The Role of DNA Polymerase ζ in Benzo[a]pyrene-induced Mutagenesis in the Mouse Lung

DNA polymerase zeta (Polζ) is a heterotetramer composed of the catalytic subunit Rev3l, Rev7 and two subunits of Polδ (PolD2/Pol31 and PolD3/Pol32), and this polymerase exerts translesion DNA synthesis (TLS) in yeast. Because Rev3l knockout results in embryonic lethality in mice, the functions of Polζ need further investigation in vivo. Then, we noted the two facts that substitution of leucine 979 of yeast Rev3l with methionine reduces Polζ replication fidelity and that reporter gene transgenic rodents are able to provide the detailed mutation status. Here, we established gpt delta mouse knocked in the constructed gene encoding methionine instead of leucine at residue 2610 of Rev3l (Rev3l L2610M gpt delta mice), to clarify the role of Polζ in TLS of chemical-induced bulky DNA adducts in vivo. Eight-week-old gpt delta mice and Rev3l L2610M gpt delta mice were treated with benzo[a]pyrene (BaP) at 0, 40, 80, or 160 mg/kg via single intraperitoneal injection. At necropsy 31 days after treatment, lungs were collected for reporter gene mutation assays. Although the gpt mutant frequency (MF) was significantly increased by BaP in both mouse genotypes, it was three times higher in Rev3l L2610M gpt delta than gpt delta mice after treatment with 160 mg/kg BaP. The frequencies of G:C base substitutions and characteristic complex mutations were significantly increased in Rev3l L2610M gpt delta mice compared with gpt delta mice. The BaP dose–response relationship suggested that Polζ plays a central role in TLS when protective mechanisms against BaP mutagenesis, such as error-free TLS, are saturated. Overall, Polζ may incorporate incorrect nucleotides at the sites opposite to BaP-modified guanines and extend short DNA sequences from the resultant terminal mismatches only when DNA is heavily damaged.

Benchmark dose analysis of multiple genotoxicity endpoints in gpt delta mice exposed to aristolochic acid I

As the carcinogenic risk of herbs containing aristolochic acids (AAs) is a global health issue, quantitative evaluation of toxicity is needed for the regulatory decision-making and risk assessment of AAs. In this study, we selected AA I (AAI), the most abundant and representative compound in AAs, to treat transgenic gpt delta mice at six gradient doses ranging from 0.125 to 4 mg/kg/day for 28 days. AAI-DNA adduct frequencies and gpt gene mutation frequencies (MFs) in the kidney, as well as Pig-a gene MFs and micronucleated reticulocytes (MN-RETs) frequencies in peripheral blood, were monitored. The dose–response (DR) relationship data for these in vivo genotoxicity endpoints were quantitatively evaluated using an advanced benchmark dose (BMD) approach with different critical effect sizes (CESs; i.e., BMD5, BMD10, BMD50 and BMD100). The results showed that the AAI-DNA adduct frequencies, gpt MFs and the MN-RETs presented good DR relationship to the administrated doses, and the corresponding BMDL100 (the lower 90% confidence interval of the BMD100) values were 0.017, 0.509 and 3.9 mg/kg/day, respectively. No positive responses were observed in the Pig-a MFs due to bone marrow suppression caused by AAI. Overall, we quantitatively evaluated the genotoxicity of AAI at low doses for multiple endpoints for the first time. Comparisons of BMD100 values across different endpoints provide a basis for the risk assessment and regulatory decision-making of AAs and are also valuable for understanding the genotoxicity mechanism of AAs.

Lack of In Vivo Mutagenicity of Acetamide in a 13-Week Comprehensive Toxicity Study Using F344 gpt Delta Rats

Acetamide, a food contaminant, has been shown to induce hepatocellular tumors in rats. However, the mode of action underlying acetamide-induced hepatocarcinogenesis remains unclear. In the current study, we aimed to examine the possible involvement of in vivo mutagenicity in hepatocarcinogenesis of acetamide and evaluate its toxicological profile using a comprehensive medium-term toxicity study in gpt delta rats. Six-week-old male F344 gpt delta rats were given a basal diet containing 0%, 0.625%, 1.25%, or 2.5% acetamide for 13 weeks. In general toxicologic assessment, hepatotoxic parameters in serum, such as aspartate aminotransferase and alanine aminotransferase were significantly changed at the 1.25% group and higher. Histopathological examination of the liver revealed that various changes related to hepatic injury were observed at the 1.25% group and higher. Interestingly, Feulgen-positive cytoplasmic inclusion was frequently observed in hepatocytes in these groups. In the hematopoietic system, red blood cell parameters in plasma, such as mean corpuscular volume and mean corpuscular hemoglobin were significantly changed at the 1.25% group and higher, and decrease of erythroblast in the spleen was observed histopathologically in the 2.5% group. Thus, the no-observed-adverse-effect level of acetamide in this study was 0.625% (equivalent to 394 mg/kg body weight/day). In vivo mutation assays showed that acetamide induced no changes in gpt and red/gam gene mutant frequencies, even at the carcinogenic target site. In contrast, Ki67-positive hepatocytes were increased significantly at carcinogenic doses. Therefore, these results suggested that cell proliferation activity, but not mutagenicity, played crucial roles in acetamide-induced hepatocarcinogenesis in rats.

Quantitative analysis of mutagenicity and carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in F344 gpt delta transgenic rats

Quantitative analysis of the mutagenicity and carcinogenicity of the low doses of genotoxic carcinogens present in food is of pressing concern. The purpose of the present study was to determine the mutagenicity and carcinogenicity of low doses of the dietary genotoxic carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Male F344 gpt delta transgenic rats were fed diets supplemented with 0, 0.1, 1, 10 or 100 ppm IQ for 4 weeks. The frequencies of gpt transgene mutations in the liver were significantly increased in the 10 and 100 ppm groups. In addition, the mutation spectra was altered in the 1, 10 and 100 ppm groups: frequencies of G:C to T:A transversion were significantly increased in groups administered 1, 10 and 100 ppm IQ in a dose-dependent manner, and the frequencies of G:C to A:T transitions, A:T to T:A transversions and A:T to C:G transversions were significantly increased in the 100 ppm group. Increased frequencies of single base pair deletions and Spi− mutants in the liver, and an increase in glutathione S-transferase placental form (GST-P)-positive foci, a preneoplastic lesion of the liver in rats, was also observed in the 100 ppm group. In contrast, neither mutations nor mutation spectra or GST-P-positive foci were statistically altered by administration of IQ at 0.1 ppm. We estimated the point of departure for the mutagenicity and carcinogenicity of IQ using the no-observed-effect level approach and the Benchmark dose approach to characterise the dose–response relationship of low doses of IQ. Our findings demonstrate the existence of no effect levels of IQ for both in vivo mutagenicity and hepatocarcinogenicity. The findings of the present study will facilitate an understanding of the carcinogenic effects of low doses of IQ and help to determine a margin of exposure that may be useful for practical human risk assessment.