Chemical-Induced Chemical-Induced

CarcinogenesisCarcinogenesis

CANCER:CANCER:““A multicausal, multistage group of diseases the mechanisms of which are still only A multicausal, multistage group of diseases the mechanisms of which are still only partially knownpartially known” (IARC Scientific Publications, 1992)” (IARC Scientific Publications, 1992)

““Cancer is a group of diseases characterized by uncontrolled growth and spread of Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells […] that can result in death”abnormal cells […] that can result in death” (American Cancer Society, 2006) (American Cancer Society, 2006)

Age-adjusted Cancer Death Rates, by Site, US, 1930-2005

http://apps.nccd.cdc.gov/uscs/

WHAT MAY CAUSE CANCER ?WHAT MAY CAUSE CANCER ? Hereditary Hereditary disordersdisorders ChemicalsChemicals VirusesViruses Chronic inflammationChronic inflammation ??????

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History of Chemical Carcinogenesis

• Chemical carcinogenesis was first suggested by clinicians 200 years ago– Scrotal cancer in chimney sweeps - Potts– Nasal cancer and snuff dipping - Hill– Today, >50 chemicals are recognized as

human carcinogens

• First experimental studies in animals were done ~80 years ago

• Large numbers of chemicals were tested for carcinogenic potential in the 1970-1990s– Maximum Tolerated Doses (MTD) were used.– 60% of rodent carcinogens were genotoxic– 40% of rodent carcinogens were nongenotoxic– Some chemicals were single site, single species

carcinogens– Others were multisite, multispecies carcinogens– Dose-response varies from <1/2 MTD to

<1/1000 MTD• Most regulations use straight mathematical

extrapolation of high dose rodent data to predict risks

History of Chemical Carcinogenesis

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The National Toxicology Program (NTP) was established in 1978 to coordinate toxicological testing programs within the Department of Health and Human Services, develop and validate improved testing methods, develop approaches and generate data to strengthen scientific knowledge about potentially hazardous substances and communicate with stakeholders.

“The NTP performs appropriate toxicity studies in part to provide dose-setting information for chronic studies and also to address specific deficiencies in the toxicology database for the chemical.”

Toxicology/Carcinogenicity studies generally fall into two categories:

1. Prechronic Toxicity Studies14-day study13 week (90 day) study

2. Two-Year Toxicology and Carcinogenesis Rodent Studiesusually - 104 wkssometimes - ~90 wks exposure followed by 10-15 wks of normal diet

14-Day Toxicity Protocol The goal of this is to provide a basis for identifying potential target organs and toxicities and to assist in setting doses for the 13-week exposure study.

Treatment: 10- to 14-day quarantine period, animals are assigned at random to groups. Five treatment groups each administered a different concentration of test article per sex/species plus a control group. For dosed-feed and dosed-water studies animals are exposed for 14 consecutive days. For inhalation, gavage and dermal studies animals are exposed for 12 treatment days, not including weekends or holidays with at least two consecutive treatment days before the terminal sacrifice day.

Observations: Animals are weighed individually on day one, after seven days, and at sacrifice. The animals are observed twice daily, at least six hours apart (before 10:00 AM and after 2:00 PM) including holidays and weekends, for moribundity and death. Animals found moribund or showing clinical signs of pain or distress are humanely euthanized. Observations are made twice daily for clinical signs of pharmacologic and toxicologic effects of the chemical. For dosed-feed or dosed-water studies, food consumption/water consumption shall be measured and recorded weekly.

Necropsy and Histopathologic Evaluation: Liver, thymus, right kidney, right testicle, heart, and lung weights are recorded for all animals surviving until the end of the study. A complete necropsy is performed on all treated and control animals that either die or are sacrificed and all tissues are saved in formalin. Histopathologic evaluation is done only on those organs/tissues showing gross evidence of treatment-related lesions to a no-effect level plus corresponding tissues are evaluated in control animals. If specific targets are required they shall be read in the control and highest treatment group and the remaining groups to a no-effect level.

In addition to obtaining toxicological data, the purpose of this study is to determine the treatments for each strain and species to be used in the 2-year toxicology/carcinogenesis study.

Treatment: 10- to 14-day quarantine period, animals are assigned at random to treatment groups. Five treatment groups plus a control group. Each group - 10 animals per sex/species. Controls receive untreated water or feed or vehicle alone in gavage and dermal studies. For dosed-feed and dosed-water studies, animals are exposed for 90 days after which they are sacrificed with no recovery period. For inhalation, gavage and dermal studies animals are exposed five times per week, weekdays only until the day prior to necropsy.

Observations: Animals are weighed individually on day 1, after 7 days, and at weekly periods thereafter. Animals are observed twice daily, at least 6 hours apart, including holidays and weekends, for moribundity and death. Formal clinical observations are performed and recorded weekly. For dosed-feed or dosed-water studies, food/water consumption is measured and recorded weekly.

Necropsy and Histopathologic Evaluation: Liver, thymus, right kidney, right testis, heart, and lung weights are recorded from all animals surviving until the end of the study. A complete necropsy is performed on all treated and control animals that die or are sacrificed.

Specific Toxicologic Parameters Evaluated in the 13-Week Study Clinical Laboratory Studies: Blood is collected from both sexes of "special study" rats, at days 4 ± 1 and 21 ± 2 and from the core study rats at the end of the study. Blood for Micronuclei: Blood samples are taken at study termination for micronuclei determinations. Sperm Morphology and Vaginal Cytology Evaluations (SMVCE)

90-Day Toxicity Protocol

Two-year Carcinogenesis “Bioassay” Protocol

WORLD HEALTH ORGANIZATIONINTERNATIONAL AGENCY FOR RESEARCH ON CANCER

IARC Monograph Evaluations

LYON, FRANCE

Slide courtesy of V. Cogliano (US EPA)

Group 1 agents with less than sufficient evidence in humans

2,3,4,7,8-Pentachlorodibenzofuran3,4,5,3’,4’-Pentachlorobiphenyl (PCB-126)4,4’-Methylenebis(2-chloroaniline) (MOCA)Alpha- and beta-particle emittersAreca nutAristolochic acid Benzidine, dyes metabolised toBenzo[a]pyreneEthanol in alcoholic beveragesEthylene oxide EtoposideIonizing radiation (all types)Neutron radiationN′-Nitrosonornicotine (NNN) and 4-(N-nitroso-

methylamino)-1-(3-pyridyl)-1-butanone (NNK)

Ultraviolet radiation

The IARC Monographs Volume 100 : The known causes of human cancer by organ site

Oral cavity Alcoholic beveragesBetel quid with tobaccoBetel quid without tobaccoHuman papillomavirus type 16Smokeless tobaccoTobacco smoking

Pharynx Alcoholic beveragesBetel quid with tobaccoHuman papillomavirus type 16Tobacco smoking

Salivary gland X-radiation, gamma-radiation

Nasopharynx Epstein-Barr virusFormaldehydeSalted fish, Chinese-styleWood dust

Nasal cavity and paranasal sinus

Isopropyl alcohol manufacture using strong acids

Leather dustNickel compoundsRadium-226 and its decay productsRadium-228 and its decay productsTobacco smokingWood dust

Larynx Acid mists, strong inorganicAlcoholic beveragesAsbestos (all forms)Tobacco smoking

Lung Aluminium productionArsenic and inorganic arsenic compoundsAsbestos (all forms)Beryllium and beryllium compoundsBis(chloromethyl)ether; chloromethyl methyl ether

(technical grade)Cadmium and cadmium compoundsChromium (VI) compoundsCoal, indoor emissions from household combustionCoal gasificationCoal-tar pitchCoke productionHaematite mining (underground)Iron and steel foundingMOPP (vincristine-prednisone-nitrogen mustard-procarbazine

mixture)Nickel compoundsPainter (occupational exposure as)PlutoniumRadon-222 and its decay productsRubber production industrySilica dust, crystallineSootSulfur mustardTobacco smoke, secondhandTobacco smokingX-radiation, gamma-radiation

Oesophagus Acetaldehyde associated with consumption of alcoholic beverages

Alcoholic beveragesBetel quid with tobaccoBetel quid without tobaccoSmokeless tobaccoTobacco smokingX-radiation, gamma-radiation

Stomach Helicobacter pyloriRubber production industryTobacco smokingX-radiation, gamma-radiation

Colon and rectum Alcoholic beveragesTobacco smokingX-radiation, gamma-radiation

Liver (hepatocytes)

AflatoxinsAlcoholic beveragesEstrogen-progestogen contraceptivesHepatitis B virusHepatitis C virusPlutoniumThorium-232 and its decay productsTobacco smoking (in smokers and in

smokers’ children)Vinyl chloride

Gall bladder Thorium-232 and its decay products

Pancreas Smokeless tobaccoTobacco smoking

Upper aerodigestive tract Acetaldehyde associated with consumption of alcoholic beverages

Uterine cervix Diethylstilbestrol (exposure in utero)Estrogen-progestogen contraceptivesHuman immunodeficiency virus type 1Human papillomavirus types 16, 18, 31, 33, 35,

39, 45, 51, 52, 56, 58, 59Tobacco smoking

Endometrium Estrogen menopausal therapyEstrogen-progestogen menopausal therapyTamoxifen

Ovary Asbestos (all forms)Estrogen menopausal therapyTobacco smoking

Vagina Diethylstilbestrol (exposure in utero)Human papillomavirus type 16

Vulva Human papillomavirus type 16

Penis Human papillomavirus type 16

Urinary bladder Aluminium production4-AminobiphenylArsenic and inorganic arsenic compoundsAuramine productionBenzidineChlornaphazineCyclophosphamideMagenta production2-NaphthylaminePainter (occupational exposure as)Rubber production industrySchistosoma haematobiumTobacco smokingortho-ToluidineX-radiation, gamma-radiation

Renal pelvis and ureter

Aristolochic acid, plants containing

PhenacetinPhenacetin, analgesic

mixtures containing

Tobacco smoking

Leukaemia/lymphoma

AzathioprineBenzeneBusulfan1,3-ButadieneChlorambucilCyclophosphamideCyclosporineEpstein-Barr virusEtoposide with cisplatin and bleomycinFission products, including Strontium-90FormaldehydeHelicobacter PyloriHepatitis C virusHuman immunodeficiency virus type 1Human T-cell lymphotropic virus type 1Kaposi sarcoma herpes virusMelphalanMOPP (vincristine-prednisone-nitrogen

mustard-procarbazine mixture)Phosphorus-32, as phosphateRubber production industrySemustine [1-(2-Chloroethyl)-3-(4-

methylcyclohexyl)-1-nitrosourea, or methyl-CCNU]

ThiotepaThorium-232 and its decay productsTobacco smokingTreosulfanX-radiation, gamma-radiation

Breast Alcoholic beveragesDiethylstilbestrolEstrogen-progestogen

contraceptivesEstrogen-progestogen

menopausal therapy

X-radiation, gamma-radiation

Skin (other malignant neoplasms)

Arsenic and inorganic arsenic compoundsAzathioprineCoal-tar distillationCoal-tar pitchCyclosporineMethoxsalen plus ultraviolet AMineral oils, untreated or mildly treatedShale oilsSolar radiationSootX-radiation, gamma-radiation

Bone PlutoniumRadium-224 and its decay productsRadium-226 and its decay productsRadium-228 and its decay productsX-radiation, gamma-radiation

Eye Human immunodeficiency virus type 1Ultraviolet-emitting tanning devicesWelding

Brain and central nervous system

X-radiation, gamma-radiation

Thyroid

Radioiodines, including iodine-131 (exposure during childhood and adolescence)

X-radiation, gamma-radiation

Mesothelioma (pleura or peritoneum)

Asbestos (all forms)ErionitePainter (occupational exposure as)

Endothelium(Kaposi sarcoma)

Human immunodeficiency virus type 1Kaposi sarcoma herpes virus

Skin (melanoma)

Solar radiationUltraviolet-emitting tanning devices

Section of the IARC Monographs (IMO)

Biliary tract Chlonorchis sinensisOpisthorchis viverrini

Multiple sites (unspecified)

CyclosporineFission products, including

Strontium-90X-radiation, gamma-radiation

(exposure in utero)

All cancers combined

2,3,7,8-Tetrachlorodibenzo-para-dioxin

Tonsil Human papillomavirus type 16

Anus Human immunodeficiency virus type 1Human papillomavirus type 16

Kidney Tobacco smokingX-radiation, gamma-radiation

http://monographs.iarc.fr/ENG/Publications/

OrganSitePoster2012.ppt

Slide courtesy of V. Cogliano (US EPA)

Cancer in humans � Sufficient evidence � Limited evidence � Inadequate evidence � Evidence suggesting lack of

carcinogenicity

Cancer in experimental animals

� Sufficient evidence � Limited evidence � Inadequate evidence � Evidence suggesting lack of

carcinogenicity

Mechanistic andother relevant data

– Identify established and likely mechanistic events

– Determine whether each mechanism could operate in humans

Overall evaluation

� Group 1 Carcinogenic to humans � Group 2A Probably carcinogenic to humans � Group 2B Possibly carcinogenic to humans � Group 3 Not classifiable as to its carcinogenicity to humans � Group 4 Probably not carcinogenic to humans

IARC has a two-step evaluation process

A tour of IARC’s classificationsPreamble, Part B, Section 6(d)

Slide courtesy of V. Cogliano (US EPA)

Group 2B(possibly

carcinogenic)

“…it is biologically plausible that agents for which there is sufficient evidence of carcinogenicity in experimental animals also present a carcinogenic hazard to humans.”

Slide courtesy of V. Cogliano (US EPA)

Caprolactam is an irritant and is mildly toxic, with an LD50 of 1.1 g/kg (rat, oral). In 1991, it was included on the list of hazardous air pollutants by the U.S. Clean Air Act of 1990. It was subsequently removed from the list in 1993.[2] In water, caprolactam hydrolyzes to amino-caproic acid, which is used medicinally.As of 2011 caprolactam had the unusual status of being the only chemical in the International Agency for Research on Cancer's lowest hazard category, Group 4 "probably not carcinogenic to humans".[3][4]

          

Group 1 Carcinogenic to humans 108 agents

Group 2A Probably carcinogenic to humans 64

Group 2B         Possibly carcinogenic to humans 272

Group 3 Not classifiable as to its carcinogenicity to humans             508

Group 4 Probably not carcinogenic to humans 1

Agents Classified by the IARC Monographs, Volumes 1–105 (monographs.iarc.fr)

http://en.wikipedia.org/wiki/Caprolactam

http://tools.niehs.nih.gov/srp/1/Resources/Arzuaga_IRIS_presentation.pdf

www.epa.gov/iris

IARC

US EPA (Cancer classification)

NTP (Report on Carcinogens)

California EPA

Globally Harmonized System of Classification and Labelling of Chemicals

Cancer Cases Attributable to Environmental Carcinogens

(Worldwide, 1990)

Infections (viruses, parasites, H. pylori) 16%Tobacco (smoked and smokeless) 14%Occupation 4%Alcohol drinking 3%

37%

Diet and dietary components including contaminants 25%Pollution 2%Reproductive factors 2%

29%

Chemical Carcinogenesis in the 21st Century

New perceptions of previously known carcinogens:

Combined effects of multiple exposures

Examples:oAlcohol drinking and aflatoxinsoAlcohol drinking and HBV/HBCoAlcohol drinking and tobacco smokingoAlcohol drinking and asbestos/arsenic/radon

Initiating

Event

Cell Proliferation

(clonal expansion)

Progression

Cell Proliferation

Cell Proliferation

Malignancy

Second Mutating

Event

"N" Mutating Event

Initiation

Promotion

Stages of CarcinogenesisStages of 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

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“Simple” genetic changes

Chemical ExposureChemical Exposure (air, water, food, etc.) (air, water, food, etc.)

Internal ExposureInternal Exposure

Metabolic ActivationMetabolic Activation

Macromolecular BindingMacromolecular Binding DetoxicationDetoxication

DNADNA RNARNA ProteinProtein

Biologically Effective DoseBiologically Effective Dose

Efficiency of MispairingEfficiency of Mispairing

Cell ProliferationCell Proliferation

XX

XXInitiationInitiation

(Biomarker)(Biomarker)

GENETIC AND EPIGENETIC MODELS OF THE CANCER INITIATION

Epigenetically reprogrammed cells

Mutator phenotype cells

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ALTERATIONS IN CELLULAR EPIGENOME

Normal cells

Cancer cells

Clonal selection and expression of initiated cells

Mutator phenotype cells

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ACQUISITION OF ADDITIONAL RANDOM MUTATIONS

Normal cells

Cancer cells

Accumulation of mutations during tumor Accumulation of mutations during tumor progressionprogression

Loeb L.A. Cancer Res. 61:3230-9 (2001)

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

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No Tumors

Tumors

No Tumors

No Tumors

Tumors

1.

2.

3.

4.

5.

X

X

X

X

Time

X = Application of Initiator = Application of Promoter

N Basophilic Focus Adenoma Carcinoma

M 1 MN

Promotion Regression Progression

Adapted from: Marsman and Popp. Carcinogenesis 15:111-117 (1994)

No Tumors

Tumors= Application of Promoter

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

From http://newscenter.cancer.gov/sciencebehind/

“Complex” genetic changes

Evolution of DNA damage during the cell cycle

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

GENOTOXIC NON-GENOTOXIC NON-GENOTOXICGENOTOXIC

Classification of Carcinogens According to Classification of Carcinogens According to the Mode of Actionthe 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

Schematic diagram showing the mechanism through which exposure to Schematic diagram showing the mechanism through which exposure to polycyclic aromatic hydrocarbons is thought to cause cancerpolycyclic aromatic hydrocarbons is thought to cause cancer

Rundle, Mutat Res 600(1-2):23-36 (2006)

Williams J.A., Carcinogenesis 22:209-14 (2001)

Classification of Carcinogens According to Classification of Carcinogens According to the Mode of Actionthe 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 CarcinogensNon-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 and Tissue Changes with Mitogenic and Cytotoxic AgentsCytotoxic 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

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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

Induction of Metabolizing EnzymesInduction of Metabolizing Enzymes

May be a reason for tissue-, and/or species-selectivity of carcinogensMay be a reason for tissue-, and/or species-selectivity of carcinogens

Metabolites may be ligands for receptorsMetabolites may be ligands for receptors

Production of reactive oxygen speciesProduction of reactive oxygen species

Nebert & Dalton Nat Rev Cancer 2006

Human Tumors and Stages of Carcinogenesis

Hussain et al., Oncogene, 2007

HALLMARKS OF CANCER1. Sustaining proliferative signaling2. Evading growth suppressors3. Resisting cell death4. Enabling replicative immortality5. Inducing aberrant angiogenesis6. Activating invasion & metastasis

Emerging Hallmarks• Reprogramming energy

metabolism• Evading immune destruction

Enabling Characteristics• Genomic instability and

mutation• Inflammation

Hanahan and Weinberg 2011 (Cell)

InitiationPromotionProgression vs Hallmarks of Cancer

Floor et al (Trends in Molecular Medicine, September 2012, Vol. 18, No. 9)