Chemical-Induced Carcinogenesis

CANCER:

“

A multicausal, multistage group of diseases the mechanisms of which are still only partially known

” (IARC Scientific Publications, 1992)

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

(American Cancer Society, 2006)

Age-adjusted Cancer Death Rates, by Site, US, 1930-2005 http://apps.nccd.cdc.gov/uscs/

WHAT MAY CAUSE CANCER ?

    

Hereditary disorders

Chemicals Viruses Chronic inflammation ???

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

History of Chemical Carcinogenesis

• 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

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.

http://ntp.niehs.nih.gov/files/Agenda_Presentations.pdf

“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 Studies

14-day study 13 week (90 day) study

2. Two-Year Toxicology and Carcinogenesis Rodent Studies

usually - 104 wks sometimes - ~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.

90-Day Toxicity Protocol

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)

Two-year Carcinogenesis “Bioassay” Protocol

WORLD HEALTH ORGANIZATION INTERNATIONAL AGENCY FOR RESEARCH ON CANCER

IARC Monograph Evaluations

LYON, FRANCE

Slide courtesy of V. Cogliano (US EPA)

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

Oral cavity Tonsil Alcoholic beverages Betel quid with tobacco Betel quid without tobacco Human papillomavirus type 16 Smokeless tobacco Tobacco smoking Human papillomavirus type 16 Pharynx Alcoholic beverages Betel quid with tobacco Human papillomavirus type 16 Tobacco smoking Nasopharynx Epstein-Barr virus Formaldehyde Salted fish, Chinese-style Wood dust Salivary gland X-radiation, gamma-radiation Thyroid Radioiodines, including iodine-131 (exposure during childhood and adolescence) X-radiation, gamma-radiation Stomach Upper aerodigestive tract

Helicobacter pylori

Rubber production industry Tobacco smoking X-radiation, gamma-radiation Acetaldehyde associated with consumption of alcoholic beverages Liver (hepatocytes) Aflatoxins Alcoholic beverages Estrogen-progestogen contraceptives Hepatitis B virus Hepatitis C virus Plutonium Thorium-232 and its decay products Tobacco smoking (in smokers and in smokers’ children) Vinyl chloride Oesophagus Gall bladder Acetaldehyde associated with consumption of alcoholic beverages Alcoholic beverages Betel quid with tobacco Betel quid without tobacco Smokeless tobacco Tobacco smoking X-radiation, gamma-radiation Thorium-232 and its decay products Biliary tract

Chlonorchis sinensis Opisthorchis viverrini

Pancreas Smokeless tobacco Colon and rectum Alcoholic beverages Tobacco smoking X-radiation, gamma-radiation Tobacco smoking Anus Human immunodeficiency virus type 1 Human papillomavirus type 16 Eye Human immunodeficiency virus type 1 Ultraviolet-emitting tanning devices Welding Brain and central nervous system X-radiation, gamma radiation Breast Nasal cavity and paranasal sinus Larynx Isopropyl alcohol manufacture using Leather dust Nickel compounds Radium-226 and its decay products Radium-228 and its decay products Tobacco smoking Wood dust Acid mists, strong inorganic Alcoholic beverages Asbestos (all forms) Tobacco smoking Mesothelioma (pleura or peritoneum) strong acids Asbestos (all forms) Erionite Painter (occupational exposure as) Lung Aluminium production Arsenic and inorganic arsenic compounds Asbestos (all forms) Beryllium and beryllium compounds Bis(chloromethyl)ether; chloromethyl methyl ether Coal gasification Coal-tar pitch Coke production (technical grade) Cadmium and cadmium compounds Chromium (VI) compounds Coal, indoor emissions from household combustion Haematite mining (underground) Iron and steel founding MOPP (vincristine-prednisone-nitrogen mustard-procarbazine mixture) Nickel compounds Painter (occupational exposure as) Plutonium Radon-222 and its decay products Rubber production industry Silica dust, crystalline Soot Sulfur mustard Tobacco smoke, secondhand Tobacco smoking X-radiation, gamma-radiation Alcoholic beverages Diethylstilbestrol Estrogen-progestogen contraceptives Estrogen-progestogen menopausal therapy X-radiation, gamma-radiation Kidney Renal pelvis and ureter Tobacco smoking Phenacetin Urinary bladder X-radiation, gamma-radiation Aristolochic acid, plants containing Phenacetin, analgesic mixtures containing Tobacco smoking Aluminium production 4-Aminobiphenyl Arsenic and inorganic arsenic compounds Auramine production Benzidine Chlornaphazine Cyclophosphamide Magenta production 2-Naphthylamine Painter (occupational exposure as) Rubber production industry

Schistosoma haematobium

Tobacco smoking

ortho

-Toluidine X-radiation, gamma-radiation Uterine cervix Endometrium Estrogen menopausal therapy Estrogen-progestogen menopausal therapy Tamoxifen Diethylstilbestrol (exposure Tobacco smoking

in utero

) Estrogen-progestogen contraceptives Human immunodeficiency virus type 1 Human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 Penis

Section of the IARC Monographs (IMO)

Vagina Vulva Diethylstilbestrol (exposure Human papillomavirus type 16 Ovary Human papillomavirus type 16 http://monographs.iarc.fr/ENG/Pub lications/OrganSitePoster2012.ppt

Human papillomavirus type 16 Asbestos (all forms) Tobacco smoking

in utero

) Estrogen menopausal therapy Multiple sites (unspecified) All cancers combin ed Cyclosporine Fission products, including Strontium-90 X-radiation, gamma-radiation (exposure

in utero

) 2,3,7,8-Tetrachlorodibenzo-

para

-dioxin Group 1 agents with less than

sufficient

evidence in humans 2,3,4,7,8-Pentachlorodibenzofuran 3,4,5,3’,4’-Pentachlorobiphenyl (PCB-126) 4,4’-Methylenebis(2-chloroaniline) (MOCA) Alpha- and beta-particle emitters Areca nut Aristolochic acid Benzidine, dyes metabolised to Benzo[

N′ a

]pyrene Ethanol in alcoholic beverages Ethylene oxide Etoposide Ionizing radiation (all types) Neutron radiation Nitrosonornicotine (NNN) and 4-( (NNK) Ultraviolet radiation

N

-nitroso methylamino)-1-(3-pyridyl)-1-butanone Skin (melanoma) Skin (other malignant neoplasms) Bone Plutonium Radium-224 and its decay products Radium-226 and its decay products Radium-228 and its decay products X-radiation, gamma-radiation Endothelium (Kaposi sarcoma) Human immunodeficiency virus type 1 Kaposi sarcoma herpes virus Solar radiation Ultraviolet-emitting tanning devices Arsenic and inorganic arsenic compounds Azathioprine Coal-tar distillation Coal-tar pitch Cyclosporine Methoxsalen plus ultraviolet A Mineral oils, untreated or mildly treated Shale oils Solar radiation Soot X-radiation, gamma-radiation Leukaemia/ lymphoma Azathioprine Benzene Busulfan 1,3-Butadiene Chlorambucil Cyclophosphamide Cyclosporine Epstein-Barr virus Etoposide with cisplatin and bleomycin Fission products, including Strontium-90 Formaldehyde

Helicobacter Pylori

Hepatitis C virus Human immunodeficiency virus type 1 Human T-cell lymphotropic virus type 1 Kaposi sarcoma herpes virus Melphalan MOPP (vincristine-prednisone-nitrogen mustard-procarbazine mixture) Phosphorus-32, as phosphate Rubber production industry Semustine [1-(2-Chloroethyl)-3-(4 methylcyclohexyl)-1-nitrosourea, or methyl-CCNU] Thiotepa Thorium-232 and its decay products Tobacco smoking Treosulfan X-radiation, gamma-radiation

IARC has a two-step evaluation process

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 and other relevant data – Identify established and likely mechanistic events – Determine whether each mechanism could operate in humans • Group 1 • Group 2A • Group 2B • Group 3 • Group 4 Overall evaluation

Carcinogenic to humans Probably carcinogenic to humans Possibly carcinogenic to humans Not classifiable as to its carcinogenicity to humans Probably not carcinogenic to humans

Slide courtesy of V. Cogliano (US EPA)

A tour of IARC’s classifications

Preamble, Part B, Section 6(d)

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)

Slide courtesy of V. Cogliano (US EPA)

Agents Classified by the IARC Monographs, Volumes 1 – 105 (

monographs.iarc.fr

)

Group 1 Carcinogenic to humans 108 agents Group 2A Group 2B Probably carcinogenic to humans Possibly carcinogenic to humans Group 3 Group 4 Not classifiable as to its carcinogenicity to humans Probably not carcinogenic to humans 64 272 508 1

Caprolactam

is an irritant and is mildly toxic, with an LD 50 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]

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

www.epa.gov/iris

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

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 ) Tobacco (smoked and smokeless) Occupation Alcohol drinking Diet and dietary components including contaminants Pollution Reproductive factors 16% 14% 4% 3% 37% 25% 2% 2% 29%

Chemical Carcinogenesis in the 21

st

Century

New perceptions of previously known carcinogens:

Combined effects of multiple exposures

Examples: o Alcohol drinking and aflatoxins o Alcohol drinking and HBV/HBC o Alcohol drinking and tobacco smoking o Alcohol drinking and asbestos/arsenic/radon

Initiating Event

Stages of Carcinogenesis

Initiation Cell Proliferation (clonal expansion) Promotion Cell Proliferation Progression Cell Proliferation Malignancy

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 Protein (Biomarker) Detoxication Biologically Effective Dose X Efficiency of Mispairing X

Cell Proliferation Initiation

GENETIC AND EPIGENETIC MODELS OF THE CANCER INITIATION Normal cells Normal cells ACQUISITION OF ADDITIONAL RANDOM MUTATIONS Clonal selection and expression of initiated cells Mutator phenotype cells Cancer cells ALTERATIONS IN CELLULAR EPIGENOME Epigenetically reprogrammed cells Mutator phenotype cells Cancer cells

Accumulation of mutations during tumor progression

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

1.

X

No Tumors

2.

X

Tumors

3.

X

Tumors

4.

X

No Tumors

5.

No Tumors

Time X = Application of Initiator = Application of Promoter

N M 1 Basophilic Focus Promotion Adenoma M N Carcinoma Regression Progression No Tumors Tumors = Application of Promoter

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

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

Evolution of DNA damage during the cell cycle

“Complex” genetic changes From http://newscenter.cancer.gov/sciencebehind/

Classification of Carcinogens According to the Mode of Action GENOTOXIC NON-GENOTOXIC

Seco nd Mu tati Even t ng Third Mut ating Even t

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

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)

Classification of Carcinogens According to the Mode of Action NON-GENOTOXIC :



Do not directly cause DNA mutation



Mechanism of action is not completely understood



Difficult to detect - requires rodent carcinogen bioassay

?

Mutation Cancer

Non-Genotoxic Carcinogens

• • • 1) Mitogens: stimulation of proliferation mutations may occur secondarily to cell proliferation may cause preferential growth of preneoplastic cells • • • 2) Cytotoxicants: cytolethal induce regenerative growth mutations may occur secondarily to cell proliferation

Tissue Changes with Mitogenic and Cytotoxic Agents

Mitogenic Agent Proliferation

Tissue

Cell Death Proliferation Cytotoxic Agent

Mechanism of Carcinogenesis: Non-Genotoxic Carcinogens

Cell proliferation (to fix “spontaneous” mutation) CANCER

Mechanisms of Non-Genotoxic Carcinogenesis (what’s in a “black box” ?)

 Increased cell proliferation  Decreased apoptosis  Changes in gene expression  Induction of metabolizing enzymes  Activation of receptors (signaling)  Oxidative stress  ???

Cell Replication is Essential for Multistage Carcinogenesis

    Decreases time available for DNA repair Converts repairable DNA damage into non-repairable mutations Necessary for chromosomal aberrations, insertions, deletions and gene amplification Clonally expands existing cell populations

  

Induction of Metabolizing Enzymes

May be a reason for tissue-, and/or species-selectivity of carcinogens Metabolites may be ligands for receptors Production of reactive oxygen species Nebert & Dalton Nat Rev Cancer 2006

Human Tumors and Stages of Carcinogenesis

Hussain

et al.,

Oncogene, 2007

HALLMARKS OF CANCER

1. Sustaining proliferative signaling 2. Evading growth suppressors 3. Resisting cell death 4. Enabling replicative immortality 5. Inducing aberrant angiogenesis 6. Activating invasion & metastasis • •

Emerging Hallmarks

Reprogramming energy metabolism Evading immune destruction • •

Enabling Characteristics

Genomic instability and mutation Inflammation Hanahan and Weinberg 2011 (Cell)

Initiation



Promotion



Progression vs Hallmarks of Cancer

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