Chemical-Induced Carcinogenesis

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Chemical-Induced Carcinogenesis
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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/
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WHAT MAY CAUSE CANCER ?

• Hereditary disorders
• Chemicals
• Viruses
• Chronic inflammation
• ???

From http//www.cancersupportivecare.com/riskintro
.html
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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, gt50 chemicals are recognized as human
  carcinogens
• First experimental studies in animals were done
  80 years ago

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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 lt1/2 MTD to lt1/1000 MTD
• Most regulations use straight mathematical
  extrapolation of high dose rodent data to predict
  risks

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http//ntp.niehs.nih.gov/files/Agenda_Presentation
s.pdf
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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.
http//ntp.niehs.nih.gov/files/Agenda_Presentation
s.pdf
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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
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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 1000 AM and
after 200 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.
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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)
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Two-year Carcinogenesis Bioassay Protocol
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WORLD HEALTH ORGANIZATION INTERNATIONAL AGENCY
FOR RESEARCH ON CANCER IARC Monograph
Evaluations LYON, FRANCE
Slide courtesy of V. Cogliano (US EPA)
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The IARC Monographs Volume 100 The known causes
of human cancer by organ site
Eye Human immunodeficiency virus type 1 Ultraviolet-emitting tanning devices Welding
Brain and central nervous system X-radiation, gamma-radiation
Lung Aluminium production Arsenic and inorganic arsenic compounds Asbestos (all forms) Beryllium and beryllium compounds Bis(chloromethyl)ether chloromethyl methyl ether(technical grade) Cadmium and cadmium compounds Chromium (VI) compounds Coal, indoor emissions from household combustion Coal gasification Coal-tar pitch Coke production 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
Oral cavity Alcoholic beverages Betel quid with tobacco Betel quid without tobacco Human papillomavirus type 16 Smokeless tobacco Tobacco smoking
Pharynx Alcoholic beverages Betel quid with tobacco Human papillomavirus type 16 Tobacco smoking
Nasal cavity and paranasal sinus Isopropyl alcohol manufacture using strong acids Leather dust Nickel compounds Radium-226 and its decay products Radium-228 and its decay products Tobacco smoking Wood dust
Nasopharynx Epstein-Barr virus Formaldehyde Salted fish, Chinese-style Wood dust
Tonsil Human papillomavirus type 16
Salivary gland X-radiation, gamma-radiation
Larynx Acid mists, strong inorganic Alcoholic beverages Asbestos (all forms) Tobacco smoking
Thyroid Radioiodines, including iodine-131 (exposure during childhood and adolescence) X-radiation, gamma-radiation
Stomach Helicobacter pylori Rubber production industry Tobacco smoking X-radiation, gamma-radiation
Mesothelioma (pleura or peritoneum) Asbestos (all forms) Erionite Painter (occupational exposure as)
Upper aerodigestive tract 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
Breast Alcoholic beverages Diethylstilbestrol Estrogen-progestogen contraceptives Estrogen-progestogen menopausal therapy X-radiation, gamma-radiation
Oesophagus 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
Urinary bladder 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
Gall bladder Thorium-232 and its decay products
Kidney Tobacco smoking X-radiation, gamma-radiation
Biliary tract Chlonorchis sinensis Opisthorchis viverrini
Pancreas Smokeless tobacco Tobacco smoking
Renal pelvis and ureter Aristolochic acid, plants containing Phenacetin Phenacetin, analgesic mixtures containing Tobacco smoking
Colon and rectum Alcoholic beverages Tobacco smoking X-radiation, gamma-radiation
Anus Human immunodeficiency virus type 1 Human papillomavirus type 16
Bone Plutonium Radium-224 and its decay products Radium-226 and its decay products Radium-228 and its decay products 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
Endometrium Estrogen menopausal therapy Estrogen-progestogen menopausal therapy Tamoxifen
Multiple sites (unspecified) Cyclosporine Fission products, including Strontium-90 X-radiation, gamma-radiation (exposure in utero)
All cancers combined 2,3,7,8-Tetrachlorodibenzo-para-dioxin
Vagina Diethylstilbestrol (exposure in utero) Human papillomavirus type 16
Uterine cervix Diethylstilbestrol (exposure 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 Tobacco smoking
Endothelium (Kaposi sarcoma) Human immunodeficiency virus type 1 Kaposi sarcoma herpes virus
Vulva Human papillomavirus type 16
Ovary Asbestos (all forms) Estrogen menopausal therapy Tobacco smoking
Penis Human papillomavirus type 16
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 Benzoapyrene Ethanol in alcoholic beverages Ethylene oxide Etoposide Ionizing radiation (all types) Neutron radiation N'-Nitrosonornicotine (NNN) and 4-(N-nitroso-methylamino)-1-(3-pyridyl)-1-butanone (NNK) Ultraviolet radiation
Skin (melanoma) Solar radiation Ultraviolet-emitting tanning devices
Skin (other malignant neoplasms) 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
Section of the IARC Monographs (IMO)
http//monographs.iarc.fr/ENG/Publications/OrganSi
tePoster2012.ppt
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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
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
Slide courtesy of V. Cogliano (US EPA)
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A tour of IARCs classificationsPreamble, 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)
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Slide courtesy of V. Cogliano (US EPA)
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           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
1105 (monographs.iarc.fr)
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".34
http//en.wikipedia.org/wiki/Caprolactam
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www.epa.gov/iris
http//tools.niehs.nih.gov/srp/1/Resources/Arzuaga
_IRIS_presentation.pdf
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NTP (Report on Carcinogens)
IARC
California EPA
US EPA (Cancer classification)
Globally Harmonized System of Classification and
Labelling of Chemicals
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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

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Chemical Carcinogenesis in the 21st Century

• New perceptions of previously known carcinogens
• Combined effects of multiple exposures
• Examples
• Alcohol drinking and aflatoxins
• Alcohol drinking and HBV/HBC
• Alcohol drinking and tobacco smoking
• Alcohol drinking and asbestos/arsenic/radon

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Stages of Carcinogenesis
Initiation
Initiating Event
Cell Proliferation (clonal expansion)
Second Mutating Event
Promotion
Cell Proliferation
Progression
"N" Mutating Event
Cell Proliferation
Malignancy
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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
From http//newscenter.cancer.gov/sciencebehind/
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Chemical Exposure (air, water, food, etc.)
Internal Exposure
Metabolic Activation
Macromolecular Binding
Detoxication
Protein
DNA
RNA
(Biomarker)
Biologically Effective Dose
X
Efficiency of Mispairing
Initiation
X
Cell Proliferation
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GENETIC AND EPIGENETIC MODELS OF THE CANCER
INITIATION
Normal cells
Normal cells
Environmental
Endogenous
Environmental
Endogenous
ALTERATIONS IN CELLULAR EPIGENOME
ACQUISITION OF ADDITIONAL RANDOM MUTATIONS
Epigenetically reprogrammed cells
Clonal selection and expression of initiated cells
Mutator phenotype cells
Mutator phenotype cells
Cancer cells
Cancer cells
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Accumulation of mutations during tumor progression
Loeb L.A. Cancer Res. 613230-9 (2001)
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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
From http//newscenter.cancer.gov/sciencebehind/
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X
1.
No Tumors
X
2.
Tumors
X
3.
Tumors
X
4.
No Tumors
No Tumors
5.
Time
Application of Initiator
Application of Promoter
X
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Basophilic Focus
N
Adenoma
Carcinoma
M
M
1
N
Promotion
Regression
Progression
No Tumors
Tumors
Application of Promoter
Adapted from Marsman and Popp. Carcinogenesis
15111-117 (1994)
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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/
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Classification of Carcinogens According to the
Mode of Action
GENOTOXIC NON-GENOTOXIC
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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
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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)
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Williams J.A., Carcinogenesis 22209-14 (2001)
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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
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Non-Genotoxic Carcinogens

• 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

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• Tissue Changes with Mitogenic and Cytotoxic Agents

Mitogenic Agent
Proliferation
Tissue
Cell Death
Proliferation
Cytotoxic Agent
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Mechanism of CarcinogenesisNon-Genotoxic
Carcinogens
Cell proliferation (to fix spontaneous mutation)
CANCER
X
APOPTOSIS
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Mechanisms of Non-Genotoxic Carcinogenesis (whats
in a black box ?)

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

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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

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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
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Human Tumors and Stages of Carcinogenesis
Hussain et al., Oncogene, 2007
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• Hallmarks of Cancer
• 1. Sustaining proliferative signaling
• 2. Evading growth suppressors
• 3. Resisting cell death
• 4. Enabling replicative immortality
• 5. Inducing aberrant angiogenesis
• Activating invasion metastasis
• Emerging Hallmarks
• Reprogramming energy metabolism
• Evading immune destruction
• Enabling Characteristics
• Genomic instability and mutation
• Inflammation

Hanahan and Weinberg 2011 (Cell)
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Initiation?Promotion?Progression vs Hallmarks of
Cancer
Floor et al (Trends in Molecular Medicine,
September 2012, Vol. 18, No. 9)