Chemical Carcinogenesis

1
Chemical Carcinogenesis

• David.bell_at_nottingham.ac.uk
• http//dmg.nott.ac.uk/teaching/msc1.ppt
• http//dmg.nott.ac.uk/teaching/cancer/cancer.html

2
Do chemicals cause cancer ?

• Epidemiology
• The study of illness in populations
• Correlate with influences on the population, eg
  diet, work, geography
• 1775
• The surgeon, Percival Potts, noted that coal
  sweeps had a high incidence of cancer, scrotal
  cancer. He hypothesised that the cancer was due
  to their intimate and prolonged exposure to coal
  dust.

3
Latency
Cancer takes 10-50 years to develop in man
1900- Cigarette smoking an accepted habit
1940- rise in male lung cancer- alarm
1960- smoking is shown to be the cause of lung
cancer
1960-2000. People who started smoking before
1960 die of lung cancer Lung cancer is the most
frequent site of cancer in UK men
4
Is cancer increasing ?
5
Epidemiology issues

• Long latency period- up to 20 years
• Poor for prediction
• Sensitive against low background
• e.g. haemangiosarcoma is vanishingly rare in the
  general population, vs high levels in workers
  exposed to vinyl chloride monomer
• Insensitive against common cancers
• Difficult to isolate the cause of cancer
• Unless you are testing for that cause
• Small studies are confounded by sampling error

6
Causes of cancer
Note that diet, tobacco, occupation, alcohol and
pollution are principally chemically-induced
cancers.
7
Screening for carcinogens

• Animal tests
• 50 animals per group
• Maximal tolerated dose (MTD), and 25
• Cost 250 000
• Time 2 years (rat or mouse)
• Definitive, identification of specific chemicals

8
Problems with bioassay

• The US National Toxicology Programme has tested gt
  500 compounds, with selection based on
  environmental relevance
• Approx 50-66 of all chemicals cause cancer

9
Nitrosamines

• Cause cancer in mouse, rat, monkey, guinea-pig,
  rabbit, fowl, newt, trout
• Wide range of organs affected
• Formed in food in the presence of nitrate, and
  acid, ie pickles
• Potent

R1
N
NO
R2
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Nitrosamines
O-H
CH2
CH3
Oxidation
N
NO
N
NO
CH3
CH3
The methyl carbonium ion is a powerful
electrophile, and reacts quickly with
macromolecules.
H2-CO
CH3-NN-OH
N2
CH3-N2
CH3
OH-
11
Electrophiles

• Chemical entities which react with centres having
  a surplus of electrons, or nucleo-philes.
• Protein, RNA and DNA contain nucleophilic sites

12
Electrophiles and DNA 2
H

• There are multiple reactions of electrophiles
  with DNA
• O6 methyl guanine is promutagenic
• The alkylated base mispairs G-T, instead of G C
• This leads to inaccurate repair, and mutations in
  DNA
• N7-G also promutagenic

N7
O
N
N
N
N
H2N
Guanine
dR
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Genotoxicity

• Toxicity to the genome- hence genotoxicity

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Benzo(a)pyrene
1
Bay region
2
10
3
9
4
8
5
7
6
K region
High electron density at Bay and K regions Planar
molecule
15
Benzo(a)pyrene

• Typical PAH
• Ubiquitous
• Coal tar, organic matter
• Formed during combustion of organics
• Cooking !
• Oils, especially used at high temperature
• Chemically inert
• Principal active agent in coal tar

16
B(a)P metabolism
Multiple sites of metabolism combination of
metabolism at multiple sites. Highly
complex metabolic pathways.
2
10
3
9
4
8
5
7
6
Hydroxylation- reactive phenols and quinones
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The ultimate carcinogen
Cytochrome P450
O
B(a)P 7,8 epoxide
Epoxide hydrolase
P450
O
OH
OH
OH
OH
((anti))B(a)P 7,8 dihydrodiol 9,10 epoxide
B(a)P 7,8 dihydrodiol
18
Is BPDE important ?

• Analysis of B(a)P- DNA adducts
• BPDE should be more carcinogenic than BP
• Dose mice at birth and autopsy at 7 months. Lung
  tumours/mouse are shown.

19
Adducts from B(a)P

• Adducts form on C10- N2 of dG using the BPDE on
  DNA in vitro
• The same adducts are formed in vivo on DNA
• Mutations in the ras oncogene with BaP are
  typified by G-T transitions at codon 12 or 13

20
DNA repair

• Methylnitrosourea (MNU) is a direct alkylating
  agent
• In rats, it has tissue-specificity, with
  brain-specific tumorigenesis
• DNA-adduct levels are similar in brain in liver
• Adducts are removed rapidly in liver, but persist
  in brain

21
DNA damage ? cancer
DNA damage is similar between the two
strains Tumour development is markedly
different Therefore other factors control the
development of cancer
22
Do adducts cause cancer ?

• Chemically synthesise DNA-adducts, insert into
  plasmid and transform into E. coli
• Measure amount of mutations in prokaryotic DNA
• Treat a cellular proto-oncogene with carcinogen
  in vitro
• Transform DNA into cells, and look for cellular
  transformation

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How many adducts for cancer ?

• For a 50 incidence of tumours in rat liver
• Estimate from 50 to 2000 adducts/ 108 nucleotides
  (2 per 100 000)
• Variation
• Measurement
• Repair
• Different compounds and adducts

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Ames assay

• Relies on the concept that DNA-damaging, or
  mutagenic, agents will cause cancer
• Uses a disabled Salmonella Typhimurium, with
  defective cell wall, allowing easy import of
  chemicals
• Look for mutations in genes for Histidine
  biosynthesis
• Mutant genes will allow survival and growth of
  the bacteria

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Ames assay 2
His- Salmonella
Histidine Deficient plates
Plate chemical
Control plate
Benefits two day assay very cheap
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Ames III

• Ames test detects direct acting mutagens
• Add liver homogenate (the S9, 9000g supernatant)
  to cells to detect mutagens which require
  activation
• Some chemicals are mutagenic, but not
  carcinogenic
• Many chemicals are carcinogenic, but not mutagenic

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The numbering of the beast
4
6
3
5
7
8
2
10
9
1
2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) One of
the most potent of a variety of related
compounds Long half-life in humans (6 years)
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Tumour incidence in rats
TCDD- ng/kg day
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TCDD and cancer

• Tumorigenic in rat, mouse, guinea pig and fish
  (everything tested)
• Primate study aborted when total dose of 2 mg/kg
  lead to death
• In rat, the lowest carcinogenic dose was 1.4
  ng/kg/day (Plt0.05) for thyroid tumours
• MTD 71ng/kg/day

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A prototypical non-genotoxin

• TCDD is a potent carcinogen
• Very weak/ no mutagenicity in Ames test
• No activity in tests of initiation activity
• Maximum estimate for binding of TCDD to rat liver
  DNA 1 molecule per 1011 bases
• 1 per 107- 105 for known carcinogens
• TCDD is a non-genotoxic carcinogen

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Rodent Bioassay Bias

• 60 of chemicals are carcinogenic
• Liver Bias
• Mouse liver tumours are 45-60 of mouse
  carcinogens
• Rat liver tumours are 20-30 of rat carcinogens
• Chemicals which induce mouse liver tumours only
  are 30 Ames positive
• Non-carcinogens are 30 Ames

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Does cancer progress ?
Model originally set up dosing mouse skin
DMBA
Vehicle control- 6 months
0 tumours
DMBA
Croton oil- 6 months
Multiple tumours
DMBA
Croton oil- 6 months
0 tumours
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Promotion

• Use subcarcinogenic dose of initiating agent
• The promoting agent is not carcinogenic per se
• The promoting agent enhances the effect of the
  genotoxic initiating agent
• Long delay possible between administration of
  initiating agent and promoting agent
• Promotion is reversible

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The active ingredient

• TPA, or tetradecanoyl phorbol acetate
• TPA causes hyperplasia in mouse skin
• TPA activates protein kinase C
• Cell-signalling receptor
• Diacyl-glycerol is an endogenous activator
• Inappropriate activation of programme of cell
  growth

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Mechanisms of promotion

• Skin promotors
• Okadaic acid
• Inhibitor of protein phosphatases
• TCDD
• Activates the Ah receptor
• Wounding

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Promotion in different tissues

• Colon
• Bile acids, high fat diet
• Bladder
• Saccharin
• Mammary gland
• Hormones, high fat diet

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Why Risk Assess ?

• Unsatisfactory to wait for epidemiology
• HAZARD
• The ability to cause cancer
• RISK
• What is the chance of it doing so ?
• Paracelsus
• All substances are poisons there is none which
  is not a poison. The right dose differentiates a
  poison and a remedy

38
Hazard Assessment

• Use of Maximal Tolerated Dose (MTD) in two-animal
  bioassay
• High dose required to obtain a significant result
• Human exposure at low dose
• How to extrapolate from high dose to low dose ?

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Extrapolation
Data
Extrapolation
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Extrapolation
US/ Europe 1 cancer in 106 is tolerable UK
guidelines more flexible
Extrapolation
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Extrapolation 3
No Observed Effect Level
NOEL
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Linear extrapolation

• Mechanism associated with genotoxicity
• 1 DNA adduct may lead to a single mutation may
  lead to cancer
• No safe dose
• Any amount of chemical will cause cancer
• Conservative

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Linear extrapolation

• Carcinogens may require metabolic activation
• Enzyme kinetics are non-linear
• Trichloroethylene produces lung tumours in mouse,
  but not rat
• TCE metabolised to chloral in mouse
• P450 mediated species differences (TCE.pdf)

44
Linear extrapolation

• Non-genotoxic carcinogens activate receptors
• Receptor activation follows non-linear kinetics

V
Substrate
45
Is linear extrapolation correct ?

• Linearity has been tested for acetyl-aminofluorene
  , which produces liver and bladder cancers- down
  to 1
• Linear for liver cancer 30-150 ppm
• Non-linear for bladder cancer
• NOEL of 60 ppm
• Increased bladder DNA synthesis above 60 ppm AAF

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Linear extrapolation

• For a chemical which causes cancer
• Extrapolate linearly to a level which causes
  cancer at 1 in 106
• i.e. MTD/ 1 000 000
• Applies to pesticides/ fungicides/ industrial
  chemicals/ etc
• Pharmaceuticals risk/ benefit balance
• UK regulation less clear

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Carcinogen exposure

• gt60 of all chemicals are carcinogens
• Food is composed of chemicals
• Plants produce a variety of secondary products
• Pesticides
• Of a selection of plant pesticides, 27/52 were
  carcinogenic

48
Carcinogen exposure

• Estimate that people eat 1.5 g of natural
  pesticides per day
• 2g of burnt food products
• 50 carcinogens
• 1 cup of coffee contains 32mgs of known
  carcinogenic chemicals ( gt1000 untested)

49
Carcinogen exposure

• Epidemiology fruit veg are beneficial
• It is not clear that there is evidence that low
  dose effects are significant for human cancer
• Compare lt0.1mg of synthetic pesticides
• Why regulate synthetic pesticides when 99.99 of
  pesticides are natural chemicals ?

50
Chemical carcinogenesis

• Chemicals are involved in human cancer
• Reaction of chemical with DNA
• Some chemicals require metabolic activation
• Non-genotoxic chemicals
• Activation of cellular receptors
• Difference between hazard and risk