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Supplementary Table 1 from Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

The lung is an organ that is sensitive to mutations induced by chemicals in ambient air, and transgenic mice harboring guanine phosphoribosyltransferase (gpt) gene as a target gene are a well-established model system for assessing genotoxicity in vivo. Transcription factor Nrf2 mediates inducible and constitutive expression of cytoprotective enzymes against xenobiotics and mutagens. To address whether Nrf2 is also involved in DNA protection, we generated nrf2+/−::gpt and nrf2−/−::gpt mice. The spontaneous mutation frequency of the gpt gene in the lung was approximately three times higher in nrf2-null (nrf2−/−) mice than nrf2 heterozygous (nrf2+/−) and wild-type (nrf2+/+) mice, whereas in the liver, the mutation frequency was higher in nrf2−/− and nrf2+/− mice than in nrf2+/+ wild-type mice. By contrast, no difference in mutation frequency was observed in testis among the three genotypes. A single intratracheal instillation of benzo(a)pyrene (BaP) increased the lung mutation frequency 3.1- and 6.1-fold in nrf2+/− and nrf2−/− mice, respectively, compared with BaP-untreated nrf2+/− mice, showing that nrf2−/− mice are more susceptible to genotoxic carcinogens. Surprisingly, mutation profiles of the gpt gene in BaP-treated nrf2+/− mice was substantially different from that in BaP-untreated nrf2−/− mice. In nrf2−/− mice, spontaneous and BaP-induced mutation hotspots were observed at nucleotides 64 and 140 of gpt, respectively. These results thus show that Nrf2 aids in the prevention of mutations in vivo and suggest that Nrf2 protects genomic DNA against certain types of mutations. [Cancer Res 2007;67(12):5643–8]

Supplementary Table 4 from Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

Supplementary Table 2 from Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

Supplementary Table 3 from Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

Background Base pair mismatches in genomic DNA can result in mutagenesis, and consequently in tumorigenesis. To investigate how mismatch repair deficiency increases mutagenicity under oxidative stress, we examined the type and frequency of mutations arising in the mucosa of the small intestine of mice carrying a reporter gene encoding guanine phosphoribosyltransferase (gpt) and in which the Msh2 gene, which encodes a component of the mismatch repair system, was either intact (Msh2+/+::gpt/0; Msh2-bearing) or homozygously knockout (KO) (Msh2−/−::gpt/0; Msh2-KO). Results Gpt mutant frequency in the small intestine of Msh2-KO mice was about 10 times that in Msh2-bearing mice. Mutant frequency in the Msh2-KO mice was not further enhanced by administration of potassium bromate, an oxidative stress inducer, in the drinking water at a dose of 1.5 g/L for 28 days. Mutation analysis showed that the characteristic mutation in the small intestine of the Msh2-KO mice was G-to-A transition, irrespective of whether potassium bromate was administered. Furthermore, administration of potassium bromate induced mutations at specific sites in gpt in the Msh2-KO mice: G-to-A transition was frequently induced at two known sites of spontaneous mutation (nucleotides 110 and 115, CpG sites) and at nucleotides 92 and 113 (3′-side of 5′-GpG-3′), and these sites were confirmed to be mutation hotspots in potassium bromate-administered Msh2-KO mice. Administration of potassium bromate also induced characteristic mutations, mainly single-base deletion and insertion of an adenine residue, in sequences of three to five adenine nucleotides (A-runs) in Msh2-KO mice, and elevated the overall proportion of single-base deletions plus insertions in Msh2-KO mice. Conclusions Our previous study revealed that administration of potassium bromate enhanced tumorigenesis in the small intestine of Msh2-KO mice and induced G-to-A transition in the Ctnnb1 gene. Based on our present and previous observations, we propose that oxidative stress under conditions of mismatch repair deficiency accelerates the induction of single-adenine deletions at specific sites in oncogenes, which enhances tumorigenesis in a synergistic manner with G-to-A transition in other oncogenes (e.g., Ctnnb1).

The safety of flavoring agents has been evaluated according to classification by chemical structure and using a decision tree approach. The genotoxic potential found in some flavoring agents has highlighted the importance of efficient toxicity studies. We performed a comprehensive toxicity analysis using reporter gene transgenic rats to assess the safety of 3-acetyl-2,5-dimethylfuran (ADF), a flavoring agent exhibiting genotoxic potential in in silico and in vitro assays. Male F344 gpt delta rats were given 0, 30, or 300 mg/kg body weight/day ADF by gavage for 13 weeks. In serum biochemistry analyses, triglyceride, total cholesterol, phospholipid, and total protein levels and albumin/globulin ratios were significantly altered in the 30 and 300 mg/kg groups. Histopathologically, nasal cavity toxicity and hepatocellular hypertrophy were observed in the 300 mg/kg group. In the livers of 300 mg/kg group, a significant increase in gpt mutant frequencies were observed along with ADF-specific DNA adduct formation. The number and area of glutathione S-transferase placental form-positive foci were significantly increased in the same group. Thus, ADF affected nasal cavity, liver, and lipid metabolism and showed genotoxicity and possible carcinogenicity in the liver. Overall, our comprehensive toxicity study using gpt delta rats provided insights into the safety evaluation of ADF.

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 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.

Although gpt delta rats, as reporter gene-transgenic rats, were originally developed for in vivo mutation assays, they have also been used to evaluate chemical carcinogenesis and comprehensive toxicity. Therefore, it is necessary to accumulate background data on carcinogenicity and general toxicity in gpt delta rats. Here, we investigated the background data of 110-week-old male and female F344 gpt delta rats and wild-type rats. There was no effect of reporter gene transfection on animal survival rates and body weights during the experiment. The relative weight of male gpt delta rat adrenals was significantly higher than that of wild-type rats, possibly due to the higher incidence of pheochromocytoma. There were no intergenotype differences in the incidence of nonneoplastic lesions in both sexes, including chronic progressive nephropathy and focus of cellular alteration in the liver, which had a higher incidence in both genotypes. Additionally, the significantly higher incidence of adrenal pheochromocytoma in male gpt delta rats than that in wild-type rats was likely incidental because of the lack of differences in the incidences of preneoplastic (male and female) and neoplastic (female) adrenal lesions in both genotypes. Other neoplastic lesions in both sexes showed no intergenotype differences in incidence rates, although large granular lymphocytic leukemia in the spleen and Leydig cell tumors in the testes of males showed higher incidence rates. Overall, there were no effects of reporter gene transfection on the spectrum of spontaneous lesions in F344 gpt delta rats, thus supporting their applicability in evaluating chemical toxicity and carcinogenicity.

2-Methylfuran (2-MF) exists naturally in foods and is used as a flavoring agent. Furan, the core structure of 2-MF, possesses hepatocarcinogenicity in rodents. Accumulation of toxicological information on furan derivatives is needed to elucidate their carcinogenic mode of action. In the current study, we examined the comprehensive toxicological studies of 2-MF using gpt delta rats. 2-MF was intragastrically administered to groups of 10 male and 10 female Sprague-Dawley gpt delta rats at a dose of 0, 1.2, 6, or 30 mg/kg/day for 13 weeks. Effects of 2-MF on the hepatobiliary system including an increase in serum alkaline phosphatase were observed in the 6 and 30 mg/kg groups, and cholangiofibrosis was found in the 30 mg/kg group. The no observed adverse effect level was set at 1.2 mg/kg/day for both sexes and 1.14 mg/kg/day was determined as the benchmark dose low. The acceptable daily intake was calculated to be 11.4 μg/kg/day. Increases in the number and areas of glutathione S-transferase placental form-positive foci in the 30 mg/kg group were apparent, suggesting the hepatocarcinogenicity of 2-MF in rats. By contrast, the lack of increase in in vivo mutagenicity in the liver implied that 2-MF hepatocarcinogenesis may not involve genotoxic mechanisms.

Chemical carcinogens to humans have been usually identified by epidemiological studies on the relationships between occupational or environmental exposure to the agents and specific cancer induction. In contrast, carcinogenic heterocyclic amines were identified under the principle that mutagens in bacterial in the Ames test are possible human carcinogens. In the 1970s to 1990s, more than 10 heterocyclic amines were isolated from pyrolysates of amino acids, proteins, meat or fish as mutagens in the Ames test, and they were demonstrated as carcinogens in rodents. In the 1980s and 1990s, we have developed derivatives of the Ames tester strains that overexpressed acetyltransferase of Salmonella typhimurium. These strains such as Salmonella typhimurium YG1024 exhibited a high sensitivity to the mutagenicity of the carcinogenic heterocyclic amines. Because of the high sensitivity, YG1024 and other YG strains were used for various purposes, e.g., identification of novel heterocyclic amines, mechanisms of metabolic activation, comparison of mutagenic potencies of various heterocyclic amines, and the co-mutagenic effects. In the 1990s and 2000s, we developed transgenic mice and rats for the detection of mutagenicity of chemicals in vivo. The transgenics were generated by the introduction of reporter genes for mutations into fertilized eggs of mice and rats. We named the transgenics as gpt delta because the gpt gene of Escherichia coli was used for detection of point mutations such as base substitutions and frameshifts and the red/gam genes of λ phage were employed to detect deletion mutations. The transgenic rodents gpt delta and other transgenics with lacI or lacZ as reporter genes have been utilized for characterization of mutagenicity of heterocyclic amines in vivo. In this review, we summarized the in vitro mutagenicity of heterocyclic amines in Salmonella typhimurium YG strains and the in vivo mutagenicity in transgenic rodents. We discussed the relationships between in vitro and in vivo mutagenicity of the heterocyclic amines and their relations to the carcinogenicity.

Chromosome aberrations (CAs), i.e. changes in chromosome number or structure, are known to cause chromosome rearrangements and subsequently tumorigenesis. However, the involvement of CAs in chemical-induced carcinogenesis is unclear. In the current study, we aimed to clarify the possible involvement of CAs in chemical carcinogenesis using a rat model with the non-mutagenic hepatocarcinogen acetamide. In an in vivo micronucleus (MN) test, acetamide was revealed to induce CAs specifically in rat liver at carcinogenic doses. Acetamide also induced centromere-positive large MN (LMN) in hepatocytes. Immunohistochemical and electron microscopic analyses of the LMN, which can be histopathologically detected as basophilic cytoplasmic inclusion, revealed abnormal expression of nuclear envelope proteins, increased heterochromatinization, and massive DNA damage. These molecular pathological features in LMN progressed with acetamide exposure in a time-dependent manner, implying that LMN formation can lead to chromosome rearrangements. Overall, these data suggested that CAs induced by acetamide play a pivotal role in acetamide-induced hepatocarcinogenesis in rats and that CAs can cause chemical carcinogenesis in animals via MN formation.

The in vivo mutagenicity of hexavalent chromium in the small intestine, the target organ of tumorgenicity, was examined by means of a transgenic mouse gene mutation assay. Sodium dichromate dihydrate was administered orally in drinking water to male gpt delta mice at a dose of 85.7 or 257.4 mg/L for 28 days or at a dose of 8.6, 28.6 or 85.7 mg/L for 90 days. No significant increase in gpt mutant frequency relative to that in control mice was observed in the small intestine in either the 28- or 90-day study, whereas 28-day oral administration of potassium bromate, a positive control substance, increased mutant frequency.

Background Previously we found that DNA adducts were accumulated in the lungs of the rats exposed to ambient air in the Tokyo metropolitan area. To examine chronological change in in vivo mutagenicity of airborne particles, extracts produced from samples of total suspended particulates (TSP) collected from urban air in 1980, 1990, and 2010 in the Tokyo metropolitan area were intratracheally administered into the lungs of gpt delta mice, and differences in mutation and mutant frequency were determined by using the gpt assay. In vivo mutations induced by the extracts were characterized and mutation hotspots were identified by DNA sequencing of the mutated gpt gene. Results Administration of the 1990 extract at a dose of 0.3 mg/animal significantly elevated total mutant frequency to 3.3-times that in vehicle control, and the in vivo mutagenicity of the extract (induced mutation frequency per milligram extract) was estimated to be 2.0- and 2.4-times higher than that of the 2010 and 1980 extract, respectively. G-to-A transition was the most common base substitution in the vehicle control mice. However, administration of the 1990 extract increased the frequency of G-to-T transversion, which is a landmark base substitution induced by oxidative stress; furthermore, when the extract was administered at a dose of 0.15 mg, the mutant and mutation frequencies of G-to-T transversion were significantly increased to frequencies comparable with those of G-to-A transition. Similar increases in the mutant and mutation frequencies of G-to-T transversion were observed after administration of the 2010 extract. Hotspots (mutation foci identified in three or more mice) of G-to-A transition mutations at nucleotides 64 and 110 were induced by the 1980, 1990, and 2010 extracts; a hotspot of G-to-T transversions at nucleotide 406 was also induced by the 2010 extract. Previously, we showed that diesel exhaust particles or their extract, as well as 1,6-dinitropyrene, administered to mice induced these hotspots of G-to-A transitions. Conclusions The results of the present study suggested that mutagenesis induced by extracts produced from TSP collected in the Tokyo metropolitan area induced in vivo mutagenicity via the same mechanism underlying the induction of in vivo mutagenicity by components of diesel exhaust.

Exposure to chemical agents is an inevitable consequence of modern society; some of these agents are hazardous to human health. The effects of chemical carcinogens are of great concern in many countries, and international organizations, such as the World Health Organization, have established guidelines for the regulation of these chemicals. Carcinogens are currently categorized into two classes, genotoxic and non-genotoxic carcinogens, which are subject to different regulatory policies. Genotoxic carcinogens are chemicals that exert carcinogenicity via the induction of mutations. Owing to their DNA interaction properties, there is thought to be no safe exposure threshold or dose. Genotoxic carcinogens are regulated under the assumption that they pose a cancer risk for humans, even at very low doses. In contrast, non-genotoxic carcinogens, which induce cancer through mechanisms other than mutations, such as hormonal effects, cytotoxicity, cell proliferation, or epigenetic changes, are thought to have a safe exposure threshold or dose; thus, their use in society is permitted unless the exposure or intake level would exceed the threshold. Genotoxicity assays are an important method to distinguish the two classes of carcinogens. However, some carcinogens have negative results in in vitro bacterial mutation assays, but yield positive results in the in vivo transgenic rodent gene mutation assay. Non-DNA damage, such as spindle poison or topoisomerase inhibition, often leads to positive results in cytogenetic genotoxicity assays such as the chromosome aberration assay or the micronucleus assay. Therefore, mechanistic considerations of tumor induction, based on the results of the genotoxicity assays, are necessary to distinguish genotoxic and non-genotoxic carcinogens. In this review, the concept of threshold of toxicological concern is introduced and the potential risk from multiple exposures to low doses of genotoxic carcinogens is also discussed.

DNA polymerase ζ (Pol ζ) is a specialized Pol that is involved in translesion DNA synthesis (TLS), in particular, in the extension of primer DNA after bypassing DNA lesions. Previously, we established human cells that express a variant form of Pol ζ with an amino acid change of leucine 2618 to methionine (L2618M) in the catalytic subunit REV3L (DNA Repair, 45, 34-43, 2016). This amino acid change made the cells more sensitive to the mutagenicity of benzo[a]pyrene diol epoxide (BPDE). In this study, we embedded BPDE-N2-guanine at a defined position in the supF gene on the shuttle plasmid and introduced it to REV3 L2618M cells or the wild-type (WT) cells to examine how far Pol ζ L2618M extends the primer DNA after bypassing the lesion. The adduct induced primarily G to T and G to C at the adducted site in both cell lines, but generated additional sequence changes such as base substitutions, deletions and additions in the extension patch much more often in REV3 L2618M cells than in the WT cells. Mutations in the extension patch in REV3 L2618M cells occurred most often within 10 bps from the adducted site. Then, the number of mutations gradually decreased and no mutations were observed between 30 and 40 bps from the lesion. We concluded that human Pol ζ L2618M and perhaps WT Pol ζ extend the primer DNA up to approximately 30 bps from the lesion in vivo. The possibility of involvement of Pol ζ L2618M in the insertion step of TLS is discussed.

Background DNA-dependent protein kinase (DNA-PK), consisting of a Ku heterodimer (Ku70/80) and a large catalytic subunit (DNA-PKcs), plays an important role in the repair of DNA double-strand breaks via non-homologous end-joining (NHEJ) in mammalian cells. Severe combined immunodeficient (scid) mice carry a mutation in the gene encoding DNA-PKcs and are sensitive to ionizing radiation. To examine the roles of DNA-PKcs in the generation of deletion mutations in vivo, we crossed scid mice with gpt delta transgenic mice for detecting mutations. Results The scid and wild-type (WT) gpt delta transgenic mice were irradiated with a single X-ray dose of 10 Gy, and Spi− mutant frequencies (MFs) were determined in the brain and spleen 2 days after irradiation. Irradiation with X-rays significantly enhanced Spi− MF in both organs in the scid and WT mice. The MFs in the brain of irradiated scid mice were significantly lower than those in WT mice, i.e., 2.9 ± 1.0 × 10− 6 versus 5.0 ± 1.1 × 10− 6 (P − 6 versus 4.8 ± 1.4 × 10− 6. Unirradiated scid and WT mice did not exhibit significant differences in MFs in either organ. Conclusions DNA-PKcs is unessential for the induction of deletion mutations in the spleen, while it plays a role in this in the brain. Therefore, the contribution of DNA-PKcs to NHEJ may be organ-specific.

1,4-Dioxane is a widely used synthetic industrial chemical and its contamination of drinking water and food is a potential health concern. It induces liver tumors when administered in the drinking water to rats and mice. However, the mode of action (MOA) of the hepatocarcinogenicity of 1,4-dioxane remains unclear. Importantly, it is unknown if 1,4-dioxane is genotoxic, a key consideration for risk assessment. To determine the in vivo mutagenicity of 1,4-dioxane, gpt delta transgenic F344 rats were administered 1,4-dioxane at various doses in the drinking water for 16 weeks. The overall mutation frequency (MF) and A:T- to -G:C transitions and A:T- to -T:A transversions in the gpt transgene were significantly increased by administration of 5000 ppm 1,4-dioxane. A:T- to -T:A transversions were also significantly increased by administration of 1000 ppm 1,4-dioxane. Furthermore, the DNA repair enzyme MGMT was significantly induced at 5000 ppm 1,4-dioxane, implying that extensive genetic damage exceeded the repair capacity of the cells in the liver and consequently led to liver carcinogenesis. No evidence supporting other MOAs, including induction of oxidative stress, cytotoxicity, or nuclear receptor activation, that could contribute to the carcinogenic effects of 1,4-dioxane were found. These findings demonstrate that 1,4-dioxane is a genotoxic hepatocarcinogen and induces hepatocarcinogenesis through a mutagenic MOA in rats. Because our data indicate that 1,4-dioxane is a genotoxic carcinogen, we estimated the point of departure of the mutagenicity and carcinogenicity of 1,4-dioxane using the no-observed effect-level approach and the Benchmark dose approach to characterize its dose–response relationship at low doses.

In regulatory genetic toxicology, it is an axiom that there is no threshold for genotoxicity of chemicals, such that genotoxic chemicals may impose carcinogenic risk on humans even at very low doses. This paradigm is counterintuitive, however, because humans possess a number of self-defense mechanisms that may suppress the genotoxicity at these low doses and therefore manifest a practical threshold. DNA polymerase zeta (Pol ζ) is a specialized Pol that plays an important role in DNA synthesis across DNA damage, thereby modulating cell survival and genotoxicity. In this study, we compared the sensitivity of three types of human cells: D2781N, L2618M, and their wild-type (WT) cells, to the low dose effects of genotoxicity of the oxidizing agents, potassium bromate (KBrO3) and sodium dichromate (Na2Cr2O7). D2781N cells express a variant form of Pol ζ, whose activity is weaker than that of the WT enzyme. L2618M cells express another variant form of Pol ζ, whose fidelity of DNA replication is lower than that of the WT enzyme. D2781N exhibited the highest sensitivity for TK gene mutation and micronucleus (MN) formation and displayed the lowest practical threshold for MN induction by KBrO3. In contrast, L2618M exhibited the lowest practical threshold for sister-chromatid exchange (SCE) induction by both chemicals. These results suggest that Pol ζ mutations have significant impacts on practical thresholds of genotoxicity; the factors affecting the practical threshold can differ depending on the endpoint of genotoxicity. Roles of the variant forms of Pol ζ in genotoxicity by the oxidizing agents are discussed.

Protein phosphatase 2A (PP2A) is a serine-threonine phosphatase that regulates cell signaling pathways. Its inactivation is correlated with tumor malignancy, possibly due to the effects on cell differentiation and malignant cell transformation. Therefore, it has been noted that PP2A could be a promising target for cancer therapy. In our previous study of the hepatocarcinogen estragole (ES), cell proliferation may be required to convert ES-specific DNA adducts to mutations. To explore the trigger for cell proliferation, gpt delta rats were administered ES by gavage at doses of 3, 30 and 300mg/kg/day for 4weeks. ES-induced cell proliferation and gene mutations were observed at only the high dose whereas ES-specific DNA adducts were detected in a dose-dependent manner. Western blot analyses revealed activation of the Akt and ERK pathways without activation of upstream regulators, such as c-Raf, PKC and, PI3K. Phosphorylation of the PP2A C subunit at Tyr307 was found along with phosphorylation of Src. The overall data might imply that PP2A inactivation is responsible for cell cycle progression through activation of the Akt and ERK pathways at high doses of ES. Based on γ-H2AX immunohistochemistry and Western blot analysis for Rad51 protein, the resultant mutation spectra showed large deletion mutations that might result from double strand breaks of DNA. Thus, it is likely that inactivation of PP2A resulted in acceleration and exacerbation of gene mutations. We conclude that PP2A might contribute to an early stage of chemical carcinogenesis, suggesting that PP2A could be a molecular target of primary cancer prevention.

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc.

Germline mutations are an important component of genetic toxicology; however, mutagenicity tests of germline cells are limited. Recent advances in sequencing technology can be used to detect mutations by direct sequencing of genomic DNA (gDNA). We previously reported induced de novo mutations detected using whole-exome sequencing in the offspring of N-ethyl-N-nitrosourea (ENU)-treated mice in a single-dose experiment (85mg/kg, i.p., weekly on two occasions). In this study, two lower doses (10 and 30mg/kg) were added, and dose-response of inherited germline mutations was analyzed. Male gpt delta transgenic mice treated with ENU in three dose groups were mated with untreated females 10 weeks after the last treatment, and offspring were obtained. The ENU-treated male mice showed dose-dependent increases in gpt mutant frequencies in their sperm, testis, and liver. gDNA of one family (parents and four offspring) from each dose group was used for whole-exome sequencing, and unique de novo mutations in the offspring were detected. Frequencies of inherited mutations increased with dosage more than 25-fold in the highest dose group. The mutation spectrum of the inherited mutations showed characteristics of ENU-induced mutations, such as A:T base substitutions. No confirmed mutations were observed in the control group. Filtering using the alternate reads ratio resulted in the mutation frequencies and spectra similar to those obtained by the Sanger sequencing confirmation. These results suggest that direct sequencing analysis may be a useful tool to investigate inherited germline mutations induced by environmental mutagens.

Background The MucA’ and MucB proteins comprise the core of DNA polymerase RI which is a strong mutator utilized in mutagenicity assays such as the standard Ames test. A close relative DNA polymerase V, composed of the homologous UmuD’ and UmuC proteins, is considered to be an ortholog of the mammalian DNA polymerase η. The catalytic subunits of these polymerases belong to the Y-family which specializes in the translesion DNA synthesis across various DNA adducts to rescue stalled chromosomal replication at the expense of mutations. Based on genetic evidence, DNA polymerase RI possesses the greatest ability to induce various types of mutations among all so far characterized members of the Y-superfamily. The exceptionally high mutagenic potential of MucA’B has been taken advantage of in numerous bacterial mutagenicity assays incorporating the conjugative plasmid pKM101 carrying the mucAB operon such as the Ames Test. Results We established new procedures for the purification of MucB protein as well as its accessory protein MucA’ using the refolding techniques. The purified MucA’ protein behaved as a molecular dimer which was fully stable in solution. The soluble monomeric form of MucB protein was obtained after refolding on a gel-filtration column and remained stable in a nondenaturing buffer containing protein aggregation inhibitors. Using the surface plasmon resonance technique, we demonstrated that the purified MucA’ and MucB proteins interacted and that MucB protein preferentially bound to single-stranded DNA. In addition, we revealed that MucB protein interacted with the β-subunit of DNA polymerase III holoenzyme of E. coli. Conclusion The MucA’ and MucB proteins can be isolated from inclusion bodies and solubilized in vitro. The refolded MucB protein interacts with its MucA’ partner as well as with DNA what suggests it retains biological activity. The interaction of MucB with the processivity subunit of DNA polymerase III may imply the role of the subunit as an accessory protein to MucB during the translesion DNA synthesis. Electronic supplementary material The online version of this article (10.1186/s41021-019-0125-8) contains supplementary material, which is available to authorized users.

Genotoxic carcinogens are regulated under the policy that there is no threshold or safe dose. It has been pointed out, however, that self-defense mechanisms, such as detoxification, DNA repair, and error-free translesion synthesis, may protect chromosome DNA from genotoxic insults, thereby constituting practical threshold. In this study, we examined dose responses of chromosome aberrations induced by three oxidative genotoxins, that is, hydrogen peroxide (H2 O2 ), menadione and paraquat, with or without DNA polymerase kappa (Polκ) activities and mismatch repair capacities in human cells. Polκ is involved in translesion synthesis across DNA damage and mismatch repair is responsible for correction of base-base mismatch in DNA. Polκ activity of the cells was inactivated either by point mutations in the catalytically essential amino acids (catalytically dead or CD) or by deletion of the POLK gene (knockout or KO). In the absence of mismatch repair, frequencies of chromosome aberrations induced by H2 O2 and menadione were not significantly different among CD, KO, and the wild type (WT) cells. In the presence of mismatch repair, however, cytotoxicity and clastogenicity were enhanced and Polκ modulated the sensitivity of the cells. No-observed-genotoxic-effect-levels (NOGELs) for H2 O2 and menadione were CD = KO < WT cells. In contrast, the sensitivities of the cells to paraquat were not significantly affected by the status of mismatch repair or Polκ activity. The results suggest that mismatch repair and Polκ coordinately modulate NOGELs for the clastogenicity of H2 O2 and menadione and also that DNA lesion(s) responsible for paraquat-induced chromosome aberrations are different from those induced by H2 O2 and menadione. Environ. Mol. Mutagen. 61:193-199, 2020. © 2019 Wiley Periodicals, Inc.

Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD>KO>WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N'-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA.

Furan has been used as an intermediate in the chemical-manufacturing industry and has been shown to contaminate various foods. Although furan induces hepatocellular tumors in rodents, equivocal results from in vitro and in vivo mutagenicity tests have caused controversy regarding the involvement of genotoxic mechanisms in furan-induced carcinogenesis. In the present study, to elucidate the possible mechanisms underlying furan-induced hepatocarcinogenesis, a comprehensive medium-term analysis was conducted using gpt delta rats treated with furan at carcinogenic doses for 13 weeks. In the liver, the frequencies of gpt and Spi- mutants derived mainly from point and deletion mutations, respectively, were not changed, and there were no furan-specific gpt mutations in furan-treated rats. In contrast, the number and area of glutathione S-transferase placental form (GST-P)- positive foci were significantly increased in the high-dose group. Also, the ratio of PCNA-positive hepatocytes was significantly elevated in the same group, as supported by significant increases in cyclin d1 and cyclin e1 mRNA levels. Thus, it is highly probable that cell proliferation, but not genotoxic mechanisms, contribute to the development of GST-P foci in furan-treated rats. Based on the close relationship between GST-P and neoplastic hepatocytes, these data allowed us to hypothesize that cell proliferation following signal transduction other than the mitogen-activated protein kinase (MAPK)/ERK pathway may play a crucial role in early-stage furan-induced hepatocarcinogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

Genotoxicity is a critical endpoint of toxicity to regulate environmental chemicals. Genotoxic chemicals are believed to have no thresholds for the action and impose genotoxic risk to humans even at very low doses. Therefore, genotoxic carcinogens, which induce tumors via genotoxic mechanisms, are regulated more strictly than non-genotoxic carcinogens, which induce tumors through non-genotoxic mechanisms such as hormonal effects, cell proliferation and cell toxicity. Although Ames bacterial mutagenicity assay is the gold standard to identify genotoxicity of chemicals, the genotoxicity should be further examined in rodents because Ames positive chemicals are not necessarily genotoxic in vivo. To better evaluate the genotoxicity of chemicals in a whole body system, gene mutation assays with gpt delta transgenic mice and rats have been developed. A feature of the assays is to detect point mutations and deletions by two distinct selection methods, ie, gpt and Spi- assays, respectively. The Spi- assay is unique in that it allows analyses of deletions and complex DNA rearrangements induced by double-strand breaks in DNA. Here, I describe the concept of gpt delta gene mutation assays and the application in food safety research, and discuss future perspectives of genotoxicity assays in vivo.

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5′-TGAA-3′ sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.

In vitro studies indicate that DNA polymerase kappa (Polκ) is able to accurately and efficiently perform DNA synthesis using templates containing various types of DNA damage, including benzo[a]pyrene (BP)-induced N2 -deoxyguanosine adducts. In this study, we examined sensitivity of inactivated Polk knock-in (Polk-/- ) mice to BP carcinogenicity in the colon by administering an oral dose of BP plus dextran sulfate sodium (DSS), an inflammation causing promoter of carcinogenesis. Although colon cancer was successfully induced by BP plus DSS, there was no significant difference in tumor incidence or multiplicity between Polk-/- and Polk+/+ mice. Malignant lymphoma was induced in thymus by the treatment only in Polk-/- mice, but it lacked statistical significance. Mutant frequencies (MFs) in the gpt reporter gene were strongly enhanced in colon; almost to the same extent in both types of mice. Micronucleus formation in bone marrow at the high dose of BP and DNA adducts in colon and lung was not significantly different between two types of mice. Surprisingly, however, Polk-/- mice exhibited significantly higher MFs in colon and lung than did Polk+/+ mice when they were treated with DSS alone. The most prominent mutation induced by DSS treatment was G:C to C:G transversion, whose specific MF in proximal colon was 30 times higher in Polk-/- than in Polk+/+ mice. DSS alone did not enhance MF at all in Polk+/+ mice. The results indicate that Polκ does not suppress BP-induced mutagenesis and carcinogenesis in the colon, but counteracts inflammation-induced mutagenesis in multiple organs. Environ. Mol. Mutagen. 60:320-330, 2019. © 2019 Wiley Periodicals, Inc.

I first became acquainted with the Ames test at the very beginning of my career in 1978, when my task at the National Institute of Health Sciences (Tokyo) was to screen for mutagenicity of food additives used in Japan, using the Ames test. I also used this test to research the metabolic activation mechanisms of chemical carcinogens, in particular, the analgesic drug, phenacetin. This chemical was not mutagenic in Salmonella typhimurium TA100 with standard 9000 × g supernatant of liver homogenates (S9) from rat but was mutagenic with hamster S9. It was revealed that hamster S9 had much higher deacetylation activities than rat S9, which accounts for the species difference. Then, my work was focused on molecular biology. We cloned the genes encoding nitroreductase and acetyltransferase in Salmonella typhimurium TA1538. Plasmids carrying these genes made strain TA98 more sensitive to mutagenic nitroarenes and aromatic amines. Because of their high sensitivity, the resulting strains such as YG1021 and YG1024 are widely used to monitor mutagenic nitroarenes and aromatic amines in complex mixtures. Later, we disrupted the genes encoding DNA polymerases in TA1538 and classified chemical mutagens into four classes depending on their use of different DNA polymerases. I was also involved in the generation of gpt delta transgenic rodent gene mutation assays, which examine the results of the Ames test in vivo. I have unintentionally developed my career under the influence of Dr. Ames and I would like to acknowledge his remarkable achievements in the field of environmental mutagenesis and carcinogenesis.

The International Association of Environmental Mutagenesis and Genomics Societies (IAEMGS) is a global organization that promotes applied and basic research on environmental mutagenesis and genomics. IAEMGS is composed of 13 national and regional societies of environmental mutagenesis and genomics and the total membership is approximately 4000. IAEMGS convenes the general assembly every four years during each International Conference on Environmental Mutagens (ICEM) for communication among members of participating societies and discussion on future directions. The latest general assembly was held during the 12th ICEM/5th Asian Conference on Environmental Mutagens (ACEM) in November 2017 in Incheon, Korea. This report summarizes the topics and discussions in the general assembly.

DNA polymerase κ (Pol κ) is a specialized DNA polymerase involved in translesion DNA synthesis. Although its bypass activities across lesions are well characterized in biochemistry, its cellular protective roles against genotoxic insults are still elusive. To better understand the in vivo protective roles, we have established a human cell line deficient in the expression of Pol κ (KO) and another expressing catalytically dead Pol κ (CD), to examine the cytotoxic sensitivity to 11 genotoxins including ultraviolet C light (UV). These cell lines were established in a genetic background of Nalm-6-MSH+, a human lymphoblastic cell line that has high efficiency for gene targeting, and functional p53 and mismatch repair activities. We classified the genotoxins into four groups. Group 1 includes benzo[a]pyrene diolepoxide, mitomycin C, and bleomycin, where the sensitivity was equally higher in KO and CD than in the cell line expressing wild-type Pol κ (WT). Group 2 includes hydrogen peroxide and menadione, where hypersensitivity was observed only in KO. Group 3 includes methyl methanesulfonate and ethyl methanesulfonate, where hypersensitivity was observed only in CD. Group 4 includes UV and three chemicals, where the chemicals exhibited similar cytotoxicity to all three cell lines. The results suggest that Pol κ not only protects cells from genotoxic DNA lesions via DNA polymerase activities, but also contributes to genome integrity by acting as a non-catalytic protein against oxidative damage caused by hydrogen peroxide and menadione. The non-catalytic roles of Pol κ in protection against oxidative damage by hydrogen peroxide are discussed.

Nitrofurans are antimicrobial compounds containing a nitro group at the 5-position of the furan ring and an amine or hydrazide side chain derivative. One member of the nitrofurans, nitrofurantoin (NFT), is a renal carcinogen in male rats despite its still controversial genotoxicity. We investigated chemical structure-related modes of action of NFT, and reporter gene mutation assays for NFT and its constituent moieties were performed. NFT, 5-nitro-2-furaldehyde (NFA), or 1-aminohydantoin (AHD) was administered to male F344 gpt delta rats by gavage for 4 or 13 weeks at a carcinogenic or the maximum tolerated dose. NFT caused a significant increase in gpt mutant frequency (MF) at 13 weeks with G-base substitution mutations. An increase in gpt MF was also observed in the NFA-treated group at 13 weeks, but not in the AHD-treated group. 8-Hydroxydeoxyguanosine (8-OHdG) levels in the kidney DNA of NFT-treated rats were significantly increased after 4 weeks. NFT caused accumulation of hyaline droplets indicated by positive immunostaining and western blot analysis for α2u-globulin in the proximal tubules. An additional study, in which female gpt delta rats were given NFT at the same dose used for males, was performed to mitigate the effect of α2u-globulin. NFT exerted the same effects on female rat kidneys to the same extent as males in terms of gpt MF and 8-OHdG level. Thus, it is highly probable that the structure of the nitro furan plays a key role in NFT-induced genotoxicity and genotoxic mechanisms including oxidative DNA damage are involved in NFT-induced renal carcinogenesis. α2u-globulin-mediated nephropathy may be a prerequisite for NFT-induced renal carcinogenesis in male rats, and additionally NFT could be a latent carcinogen in female rats and other animal species.

Transgenic rodents carrying reporter genes to detect organ-specific in vivo genetic alterations are useful for risk assessment of genotoxicity that causes cancer. Thus, the Organization for Economic Co-operation and Development has established the guideline for genotoxicity tests using transgenic animals, which may be combined with repeated-dose toxicity studies. Here, we provide evidence to support equivalence of gpt delta and wild type (WT) rats in terms of toxicological responses to a genotoxic hepatocarcinogen, N-nitrosodiethylamine (DEN), and a non-genotoxic hepatocarcinogen, di(2-ethylhexyl)phthalate (DEHP). gpt delta rats treated with DEHP showed similar increases in liver and kidney weights, serum albumin, albumin/globulin ratios, and incidence of diffuse hepatocyte hypertrophy compared to WT F344 and Sprague-Dawley (SD) rats. DEN-treated gpt delta rats showed equivalent increases in the number and area of precancerous GST-P-positive foci in the liver compared to WT rats. The livers of DEN-treated gpt delta rats also showed increased frequencies of gpt and Spi(-) mutations; such changes were not observed in DEHP-treated gpt delta rats. These results indicated that gpt delta rats (both F344 and SD backgrounds) showed comparable DEHP-induced toxicity and DEN-induced genotoxicity to those observed in WT rats. With regard to the administration period, the general toxicity of 1.2% DEHP was evident throughout the experimental period, and the genotoxicity of 10 p.p.m. DEN could be detected after 2 weeks of administration and further increased at 4 weeks. These results suggested that combined assays using gpt delta rats could detect both general toxicity and genotoxicity by the canonical 4-week administration protocol. Therefore, this assay using gpt delta rats would be applicable for risk assessment including early detection of genotoxic carcinogens and ultimately serve to reduce cancer risks in humans from environmental chemicals.

Carcinogenic doses of ochratoxin A (OTA) cause increases of mutant frequencies (MFs) of the red/gam gene (Spi(-)) in the kidneys of p53-deficient gpt delta mice, but not in p53-proficient mice. Here, we investigated the role of p53 in the progression from OTA-induced DNA damage to gene mutations. To this end, p53-proficient and -deficient mice were administered 5 mg/kg OTA for 3 days or 4 weeks by gavage. After 3 days of administration, comet assays were performed and there were no differences in the degrees of OTA-induced DNA damage between p53-proficient and -deficient mice. However, the frequencies of γ-H2AX-positive tubular epithelial cells in p53-deficient mice were significantly higher than those in p53-proficient mice, implying that p53 inhibited the progression from DNA damage to DNA double-strand breaks (DSBs). Evaluation of global gene expression and relevant mRNA/protein expression levels demonstrated that OTA increased the expression of Cdkn1a, which encodes the p21 protein, in p53-proficient mice, but not in p53-deficient mice. Moreover, in p53-deficient mice, mRNA levels of cell cycle progression and DSB repair (homologous recombination repair [HR])-related genes were significantly increased. Thus, G1/S arrest due to activation of the p53/p21 pathway may contribute to the prevention of DSBs in p53-proficient mice. In addition, single base deletions/insertions/substitutions were predominant, possibly due to HR. Overall, these results suggested that OTA induced DSBs at the carcinogenic target site in mice and that p53/p21-mediated cell cycle control prevented an increase in the formation of DSBs, leading to gene mutations.

The kidney is a major target site of chemical carcinogenesis. However, a reliable in vivo assay for rapid identification of renal carcinogens has not been established. The purpose of this study was to develop a new medium-term gpt delta rat model (the GNP model) to facilitate identification of renal carcinogens. In this model, we carried out an in vivo mutation assay using unilaterally nephrectomized kidney tissue and a tumor-promoting assay using residual kidney tissue, with diethylnitrosamine (DEN) as the renal tumor initiator. To clarify the optimal time of DEN injection after nephrectomy, time-dependent changes in bromodeoxyuridine-labeling indices in the tubular epithelium of nephrectomized rats were examined. The optimal dose of DEN injection and sufficient duration of subsequent nitrilotriacetic acid treatment were determined for detection of renal preneoplastic lesions. The standard protocol for the GNP model was determined as follows. Six-week-old female gpt delta rats were treated with test chemicals for 4 weeks, followed by a 2-week washout period, and 40 mg/kg DEN was administered intraperitoneally to initiate renal carcinogenesis. Unilateral nephrectomy was performed 48 h before DEN injection, followed by gpt assays using excised kidney tissues. One week after DEN injection, rats were further exposed to test chemicals for 12 weeks, and histopathological analysis of renal preneoplastic lesions was performed as an indicator of tumor-promoting activity in residual kidney tissue. Validation studies using aristolochic acid, potassium dibasic phosphate, phenylbutazone, and d-limonene indicated the reliability of the GNP model for predicting renal carcinogens and the underlying mode of action.