogenic

Ames and Gold Ames and Gold Mutat ResMutat Res 447:3-13, 2000 447:3-13, 2000

What do we do now?What do we do now?

Look at the “larger picture”Look at the “larger picture”

Probe human relevance of animal dataProbe human relevance of animal data

Continue research on the mechanismsContinue research on the mechanisms

Change/improve current test used for detection of Change/improve current test used for detection of

carcinogenicitycarcinogenicity