ACB-PCR measurement of K-ras codon 12 mutant fractions in livers of Big Blue® rats treated with N-hydroxy-2-acetylaminofluorene

Abstract

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 × 10-6 (95% CI: 27 × 10-6, 95 × 10-6) and for four treated rats was 165 × 10-6 (95% CI: 87 × 10-6, 312 × 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 × 10-6 (95% CI: 498 × 10-6, 3500 × 10-6) and for treated rats was 8450 × 10-6 (95% CI: 3180 × 10-6, 22400 × 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.