Genotoxicity

1
Genotoxicity

• Chemicals, Risk Cancer 2
• David R. Bell

2
Do chemicals cause cancer ?

• Dose animals with a chemical, or vehicle control
• maintain animals on the Maximum Tolerated Dose
  until the end of their lives or 2 years
• Kill animals, and necropsy
• Detailed account of tumour incidence
• DiMethylBenzAnthracene (DMBA from coal dust) was
  found to give rise to cancer
• Systematic investigation of chemicals which cause
  cancer

3
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
4
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-
5
Electrophiles

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

6
Electrophiles and DNA
H
CH3
H
CH3
..
O
O
N
N
N
N
N
N
N
N
H2N
H2N
dR
dR
Guanine
O6-methyl Guanine
7
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
8
Methylnitrosourea
CH3
NO
NH2CO2H
H2O
N
CH3-NH-NO
C
O
H2N
OH-
CH3-N?N
Electrophiles
CH3
N2
9
Genotoxicity

• Electrophiles react most with protein, then RNA,
  then DNA- based on abundance
• O6-Me dG read as dA during replication, and
  mis-paired to dT
• Leads to a G-A transition, or point mutant
• Toxicity to the genome- hence genotoxicity

10
Genotoxicity and cell division

• Cell division greatly enhances the mutagenic
  effects of DNA alkylation

Two weeks promotion
3 hrs
7 days
Kill
Partial hepatectomy
MNU
11
Effect of cell division
12
Consequences of DNA alkylation

• Large amounts of endogenous methylation
• 5MeA, 6MeC
• Specific repair enzyme- O6 MeG methyltransferase
  (MGMT) in mammalian cells
• MGMT is induced by genotoxic carcinogens, or
  radiation, i.e. DNA damage

13
MGMT

• MGMT removes O6-Methyl from O6MedG in a one-step
  suicide reaction
• No cofactors
• The Methyl group transfers to Cys145 of MGMT
• MGMT inactivated and rapidly degraded
• High cost to cell

14
Is MGMT important ?

• Overexpression of bacterial MGMT in cells makes
  the cells resistant to MNU killing
• Gene KO of MGMT makes mice sensitive to MNU in
  terms of death, and cancer
• MGMT-/-, MLH1 -/- mice remain sensitive to
  MNU-induced tumours, but not to MNU induced
  killing.
• Mismatch repair of damaged DNA leads to cell death

15
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

16
Consequences of mutation

• MNU mutates codon 12, base 2, G-A in 100 of
  H-ras oncogene in mammary tumours
• Codons 12, 13, 16, 59, 61, 116 activate Ras
• Specific sequences are particularly susceptible
  to mutation
• Specific sequences are refractory to repair
• Organ-specific effects on oncogene activation

17
DNA damage ? cancer
DNA damage is similar between the two
strains Tumour development is markedly
different Therefore other factors control the
development of cancer
18
DNA adducts

• Each alkylating agent produces a spectrum of
  adducts
• Measurement of adducts
• Which is the important adduct ?
• Very high levels of some endogenous adducts
  (methyl, ethyl, estrogens, etc)
• Adducts are repaired/ fixed
• What is important- the adducts remaining, or the
  adducts which caused a mutagenic event ?

19
DNA adducts

• Hydrolyse DNA
• Chemical or enzymatic
• Chemical stability of adduct
• Label with 32P
• Thin layer chromatography, and autorad
• GC, or LC, -Mass Spectrometry

20
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

21
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

22
Molecular Dosimetry

• Target tissue DNA is difficult to obtain
• Protein adducts can be abundant
• Measure DNA in a protein proxy for DNA
• Haemoglobin has a half life of 120 days in human
• Blood is easy to sample
• Successfully used for ethylene oxide
• Detect endogenous production of EtO and ethylene