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Mutagenesis Advance Access published online on September 29, 2006
Mutagenesis, doi:10.1093/mutage/gel041
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Published by Oxford University Press 2006
Received May 26, 2006
Revised August 18, 2006
Accepted August 21, 2006

Original article

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

Page B. McKinzie 1 *, Robert R. Delongchamp 2, Tao Chen 1, and Barbara L. Parsons 1

1 Division of Genetic and Reproductive Toxicology HFT-120, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA
2 Division of Biometry and Risk Assessment, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA

* To whom correspondence should be addressed.
Page B. McKinzie, E-mail: page.mckinzie{at}fda.hhs.gov


  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 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.


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