The in vitro chromosome aberration assay is very sensitive to chemical mutagens

1
The in vitro chromosome aberration assay is very
sensitive to chemical mutagens
Note lack of cytotoxicity
2
Chromosome aberrations in vitro in CHO cells
With S-9
Without S-9
Numbers on top of bars are cell counts as
control.
3
Negative genotoxicity assays of Angiotensin II
receptor antagonist and analogs

• Microbial mutagenicity in Salmonella and E.Coli
• Alkaline elution (DNA strand breaks) in rat
  hepatocytes
• Mammalian cell mutagenicity hprt in V79 cells
• In vivo
• Chromosome abs in M F mice (1500 mg/kg)
• Alkaline elution in F rat liver (1978 mg/kg)
• Additional negative tests on analogs
• DNA adducts in calf thymus DNA
• phage DNA fragmentation
• unscheduled DNA synthesis

4
Inhibition of DNA synthesis by AIIRAs
5
Significance of aberrations associated with
cytotoxicity

• Potential threshold
• no structural alert
• no mutation
• no DNA binding
• no aberrations in vivo
• likely secondary to toxicity not relevant at
  human exposure levels
• In vitro positive at 1400 µg/ml
• In vivo mouse exposure at 500 mg/kg/d x8
• C Max 74 µg/ml AUC 90 µg/ml.hr
• Human 40mg oral
• C Max 0.2 µg/ml AUC 0.4 µg/ml.hr

6
Mechanisms of chromosome aberrations

• Aberrations result from DNA strand breaks
• Strand breaks induced by e.g.
• ionizing radiation
• processing of lesions/adducts/abnormal bases
• but also by
• inhibition of DNA synthesis
• inhibition of topoisomerases
• Cytotoxicity
• Mis-incorporation altered bases
• nucleoside analogs ribonucleotide reductase
  inhibitors folate antagonists
• DNA synthesis inhibition chain termination
  mis-pairs gaps

7
Mechanisms of genotoxicity that may have threshold

• Disruption of cell division
• Disruption of chromosome segregation
• Inhibition of DNA synthesis
• Inhibition of topoisomerases
• Nucleotide pool imbalance
• Overloading of oxidative defence mechanisms
• Metabolic overload (phase II enzymes etc)
• Ion chelation disturbance of metal homeostasis
• Extremes of pH/osmolality
• Cytotoxicity
• (adapted from Henderson Albertini Aardema
  Mutat Res 464 123-128 2000)

8
Possible mechanisms for chromosome aberration
induction by nucleoside analogs

• DNA synthesis inhibition
• incorporation
• chain termination
• attempted repair of mispaired bases
• pool imbalance
• DNA synthesis inhibition
• polymerase errors- attempted repair

9
Genetic Toxicology of Antivirals/Nucleosides

• All are negative in AMES (no data on adenosine)

Wutzler and Thust. 2001. Antiviral Research.
49 55-74 Phillips et al Env Molec Mutagen 18
168-183 2001
10
Risk evaluation for nucleoside analog

• Specificity for viral vs mammalian enzyme
• Is it incorporated into DNA RNA
• If incorporated is it pro-mutagenic (AZT)
• If not incorporated High dose effect.
• Possible mechanisms
• pool imbalance (try to restore)
• DNA synthesis inhibition (demonstrate)
• Genotoxicity may have threshold
• in vivo cytogenetic assay negative
• good safety margin (exposure)
• May not be genotoxic risk to humans.

11
Strategy for Genotoxicity Follow-Up Testing for
Unique in vitro Positive
In vitro Chromosome Aberrations
Indirect DNA Synthesis Inhibition
Direct DNA DamageDNA adducts or neg Ames Alk
elut
Acute Mouse Bone Marrow Micronucleus
Human (TK6)Cell Mutation
-
Measure Exposure
2nd in vivo test if exposure appropriate
No Go
yes
-
-
Good Margin
OK for single or multiple dose trials in normal
volunteers.
12
Risk evaluation when mechanism is indirect

• Chromosome aberrations may be induced only above
  a threshold dose.
• Therefore if
• in vivo cytogenetic assay negative
• second in vivo assay negative
• good safety margin (exposure)
• May not be genotoxic risk to humans.