Accumulation of K‐Rascodon 12 mutations in the F344 rat distal colon following azoxymethane exposure

Abstract

Azoxymethane (AOM) administration to F344 male rats is a widely used model of human colon carcinogenesis. The current study investigates quantitatively the accumulation of K-Ras codon 12 mutations following AOM exposure. Male, 6-week-old F344 rats were treated subcutaneously with 30 mg/kg body weight of AOM, and colon tissue was collected at 1, 8, 24, and 32 weeks after treatment. The K-Ras codon 12 GGT to GAT and GGT to GTT mutant fractions (MFs) were measured using allele-specific competitive blocker polymerase chain reaction (ACB-PCR). Between 1 and 32 weeks after AOM treatment, the K-Ras codon 12 GGT to GAT geometric mean MF in the rat colon increased significantly from 12.9 × 10(-5) to 145 × 10(-5) , and the GGT to GTT geometric mean MF increased significantly from 5.26 × 10(-5) to 180 × 10(-5) . K-Ras codon 12 GGT to GAT MF also increased significantly in AOM-treated rat colon tissue at 1 week compared to controls (4.44 × 10(-5) ). The accumulation of the GGT to GAT MF long after the DNA adduct repair phase suggests that a portion of cells containing this mutation have a proliferative advantage, allowing them to accumulate as nascent tumors progress. Also, the GGT to GAT background MF increased in untreated rats, indicating that there is accumulation with age. The ACB-PCR assay generates quantitative data of cancer-related mutations and thus provides insight into pathological processes following carcinogen exposure.

Ovarian effects of prenatal exposure to benzo[a]pyrene: Roles of embryonic and maternal glutathione status

“…Evaluation of more than one mutation by ACB-PCR has been used as a paradigm to detect chemical-specific effects on mutation frequency. In the rat colon cancer model, for example, azoxymethane induced Kras codon 12 GAT mutations, but not codon 12 GTT mutations, which was consistent with the mutational specificity expected for azoxymethane [54]. Conversely, Big Blue rats treated with N -hydroxy-2-acetylaminofluorene ( N -OH-AAF) had significantly increased levels of both mutations in liver DNA, even though induction of G to T mutation was the primary mutational specificity observed in the LacI neutral reporter gene of the liver DNA from the same rats.…”

“…Comparisons of KRAS codon 12 GAT (G12D) and GTT (G12V) MF in a variety of human and rodent tissues has demonstrated that the KRAS codon 12 GAT mutation is generally the more abundant spontaneous mutation, often present in normal human or control rodent tissues at frequencies up to ten-fold greater than the KRAS codon 12 GTT (G12V) mutation [37, 38, 54, 55]. In contrast, we observed significantly higher levels of GTT mutations than GAT mutations in the mouse ovary in the present study.…”

Oncomutations as biomarkers of cancer risk

“…In the ACB-PCR studies described above, as well as other studies [McKinzie et al, 2006;McKinzie et al, 2007], it has been possible to measure the induction of oncomutations at relatively short times after in vivo carcinogen exposure. Additional studies with a variety of chemicals will be needed to establish whether and how carcinogen effect can be monitored using such methods as part of 28-or 90-day repeat dose toxicity testing.…”

Low-Frequency Kras Mutations are Prevalent in Lung Adenocarcinomas

“…Allele-specific competitive blocker PCR (ACB-PCR) is a DNA-based mutation detection method that can quantify specific base pair substitutions present in DNA samples with allele frequencies as low as 1 mutant per 100,000 wild-type sequences [37,38,42–44]. Unlike approaches for mutation detection that report the presence or absence of mutation, the output of ACB-PCR is a measured mutant fraction (MF), which is the ratio of mutant allele to wild-type allele within a given DNA sample.…”