Simulated solar light‐induced p53 mutagenesis in SKH‐1 mouse skin: A dose–response assessment

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Cancer risk assessment impacts a range of societal needs, from the regulation of chemicals to achieving the best possible human health outcomes. Because oncogene and tumor suppressor gene mutations are necessary for the development of cancer, such mutations are ideal biomarkers to use in cancer risk assessment. Consequently, DNA-based methods to quantify particular tumor-associated hotspot point mutations (i.e., oncomutations) have been developed, including allele-specific competitive blocker-PCR (ACB-PCR). Several studies using ACB-PCR and model mutagens have demonstrated that significant induction of tumor-associated oncomutations are measureable at earlier time points than are used to score tumors in a bioassay. In the particular case of benzo[a]pyrene induction of K-Ras codon 12 TGT mutation in the A/J mouse lung, measurement of tumor-associated oncomutation was shown to be an earlier and more sensitive endpoint than tumor response. The measurement of oncomutation by ACB-PCR led to two unexpected findings. First, oncomutations are present in various tissues of control rodents and "normal" human colonic mucosa samples at relatively high frequencies. Approximately 60% of such samples (88/146) have mutant fractions (MFs) >10(-5), and some have MFs as high as 10(-3) or 10(-4). Second, preliminary data indicate that oncomutations are present frequently as subpopulations in tumors. These findings are integrated into a hypothesis that the predominant preexisting mutations in particular tissues may be useful as generic reporters of carcinogenesis. Future research opportunities using oncomutation as an endpoint are described, including rodent to human extrapolation, dose-response assessment, and personalized medicine.

Section: Lessons Learned During Initial Validation Of Oncomutation Asmentioning

confidence: 79%

Section: Advantages and Disadvantages Of The Acb-pcr Approach And Mutmentioning

confidence: 95%

Section: Validation Of Oncomutations As Endpoints For Assessing Chemimentioning

confidence: 99%

Section: Future Directions Research Strategies and Potential Uses Fmentioning

confidence: 99%

Section: Validation Of Oncomutations As Endpoints For Assessing Chemimentioning

confidence: 99%

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Oncomutations as biomarkers of cancer risk

Parsons

Myers

Meng

et al. 2010

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K‐Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose

Meng

Knapp

Green

et al. 2009

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K-Ras mutant fraction (MF) was measured to examine the default assumption of low-dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of 10 male A/J mice (7- to 9-weeks old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P and were sacrificed 28 days after treatment. K-Ras codon 12 TGT and GAT MFs in lung DNAs were measured using Allele-specific Competitive Blocker-PCR (ACB-PCR). The K-Ras codon 12 TGT geometric mean MF was 3.88 x 10(-4) in controls, indicating an average of 1 mutation in every approximately 1,288 lung cells. The K-Ras codon 12 TGT geometric mean MFs were as follows: 3.56 x 10(-4); 6.19 x 10(-4); 2.02 x 10(-3), and 3.50 x 10(-3) for the 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The 5 and 50 mg/kg dose groups had TGT MFs significantly higher than did controls. Although 10(-5) is considered as the limit of accurate ACB-PCR quantitation, K-Ras codon 12 GAT geometric mean MFs were as follows: 8.38 x 10(-7), 1.47 x 10(-6), 2.19 x 10(-6), 5.71 x 10(-6), and 8.99 x 10(-6) for the 0, 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The K-Ras TGT and GAT MFs increased in a B[a]P-dose-dependent manner, with response approximately linear over the 0.05 to 5 mg/kg dose range. K-Ras MF increased with B[a]P adduct burden measured for identical doses in a separate study. Thus, ACB-PCR may be useful in characterizing the shape of a dose-response curve at low doses and establishing relationships between DNA adducts and tumor-associated mutations.

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

McKinzie

Parsons

2011

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.