ACB-PCR measurement of K-ras codon 12 mutant fractions in livers of Big Blue rats treated with N-hydroxy-2-acetylaminofluorene.
K-ras codon 12 GGT-->GAT and GGT-->GTT mutations are the most frequently observed K-ras point mutations in human and rodent tumors and therefore are implicated in carcinogenesis for many tissues. Measurement of these mutations in rat models and human tissue could facilitate a more logical extrapolation of rodent tumorigenesis data to human disease. We have developed allele-specific competitive blocker PCR (ACB-PCR) assays for rat K-ras codon 12 GGT-->GTT and GGT-->GAT mutations that parallel the already published assays for human K-ras codon 12 mutations. Liver K-ras codon 12 mutant allele fractions were measured in vehicle-treated and N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-treated Big Blue rats. The average K-ras codon 12 GGT-->GTT mutant fraction (MF) for four control rats was 50 x 10(-6) (95% CI: 27 x 10(-6), 95 x 10(-6)) and for four treated rats was 165 x 10(-6) (95% CI: 87 x 10(-6), 312 x 10(-6)), indicating a 3.3-fold increase with treatment (95% CI: 1.3-8.1). The average MF of K-ras codon 12 GGT-->GAT for control rats was 1320 x 10(-6) (95% CI: 498 x 10(-6), 3500 x 10(-6)) and for treated rats was 8450 x 10(-6) (95% CI: 3180 x 10(-6), 22 400 x 10(-6)), indicating a 6.4-fold increase with treatment (95% CI: 1.6-25.4). These transgenic rats were part of a study that included analysis of liver lacI mutations. Although data from lacI determinations show that this compound induces mostly G-->T mutations, using the ACB-PCR method both K-ras codon 12 GGT-->GTT and GGT-->GAT MFs were significantly increased in treated rats versus control rats. This data raises the possibility that N-OH-AAF may not only induce mutations by a genotoxic mechanism, but also by amplification of both de novo and pre-existing K-ras mutation.
McKinzie
Page B
PB
Division of Genetic and Reproductive Toxicology, HFT-120, 3900 NCTR Road, Jefferson, AR 72079, USA. page.mckinzie@fda.hhs.gov
Delongchamp
Robert R
RR
Chen
Tao
T
Parsons
Barbara L
BL
D000483:Alleles; D000818:Animals; D002273:Carcinogens / Q000633:toxicity*; D003062:Codon; D011905:Genes, ras / Q000235:genetics*; D006881:Hydroxyacetylaminofluorene / Q000633:toxicity*; D008099:Liver / Q000187:drug effects*; D008297:Male; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D051381:Rats; D011916:Rats, Inbred F344
D002273:Carcinogens; D003062:Codon; D006881:Hydroxyacetylaminofluorene