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ACB-PCR MEASUREMENT OF K-RAS CODON 12 MUTATION IN A/J MOUSE LUNG EXPOSED TO BENZO[A]PYRENE: A DOSE-RESPONSE ASSESSMENT
Citation:
MENG, F., G. W. KNAPP, T. B. GREEN, J. A. ROSS, AND B. PARSONS. ACB-PCR MEASUREMENT OF K-RAS CODON 12 MUTATION IN A/J MOUSE LUNG EXPOSED TO BENZO[A]PYRENE: A DOSE-RESPONSE ASSESSMENT. Presented at Environmental Mutagen Society 38th Annual Meeting, Atlanta, GA, October 20 - 24, 2007.
Impact/Purpose:
The specific aims of this study were to determine the background level and inter-animal variability in K-Ras codon 12 TGT mutation in A/J mouse lung tissue.
Description:
Benzo[a]pyrene (B[a]P) is a known human carcinogen and environmental contaminant. The direct measurement of K-Ras mutant fraction (MF) was developed as a metric with which to examine the default assumption of low dose linearity in the mutational response to B[a]P. The specific aims of this study were to determine the background level and inter-animal variability in K-Ras codon 12 TGT mutation in A/J mouse lung tissue, as well as the levels of mutation induced by exposure to B[a]P. Male A/J mice (n=10 mice, 7-9 weeks of age) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P. The mice were sacrificed 28 days after treatment and DNA isolated from a single lung of each mouse was used in a high-fidelity PCR to amplify K-Ras DNA sequence. The level of codon 12 TGT mutation in each PCR product was then determined by three replicate allele-specific competitive blocker-PCR measurements. This mutational endpoint was chosen because K-Ras codon 12 TGT mutation is frequently found in A/J mouse lung tumors induced by B[a]P. The A/J mouse lung contained relatively high spontaneous levels of K-Ras mutations. The lung tissue from control animals had a geometric mean MF of 3.88 X 10-4, meaning on average 1 in every ~1,288 lung cells carries the mutation. The inter-animal variability in K-Ras TGT geometric mean MF was large, with the control animals having MFs ranging from 3.50 x 10-5 to 3.24 x 10-3 . The geometric mean MFs for the B[a]P treatment groups were: 3.56 x 10-4,0.05 mg/kg; 6.19 x 10-4, 0.5 mg/kg; 2.02 x 10-3, 5 mg/kg; and 3.50 x 10-3, 50 mg/kg. Although statistically significant differences between dose groups were observed (ANOVA, P <0.001, with Holm-Sidak method for multiple comparisons), only the 5 and 50 mg/kg dose groups were significantly higher than controls. While the data demonstrate that the K-Ras codon 12 TGT mutational response to B[a]P occurs at early times after treatment, larger treatment groups will be needed to determine the shape of the K-Ras MF dose-response at low B[a]P doses.