Genetic Toxicity and Cancer

From:Mitchell L. Cohen, 2000. Nature 406, 762-767. Changing patterns of infectious disease.

cancer

cancer

Molecular Biology of Cancer

Events in ToxicosisBiochemical Developmental Genetic

Access to the organism

Absorption into bloodstream

Activation

Transport to target Various tissues Embryo DNA

Binding to target /Alteration of target

Various cell constituents

Embryonic cells and tissues

DNA

Inactivation No additional damage

Too late? Prevents damage to additional DNA molecules

Excretion No additional damage

Too late? Damage propagates regardless

Repair Significant Limited -- includes cell death. Damage often irreversible

In 1st replication or not at all.

Events in Genetic Toxicity

• Access to the organism• Absorption into the bloodstream• Transport in bloodstream• Entry to target cell

– All cells are potential targets• Binding to (altering) target

– DNA for genotoxic compounds– Variable for nongenotoxic cpds

• Activation• Inactivation

– May be irrelevant if DNA damage has occurred

• Excretion• Repair

– DNA repair ordinarily very efficient

• Critical Difference:

– DNA is self-replicating.

– Damage to DNA is propagated to all daughter cells.

– Damage can increase for decades after toxicant is gone

Reversible illness

Irreversible effects:(Neuro, Developmental, Organ failure,Diabetes ….)

Genetic Toxicity

Functional effects (e.g.: Enzyme inhibition)

Death

DNA Damage

Germcells

Somaticcells

Cancer

Cytoplasmic toxicity

Inheriteddisease

Alteredmutationrate

Cell death

Comparison of cytoplasmic and genetic toxicities

Birth defects

Premature aging

Evidence for Genetic Origins of Cancer • Historical arguments:

– Inbred mouse lines– High-cancer families

• p53: Li-Fraumeni families• BRCA2: breast/ovarian cancer

– Genetic diseases carrying a high risk of cancer• Xeroderma pigmentosa • Downs’ syndrome• Ataxia telangiectasia• Bloom’s syndrome• Fanconi’s anemia

– Correlation between carcinogenic and mutagenic chemicals

• Molecular evidence– Beginning in 1950s– Overwhelming by 1980

Cancer is a Genetic Disease• Origins

– Errors of DNA replication to somatic cells• = somatic mutations

• Nature of mutations leading to cancer– Cell must survive– Initial mutation enhances rate of cell division

• Turns off (inactivates) gene that blocks cell division– Tumor suppressor gene

• Turns on (activates) gene that increases cell division– Oncogene

• Multiple changes needed to produce most malignant tumors.– And, because the probability that both of 2 independent events

occur is the product of their individual probabilities,– It takes a long time to accumulate all the changes that make a cell

a cancer cell.

Digression: Tumors

• Tumor– Overgrowth of cells– Benign tumors

• Cells – Replicate autonomously– Do not invade neighboring tissue– Do not migrate via bloodstream or lymph

• Chromosome complement usually looks normal

– Malignant tumors• Cells

– Replicate autonomously– Invade neighboring tissues– May migrate via bloodstream or lymph to

distant parts of body– Lose characteristics of parent tissue (look

primitive or dedifferentiated)• Chromosome complement often abnormal

Progression of tumor formation

Cancer

• Cancer consists of cells that have escaped growth regulation, and that have the additional properties of– Invasiveness– Metastasis

• Tumors are clonal– They originate from a single cell– That has suffered DNA damage, causing it to divide

more rapidly.– Because DNA is self-replicating, this damage is

propagated to all progeny of the original cell.

Most Errors Occur During DNA Replication

• Semiconservative replication allows for checking of accuracy.• Uncorrected error rate ~ 1x10-6.• After proofreading, error rate ~ 1x10-9.

• Why do we not all succumb to consequences of errors of DNA replication?– Code is degenerate.– Not all genes are turned on in all cells.– Many errors lead to cell death ± replacement.

Summary

• Cancer is a genetic disease.• Cancer results from one or more genetic alterations that

– Increase rate of cell division– Allosw tumor to invade adjacent tissues– Allow cells to migrate through blood or lymph to new sites

• If a tumor requires only 1 or 2 “hits” (genetic alterations)– It will often occur in children– Example: retinoblastoma

• Sporadic = 2-hit• Familial = 1-hit

• If a tumor requires multiple ‘hits’– It will occur in adults– Incidence will increase with increasing age

Cancer is a disease of aging

• Most adult cancers require more than one alteration to DNA – All changes must occur in a single cell or its progeny

• It takes time to accumulate multiple changes in DNA• Cancers that require only a single ‘hit’ to DNA tend to

occur in children– Retinoblastoma– Leukemia

Multistage Carcinogenesis: Colon Cancer

Normal colon cell

Increased cell growth

Early Adenoma

Adenoma 2

Adenoma III

Carcinoma

Metastasis

Initiation: loss of APC on 5q

DNA demethylation

Ras gene mutationon 12p

Chromosome 18 loss

Chromosome 17 loss: p53

Other chromosome

losses

after Fearson and Vogelstein, Cell 61:759-767, 1990

How DNA Damage Leads to Cancer• 1. Translocation

– Burkitt’s lymphoma• c-myc from chromosome 8 to chromosome 14• antibody genes from chromosome 2 or 22 to chromsome 8

– Mantle cell lymphoma• t(11;14)(q13;q32) translocation• Moves bcl-1 to Ig controller• Deregulation of cyclin D1• compare to CLL (chronic lymphocytic leukemia)

• 2. Amplification of oncogene– By transfer from viral genome– c-ras in culture

• 3. Deletion, insertion or point mutation leading to

– a. Overexpression of oncogene– b. Inactivation of tumor suppressor gene

• Retinoblastoma in children– Loss of segment of chromosome 13 on both copies of the chromosome

There are also 240 subtypes of leukemia

Immunophenotype of B-cell chronic lymphocytic leukemia (B-CLL) and lymphomas that resemble it.

Antigen B-CLL Mantle Cell SLVL Follicular Lymphoma

slg Weak ++ ++ ++

CD5 ++ ++ - -

CD19 ++ ++ ++ ++

CD20 + ++ ++ ++

CD22 Weak or - ++ ++ ++

CD23 ++ - - -

CD79b Weak or - ++ ++ ++

FMC7 - ++ ++ ++

CD10 - - - ++

low-grade aggressive low-grade low-grade

Summary

• Cancer is a genetic disease.• Cancer results from one or more genetic alterations that

– Increase rate of cell division– Allosw tumor to invade adjacent tissues– Allow cells to migrate through blood or lymph to new sites

• If a tumor requires only 1 or 2 “hits” (genetic alterations)– It will often occur in children– Example: retinoblastoma

• Sporadic = 2-hit• Familial = 1-hit

• If a tumor requires multiple ‘hits’– It will occur in adults– Incidence will increase with increasing age

Cancer Is An Environmental Disease:• Differences between countries

– could be genetic or cultural– Look at immigrants

• Israel– Ashkenazi (European) Jews– Sephardic (African and Middle Eastern) Jews– Gentiles

• USA– Immigrants, their children and grandchildren

• Diffferences within countries– Hawaii

• Japanese/nonJapanese– occupation – ambient environment– cutural habits

Cancer in Israelis: 1961-1965

Group Population

Annual Incidence per 100,000 in population:Males

Annual Incidence per 100,000 in population:Females

non-Jews (Mostly Arab ethnicity) 140,000 179 93

Jews born in Israel 425,000 193 195

Jews born in Afirca or Asia(Sephardic)

291,000 208 167

Jews born in the US or Europe(Ashkenazi)

352,000 294 313

U.S. Population300-400 300-400

Cancer in Japanese: Japan and California

Cancer Site Japanese in Japan Japanese Immigrants to California

Sons of Japanese Immigrants

Stomach 6.5 4.6 3.0

Liver 3.7 2.1 2.2

Colon 0.2 0.8 0.9

Prostate 0.1 0.5 1.0

Richard Doll: Environmental Causes of Cancer(JNCI 66:123,1981)

Occupational Medical Life-Style Ambient

Aromatic amines Alkylating agents Smoking Aflatoxin

Benzene Chlornaphazine Alcohol Asbestos

Asbestos Immunosuppressants Anabolic steroids Benzene

Bis(chloromethyl) ether Busulfan Chewing tobacco Asbestos

Cadmium Estrogens Chewing lime Schistosomiasis

Chromium Phenacetin Chewing betel Parasites

Furniture-making Ionizing radiation Contraceptives?

Ionizing radiation Hormones (DES) Overeating/Obesity

Isopropanol Mfg Chemotherapy Reproductive history

Leather Mfg Sexual promiscuity

Mustard gas Dietary ingredients

Nickel (some forms)

Vinyl chloride

PAHs

UV light PAHs PAHs PAHs

Arsenic UV light UV light UV light

Arsenic Arsenic

Hepatitis b Hepatitis b Hepatitis b

Occupations with High Cancer IncidenceOccupation Type of Cancer Probable Chemical Carcinogen

Chemist brain, pancreas, bladder, lymphatic system

Coal miner stomach

Foundry worker lung benzpyrene, metals

Petrochemicals industry brain, blood, lung, bone, stomach, esophagus, blood

benzene

Painters blood solvents

Printers lung, mouth, pharynx metals? solvents?

Rubber industry bladder, blood, lung, prostate, stomach

organic volatiles

Textile industry nasal cavity, sinuses formaldehyde

Woodworkers lymphatic system terpenes, wood oils, formaldehyde

Farmers skin, lymphatic system UV light, pesticides?

Sailors skin UV light

Radiologist bone marrow gamma radiation

True or False ??

• “If you get cancer, it’s your own fault. You smoked, or ate too much, or drank alcohol.”– speaker from the American Cancer Society at a toxicology

meeting in Florida, ca 1978.

Cancer Epidemiologyand

Identifying Carcinogensand

A Digression Into Statistics

Contrast between cancer incidence and mortality, by state, 2011

http://www.cdc.gov/cancer/dcpc/data/state.htm

http://www3.cancer.gov/atlasplus/charts.html

ALL SITES:1950-1994

US, black lines

IL, tan, left;LA, tan, right

US by race & sex: all sites US by race and sex:non-Hodgkins lymphoma

colon cancer

melanoma

Brain & nervous systemStomach

Cervix Testis

Cancer Statistics

• For an idea of how convoluted the arguments can (and do!) become, see– http://katatrepsis.com/2012/10/30/why-does-breast-cancer-

research-receive-more-research-funding-than-prostate-cancer/

– (optional!!!)

Assays for carcinogenicity

• In vitro or cell-based– Ames assay in S. typhimurium– CHO cells– Other mammalian cells– Drosophila melanogaster mutagenesis– COMET assay

• In vivo or animal bioassays– Rats – Mice

• Epidemiology– Prospective– Retrospective– Ecological

COMET Assay (Single Cell Electrophoresis)

• Expose cells to mutagen – In vivo or in vitro– Can use bacteria, mammalian cells, human cells

• White blood cells from smokers vs nonsmokers

• Fix cells appropriately• Electrophoresis of single cells

– Observe migration of DNA in single exposed cells– Compare to unexposed cells

Control

Low Dose

High Dose

Example of COMET Assay Results

Mammalian Bioassays

• 2 species• 2 sexes per species• 50 animals per dose• 3 doses + vehicle control• Begin dosing animals soon after weaning• Dose for 18 months• Hold for 6 months• Examine every tissue and organ for signs of tumors

– Benign– Malignant

• Interpretation– Not without problems

Mammalian Bioassays

• Advantages:– Reliability

• Adequate data exist on 400 chemicals– Carried out by NTP

• Problems:– Discordance between species or sexes

• Fluoride– Bone cancer in male rats

• Disadvantages:– Cost

• $5,000,000 ??– Time

• 2-3 years– Manpower

• Histopathology!• Animal care• Record keeping

– Extrapolation • Between species• To lower doses

Extrapolation Outside of Dosing Levels

• Humans are exposed to many chemicals at low levels– ppm – ppb

• There are 300,000,000 people in the US today– An incidence of “1 in a million”

= 300 cases• How many mice and rats can we test?

– 50 per dose per sex– 2 sexes x 2 species x 4 doses = 16 x 50 – = 800 animals / bioassay

xx

x

% Response

Log Dose

Laboratory studies maximize dose because small numbers of animals are treated.Extrapolations have to made outside of these dosing levels to the actual exposure of 300,000,000 people.

x

How do you extend the line from dosing levels to control?

experimental doses

Control

human exposurelevel

xx

x

% Response

Log Dose

Linear response; no threshold

xx

x

% Response

Log Dose

Linear response with threshold(a linear interpolation using the best fit to the 3 points.)

xx

x

% Response

Log Dose

Nonlinear response, no threshold

xx

x

% Response

Log Dose

Nonlinear response with threshold

Human Epidemiology Studies

• Types– Retrospective– Prospective– Ecological

• Advantages of each– cost– time– reliability

• Disadvantages– Cost

• financial• human

– time– reliability

Lies, Damned Lies, and………..…

Statistics

Statistics are like a bikini: what is revealed is interesting; what is concealed is crucial.

A. R. Feinstein, Science 242:1257-1263, 1988

…. Some Data

Article: case reports– 6 cases of probable DEET-induced convulsions – all in girls under 6 years of age– 3 girls died

• What conclusions can we draw?

…Some More Data …

• Bendectin was sold by the same company that wanted to market thalidomide in the US.

• Bendectin was reportedly effective against nausea of pregnancy.

• In the U.S. in 1980, numerous children whose mothers took bendectin during pregnancy were born with limb defects.

• The lawsuits argued that bendectin caused limb defects, and eventually Merrill took it off the market.

• What is wrong here?

Bendectin and Birth Defects

• Births in the US in 1980 = 3,612,258• Prescriptions for bendectin = 903,064• Incidence of limb defects (reduction):

– 5 per 10,000 livebirths– = ~ 451 limb reduction defects in 1980

• If 1/4 of pregnant women took bendectin• And if bendectin does NOT cause limb defects:

– then an estimated 100 women who took bendectin had children with limb defects.

And it might be true…. ?

• My friend Beverly has compiled a list of 20 people in her neighborhood who have cancer.

• When she was young, she knew of only 1 case of cancer in her entire town, so clearly cancer is on the rise.

• Pesticides came in as she was growing up, and everyone in the neighborhood uses pesticides on their lawns.

• Beverly is convinced that lawn pesticides cause cancer.

Tricky Percentages: Absolute vs Relative Risk

• The headline says:• “TAMOXIFEN REDUCES RISK OF BREAST

CANCER RECURRENCE BY ALMOST 50%”

• A woman with an invasive tumor < 1 cm in diameter, ER+PR+ HR-, treated with either mastectomy or with lumpectomy plus radiation, has a recurrence risk of 17% over 10 years.

• If she takes tamoxifen for 5 years, her recurrence risk drops to 9%.

• What is the risk reduction: 8% or 47% ???

And what about the controls?

• At Love Canal, the incidence of chromosome abnormalities was significantly higher than the U.S. “background” level.

• The data were not allowed because the investigators did not use matched controls from the area around the affected Love Canal neighorhood.

• Question: are people living in somewhat-less-polluted neighborhoods of a very polluted city really the right controls to see if a subset of neighborhoods in that city are unfit for human habitation?

One Last Example …

• When computers were still quite new, and scientists reveled in their new ability to test multiple factors at once, an ecologist looked at 500 factors affecting tree growth.

• He gathered data for 10 years, put all the numbers into the computer, and found – 5 factors that were significant at the

0.01 level - which is highly significant.

• Should we be impressed?

And the moral is ….?

• Don’t believe anything you read.

• There are lies, damned lies, and statistics.

• Environmentalists are all crazy.

• Really good results are obvious enough that you don’t need statistics.

Critical Reviews of Health Data

• How do you know if one treatment is better than another?• How do you know if a test will do more harm than good?

• Cochrane Reviews– http://community.cochrane.org/cochrane-reviews

• Bandolier– http://www.medicine.ox.ac.uk/bandolier/band71/b71-2.html

ASSIGNMENT – DUE Monday 3/2/15Select one of the following topics:

Breast Cancer Prostate Cancer

• Discuss the benefits and costs of the PSA assay to identify early prostate cancer.

• Under what circumstances is this test advisable?

• Discuss the benefits and costs of yearly mammograms to identify early breast cancer.

• Under what circumstances are yearly mammograms advisable?