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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).

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.

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.

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.

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.

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.

Translesion DNA synthesis (TLS) is an important pathway that avoids genotoxicity induced by endogenous and exogenous agents. DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in TLS but its protective roles against DNA damage in vivo are still unclear. To better understand these roles, we have established knock-in mice that express catalytically-inactive Polk and crossbred them with gpt delta mice, which possess reporter genes for mutations. The resulting mice (inactivated Polk KI mice) were exposed to mitomycin C (MMC), and the frequency of point mutations, micronucleus formation in peripheral erythrocytes, and γH2AX induction in the bone marrow was determined. The inactivated Polk KI mice exhibited significantly higher frequency of mutations at CpG and GpG sites, micronucleated cells, and γH2AX foci-positive cells than did the Polk wild-type (Polk + ) mice. Recovery from MMC-induced DNA damage, which was evaluated by γH2AX induction, was retarded in embryonic fibroblasts from the knock-in mice when compared to those from the Polk + mice. These results suggest that Polk mediates TLS, which suppresses point mutations and DNA double-strand breaks caused by intra- and interstrand cross-links induced by MMC treatment. The established knock-in mice are extremely useful to elucidate the in vivo roles of the catalytic activity of Polk in suppressing DNA damage that was induced by a variety of genotoxic stresses.

Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.

We have developed a new medium-term animal model, "GPG", in which an in vivo mutation assay in partially hepatectomized tissue and a tumor-promoting assay were performed. The tumor-promoting assay measures glutathione S-transferase placental form positive foci induced by diethylnitrosamine (DEN) in the residual tissue. Given that a limitation of the original protocol is the potential interaction between the test chemical and DEN, the present study establishes a modified protocol that includes a test chemical washout period. Using CYP2E1 inhibitor and CYP1A or CYP2B inducers, a period of 2 weeks after cessation of exposure to the chemicals was confirmed to be sufficient to return their enzymatic activities to normal levels. Additionally, to avoid the effects of DEN on the pharmacokinetics of the test chemical, re-exposure to the test chemical started 1 week after DEN injection, in which tumor-promoting activities were clearly detected. Consequently, a modified protocol has been established with 2- and 1-week washout periods before and after DEN injection, respectively. The applicability of the modified protocol was demonstrated using the genotoxic hepatocarcinogen, estragole (ES), the genotoxic renal carcinogen, aristolochic acid (AA), and the non-genotoxic hepatocarcinogens, β-naphthoflavone and barbital. Furthermore, the increase of cell cycle-related parameters in ES-treated livers, but not in AA-treated livers, may indicate that the liver is not the carcinogenic target site of AA despite its genotoxic role. Thus, since various parameters related to carcinogenesis can be evaluated concurrently, the GPG model could be a rapid and reliable assay for the assessment of human cancer hazards.

3-Monochloropropane-1,2-diol (3-MCPD) is regarded as a rat renal and testicular carcinogen and has been classified as a possible human carcinogen (group 2B) by International Agency for Research on Cancer. This is potentially of great importance given that esters of this compound have recently found to be generated in many foods and food ingredients as a result of food process- ing. There have been a few reports about their toxicity, although we have recently found that the toxicity profile of 3-MCPD esters was similar to that of 3-MCPD in a rat 13-week repeated dose study, except for the acute renal toxicity seen in 3-MCPD-treated females. In the pre- sent study, to examine in vivo genotoxicity we adminis- tered equimolar doses of 3-MCPD or 3-MCPD fatty acid esters (palmitate diester, palmitate monoester and oleate diester) to 6-week-old male F344 gpt delta rats carrying a reporter transgene for 4 weeks by intragastric admin- istration. In vivo micronucleus, Pig-a mutation and gpt assays were performed, as well as investigations of major toxicological parameters including histopathological fea- tures. As one result, the relative kidney weights of the 3-MCPD and all three ester groups were significantly increased compared with the vehicle control group. However, the frequency of micronucleated reticulocytes and Pig-a mutant red blood cells did not differ among groups. Moreover, no changes were observed in mutant frequencies of gpt and red/gam (Spi − ) genes in the kidney and the testis of 3-MCPD and 3-MCPD-fatty-acid-esters- treated rats. In histopathological analyses, no treatment related changes were observed, except for decrease of eosinophilic bodies in the kidneys of all treated groups. These results suggest that 3-MCPD and its fatty acid esters are not in vivo genotoxins, although they may exert renal toxicity.

Ochratoxin A (OTA) is a carcinogen targeting proximal tubules at the renal outer medulla (ROM) in rodents. We previously reported that OTA increased mutant frequencies of the red/gam gene (Spi(-)), primarily deletion mutations. In the present study, Spi(-) assays and mutation spectrum analyses in the Spi(-) mutants were performed using additional samples collected in our previous study. Spi(-) assay results were similar to those in our previous study, revealing large (>1kb) deletion mutations in the red/gam gene. To clarify the molecular progression from DNA damage to gene mutations, in vivo comet assays and analysis of DNA damage/repair-related mRNA and/or protein expression was performed using the ROM of gpt delta rats treated with OTA at 70, 210 or 630 µg/kg/day by gavage for 4 weeks. Western blotting and immunohistochemical staining demonstrated that OTA increased γ-H2AX expression specifically at the carcinogenic target site. In view of the results of comet assays, we suspected that OTA was capable of inducing double-strand breaks (DSBs) at the target sites. mRNA and/or protein expression levels of homologous recombination (HR) repair-related genes (Rad51, Rad18 and Brip1), but not nonhomologous end joining-related genes, were increased in response to OTA in a dose-dependent manner. Moreover, dramatic increases in the expression of genes involved in G2/M arrest (Chek1 and Wee1) and S/G2 phase (Ccna2 and Cdk1) were observed, suggesting that DSBs induced by OTA were repaired predominantly by HR repair, possibly due to OTA-specific cell cycle regulation, consequently producing large deletion mutations at the carcinogenic target site.

Citrinin (CTN) is a food-contaminating mycotoxin that efficiently induces renal tumors in rats. However, the modes of carcinogenic action are still unknown, preventing assessment of the risks of CTN in humans. In the present study, the proliferative effects of CTN and its causal factors were investigated in the kidneys of gpt delta rats. In addition, three in vivo genotoxicity assays (reporter gene mutation using gpt delta rats and comet and micronucleus assays using F344 rats) were performed to clarify whether CTN was genotoxic in vivo. CTN was administrated at 20 and 40mg/kg/day, the higher dose being the maximal tolerated dose and a nearly carcinogenic dose. In the kidney cortex of gpt delta rats, significant increases in the labeling indices of proliferating cell nuclear antigen (PCNA)-positive cells were observed at all doses of CTN. Increases in the mRNA expression levels of Ccna2, Ccnb1, Ccne1, and its transcription factor E2f1 were also detected, suggesting induction of cell cycle progression at all tested doses of CTN. However, histopathological changes were found only in rats treated with the higher dose of CTN, which was consistent with increases in the mRNA expression levels of mitogenic factors associated with tissue damage/regeneration, such as Hgf and Lcn2, at the same dose. Thus, the proliferative effects of CTN may result not only from compensatory reactions, but also from direct mitogenic action. Western blot analysis showed that ERK phosphorylation was increased at all doses, implying that cell cycle progression may be mediated by activation of the ERK pathway. On the other hand, in vivo genotoxicity analyses were negative, implying that CTN did not have the potential for inducing DNA damage, gene mutations, or chromosomal aberrations. The overall data clearly demonstrated the molecular events underlying CTN-induced cell cycle progression, which could be helpful to understand CTN-induced renal carcinogenesis.

Ochratoxin A (OTA) is a renal carcinogen primarily affecting the S3 segment of proximal tubules in rodents. In our previous study, we reported that OTA induces reporter gene mutations, primarily deletion mutations, in the renal outer medulla (OM), specifically in the S3 segment. In the present study, to identify genes involved in OTA-induced genotoxicity, we conducted a comparative analysis of global gene expression in the renal cortex (COR) and OM of kidneys from gpt delta rats administered OTA at a carcinogenic dose for 4 weeks. Genes associated with DNA damage and DNA damage repair, and cell cycle regulation were site-specifically changed in the OM. Interestingly, genes that were deregulated in the OM possessed molecular functions such as DNA double-strand break (DSB) repair (Rad18, Brip1, and Brcc3), cell cycle progression (Cyce1, Ccna2, and Ccnb1), G(2)/M arrest in response to DNA damage (Chek1 and Wee1), and p53-associated factors (Phlda3 and Ccng1). Significant increases in the mRNA levels of many of these genes were observed in the OM using real-time RT-PCR. However, genes related to oxidative stress exhibited no differences in either the number or function of altered genes in both the OM and COR. These results suggested that OTA induced DSB and cell cycle progression at the target site. These events other than oxidative stress could trigger genotoxicity leading to OTA-induced renal tumorigenicity.

In this study, the potential for development of an animal model (GPG46) capable of rapidly detecting chemical carcinogenicity and the underlying mechanisms of action were examined in gpt delta rats using a reporter gene assay to detect mutations and a medium-term rat liver bioassay to detect tumor promotion. The tentative protocol for the GPG46 model was developed based on the results of dose-response exposure to diethylnitrosamine (DEN) and treatment with phenobarbital over time following DEN administration. Briefly, gpt delta rats were exposed to various chemicals for 4 weeks, followed by a partial hepatectomy (PH) to collect samples for an in vivo mutation assay. The mutant frequencies (MFs) of the reporter genes were examined as an indication of tumor initiation. A single intraperitoneal (ip) injection of 10 mg/kg DEN was administered to rats 18 h after the PH to initiate hepatocytes. Tumor-promoting activity was evaluated based on the development of glutathione S-transferase placental form (GST-P)-positive foci at week 10. The genotoxic carcinogens 2-acetylaminofluorene (2-AAF), 2-amino-3-methylimidazo [4,5-f] quinolone (IQ) and safrole (SF), the non-genotoxic carcinogens piperonyl butoxide (PBO) and phenytoin (PHE), the non-carcinogen acetaminophen (APAP) and the genotoxic non-hepatocarcinogen aristolochic acid (AA) were tested to validate the GPG46 model. The validation results indicate that the GPG46 model could be a powerful tool in understanding chemical carcinogenesis and provide valuable information regarding human risk hazards.

Background: Although radiation-induced bystander effects have been confirmed using a variety of endpoints, the mechanism(s) underlying these effects are not well understood, especially for in vivo study. Methods: A 1-cm2 area (1 cm × 1 cm) in the lower abdominal region of gpt delta transgenic mice was irradiated with 5 Gy of 300 keV X-rays, and changes in out-of-field lung and liver were observed. Results: Compared with sham-treated controls, the Spi− mutation frequency increased 2.4-fold in non-targeted lung tissues at 24 h after partial body irradiation (PBIR). Consistent with dramatic Cyclooxygenase 2 (COX-2) induction in the non-targeted bronchial epithelial cells, increasing levels of prostaglandin, together with 8-hydroxydeoxyguanosine, in the out-of-field lung tissues were observed after PBIR. In addition, DNA double-strand breaks and apoptosis were induced in bystander lung tissues after PBIR. Conclusion: The PBIR induces DNA damage and mutagenesis in non-targeted lung tissues, especially in bronchial epithelial cells, and COX-2 has an essential role in bystander mutagenesis.

Rats are a standard experimental animal for cancer bioassay and toxicological research for chemicals. Although the genetic analyses were behind mice, rats have been more frequently used for toxicological research than mice. This is partly because they live longer than mice and induce a wider variety of tumors, which are morphologically similar to those in humans. The body mass is larger than mice, which enables to take samples from organs for studies on pharmacokinetics or toxicokinetics. In addition, there are a number of chemicals that exhibit marked species differences in the carcinogenicity. These compounds are carcinogenic in rats but not in mice. Such examples are aflatoxin B1 and tamoxifen, both are carcinogenic to humans. Therefore, negative mutagenic/carcinogenic responses in mice do not guarantee that the chemical is not mutagenic/carcinogenic to rats or perhaps to humans. To facilitate research on in vivo mutagenesis and carcinogenesis, several transgenic rat models have been established. In general, the transgenic rats for mutagenesis are treated with chemicals longer than transgenic mice for more exact examination of the relationship between mutagenesis and carcinogenesis. Transgenic rat models for carcinogenesis are engineered mostly to understand mechanisms underlying chemical carcinogenesis. Here, we review papers dealing with the transgenic rat models for mutagenesis and carcinogenesis, and discuss the future perspective.

Currently, chemical carcinogens are classified into two groups, that is, genotoxic carcinogens and nongenotoxic carcinogens. The former are chemicals that induce tumors through genotoxic mechanisms such as mutations, while the latter are those that induce tumors through nongenotoxic mechanisms such as hormonal effects or cell toxicity. This classification has strong implications in regulation of chemicals because neither thresholds nor acceptable daily intake levels are accepted for genotoxic carcinogens. However, this regulatory policy, that is, a linear no-threshold model for genotoxic carcinogens, has been challenged recently. Humans possess a number of self-defense mechanisms, which may suppress genotoxicity of chemicals at low doses to spontaneous levels, thereby establishing practical thresholds. In addition, it is not simple to distinguish genotoxic from nongenotoxic carcinogens. In this chapter, we argue the challenges in the identification of genotoxicity of chemicals and discuss possible roles of DNA repair and translesion DNA synthesis in the practical thresholds for genotoxicity.

Oxidative stress is thought to participate in chemical carcinogenesis and may trigger gene mutations. To accurately assess the carcinogenesis risk posed to humans by chemical exposure, it is important to understand the pathways by which reactive oxygen species (ROS) are generated and the effects of the resulting oxidative stress. In the present study, p53-proficient and -deficient gpt delta mice were given pentachlorophenol (PCP), phenobarbital (PhB) or piperonyl butoxide (PBO), which are classified as non-genotoxic hepatocarcinogens in rodents, at the respective carcinogenic doses for 13 weeks. Exposure to PCP or PBO, but not PhB, invoked significant increases in liver DNA 8-hydroxydeoxyguanosine (8-OHdG) levels. Treatment with PCP significantly increased mRNA levels of the gene encoding NAD(P):quinone oxidoreductase 1 (NQO1) in the liver, suggesting that redox cycling of the PCP metabolite tetrachlorohydroquinone gave rise to ROS. Exposure to PhB or PBO significantly elevated CYP 2B10 mRNA levels while NQO1 levels were also significantly increased in PBO-treated mice. Therefore, in addition to involvement of the CYP catalytic pathway in the ROS-generated system of PBO, catechol derivatives produced from the opening of the PBO functional group methylenedioxy ring probably resulted in ROS generation. However, PCP, PBO and PhB failed to increase gpt and red/gam gene mutations in the liver independently of p53. Overall, the action of oxidative stress by ROS derived from the metabolism of these carcinogens might be limited to cancer-promoting activity, which supports the previous classification of these carcinogens as non-genotoxic. Copyright © 2012 John Wiley & Sons, Ltd.

Estragole (ES) is a natural organic compound used frequently as a flavoring food additive. Although it has been reported to be tumorigenic and induce DNA adducts in the mouse liver, there have been no reports regarding ES hepatocarcinogenicity in rats. In the current study, we therefore examined potent carcinogenicity, DNA adduct formation and in vivo genotoxicity of ES in the livers of wild and reporter gene-carrying F344 rats. Males were administered 600 mg/kg bw ES by gavage and sequentially sacrificed at weeks 4, 8 and 16 for GST-P and PCNA immunohistochemistry and measurement of ES-specific DNA adducts by LC-MS/MS in the livers. GST-P-positive foci increased with time in ES-treated rats from week 4, PCNA-labeling indices being similarly elevated at both weeks 4 and 8. ES-specific DNA adducts such as ES-3'-N(2)-dG, 3'-8-dG and 3'-N(6)-dA were consistently detected, particularly at week 4. In a second study, male F344 gpt delta rats were administered 0, 22, 66, 200 or 600 mg/kg bw ES for 4 weeks. Gpt mutant frequency in the liver was increased in a dose-dependent manner, with significance at 200 and 600 mg/kg bw in good correlation with PCNA-labeling indices. Mutation spectra analysis showed A:T to G:C transitions to be predominantly increased in line with the formation of ES-3'-N(6)-dA or 3'-8-dG. These results indicate that ES could be a possible genotoxic hepatocarcinogen in the rat, at least when given at high doses.

Kiyoji Tanaka and colleagues report mutations of UVSSA causing a third complementation group of UV-sensitive syndrome. UVSSA deficiency results in defective transcription-coupled nucleotide-excision repair and failure to resolve stalled RNA polymerase IIo at DNA damage sites.

Aristolochic acid (AA) is known to be a potent mutagen and carcinogen. Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of AA, differ from each other by a single methoxy group. However, their individual mutagenic characteristics in vivo are unclear. In the present study, we compared their DNA adduct formation and mutagenicities in the gpt delta transgenic mouse kidney. The dA-AAI, dG-AAI, dA-AAII and dG-AAII were identified in the kidney two days after intragastric administration of AAI or AAII at 5mg/kg. The concentration of DNA adducts formed by AAII was approximately 2.5-fold higher than that formed by AAI (p0.05). The mutant frequency induced by AAII was nearly two-fold higher than that induced by AAI (p0.05) following administration of 5mg/kg AAI or AAII, five times per week for six weeks. Investigation of the mutation spectra showed no statistically significant difference between AAI- and AAII-treated mice (p0.05). A:T to T:A transversion was the predominant type of mutation in both treated groups, the GC-associated mutation rates, however, differed between the AAI and AAII treatments. The in vivo metabolic pathways of AAI and AAII are different, and this may affect their mutagenicity. In the present study, we measured the levels of AAI and AAII in the kidney and plasma of gpt delta transgenic mice at multiple time points after a single intragastric dose of 1 or 5mg/kg of either component. Our results showed that the levels of AAII in both kidney and plasma were considerably higher than those of AAI (p0.01). The present study indicated that AAII showed more carcinogenic risk than AAI in vivo, and this may be, at least partly, the result of its increased levels in kidney and plasma.

This study was conducted to evaluate the effectiveness of a transgenic rat mutation assay using F344 gpt delta rats. We investigated the mutagenic potential in the lung of nickel subsulfide (Ni3S2), an insoluble fine-crystalline-metallic compound and a carcinogen in the rodent and human lung. Ni3S2 carcinogenicity has been proposed to act via both genotoxic and non-genotoxic mechanisms. Ni3S2 was intratracheally instilled into male gpt delta rats at doses of 0.5 and 1 mg/animal once a week for four weeks; these doses of Ni3S2 are high enough to induce inflammation in the lung. Following a period of 28 and 90 days after the first administration, the gpt mutant frequencies (MFs) in lung were determined in four independent laboratories, and Spi− selection for larger deletion mutations was done in one laboratory. The gpt MFs of the rats treated with Ni3S2 were not increased: all four laboratories obtained similar results with no statistical differences. The Spi− MFs were also not increased by exposure to Ni3S2. These results indicate that intratracheally instilled Ni3S2 is non-mutagenic in the lung of gpt delta transgenic rats; however, whether Ni3S2 is non-mutagenic in the lung or it induces mutations which are not detectable by transgenic rodent mutation assays requires further investigation.

1-Methylnaphthalene (1-MN), a constituent of the polycyclic aromatic hydrocarbons (PAHs), is a lung carcinogen in mice. However, conventional genotoxicity tests such as the Ames test and sister chromatid exchange (SCE) test have yielded equivocal results. In the present study, the in vivo genotoxicity of 1-methylnaphthalene (1-MN) together with its toxicological profile was investigated in a 13-week repeated dose toxicity study of 1-MN using B6C3F1 gpt delta mice. In the serum biochemistry, significant increases in AST and ALP were observed in males of the 0.15% 1-MN group. From histopathological examination, the incidence of single cell necrosis in the liver was significantly increased in males of the 0.15% 1-MN group; however, no changes were observed in the lungs, the target organ of 1-MN. In an in vivo mutation assay, no changes in mutant frequencies of gpt and red/gam (Spi-) in lung DNA of 1-MN treated mice were observed at 13 weeks. In addition, there were no significant differences in the proliferating cell nuclear antigen (PCNA)-positive ratios in bronchiolar epithelial cells among the groups for either sex. These results suggest that 1-MN at a carcinogenic dose not induce overt toxicity for any organs and has no in vivo genotoxicity in the lungs.

The transgenic rodent (TGR) assay has been widely used to study in vivo gene mutations by chemicals or radiation; however, an optimal protocol has not yet been established to assess unknown genotoxic potential. The International Workshop on Genotoxicity Testing (IWGT) strongly recommends a repeated-dose regimen for the TGR assay protocol for regulatory safety assessment as follows: a treatment period of 28 days and a sampling time of 3 days following the final treatment. In this study, TGR assays using F344 gpt delta transgenic rats were conducted at three laboratories to evaluate the validity of the IWGT protocol, as part of a collaborative study of the transgenic rat mutation assay. Male F344 gpt delta transgenic rats were orally treated with 2,4-diaminotoluene (2,4-DAT; hepatic carcinogen in rodents; 10 and 30 mg/kg/day) or 2,6-diaminotoluene (2,6-DAT; non-carcinogen in rodents; 60 mg/kg/day) once daily for 28 days. Rats were euthanized 3 days after the last dosing, and then mutant frequencies (MFs) of the gpt gene in the livers were studied. As a result, a significant increase in the MF was observed at 30 mg/kg in the 2,4-DAT-treated group, but not in the 2,6-DAT-treated group. These results were commonly observed among the three laboratories. In addition, the overall results from the three laboratories were in general agreement. These results indicate that 2,4-DAT induces gene mutation in the liver of gpt delta rats, but 2,6-DAT does not. These results also indicate that the F344 gpt delta transgenic rat mutation assay can distinguish differences in the in vivo mutagenic potential between a hepatic carcinogen and a non-carcinogen. Results from one laboratory showed more variability than those from the other two laboratories, and this appearance was due to the smaller number of colonies scored. Thus, these results demonstrate that the IWGT protocol for the TGR assays is valid, and show that consistent results are obtained among different laboratories.

Transgenic rat gene-mutation assays can be used to assess genotoxicity of chemicals in target organs for carcinogenicity. Since gene mutations in transgenes are genetically neutral and thus accumulate along with treatment periods, the assays are suitable for genotoxicity risk assessment of chemicals using repeated-dose treatment methodologies. However, few studies have been conducted to examine the suitability of the assays in repeat-dose treatment protocols. In order to prove the utility of the transgenic rat assays, we treated gpt delta rats with aristolochic acid at 0.3 and 1 mg/kg by gavage daily for 28 days, and autopsied the rats 3 days after the final treatment, which is a protocol recommended by the International Workshop on Genotoxicity Testing (IWGT). Aristolochic acid exists in herbs and some other plants, and is carcinogenic in the kidney, bladder and stomach in rats. The mutant frequency (MF) in both the kidney and the liver increased significantly in a dose-dependent manner when the rats were treated with aristolochic acid. We concluded that the gpt delta rat assay is sensitive enough to detect gene mutations induced by aristolochic acid and also that the 28-day repeated-dose protocol is suitable for assessing genotoxicity of chemicals.

Since the disaster at the Fukushima No. 1 nuclear power plant in March 2011, people became concerned about adverse eSects of radiation, in particular those of low dose radiation strongly. The eSects of radiation on chromosome DNA are stochastic events, and thus it is thought that radiation poses cancer risk to humans even at very low doses. Likewise, genotoxic compounds, which interact with DNA and induce mutations, are assumed to have no thresholds for their action. These compounds are used to be called ``radiomimetic compounds''. Hence, genotoxic carcinogens, which induce cancer via genotoxic mechanisms such as mutations, are regulated based on a paradigm that they have no thresholds for the cancer risk. Recently, however, the paradigm has been challenged by research on analyzes of carcinogenicity and genotoxicity of chemicals at low doses. In addition, organisms including humans possess various self-defense mechanisms, such as detoxication metabolism, DNA repair, error-free translesion DNA synthesis and apoptosis etc, which may suppress genotoxicity of chemicals at low doses and reduce the mutation frequency and cancer risk to spontaneous levels. These self defense mechanisms may constitute ``apparent'' or ``practical'' thresholds for genotoxic carcinogens. To discuss the low dose eSects of genotoxic and carcinogenic compounds and the implication in regulatory toxicology, the second international symposium on genotoxic and carcinogenic thresholds was held on November 23, 2011 in Tokyo. In this symposium, six and four experts of genotoxicity and chemical carcinogenicity were invited from inside and outside of Japan, respectively, to discuss genotoxicity and carcinogenicity of chemicals at low doses and the regulatory policies. This symposium follows the precedent symposia ``International symposium—threshold of carcinogenicity and genotoxicity'' in Kobe in Japan in 2006, and ``the 1st International symposium on genotoxic and carcinogenic thresholds'' in Tokyo in 2008. Here, we summarize the presentations of the symposium to discuss future perspectives of research on genotoxic and carcinogenic thresholds.

As humans are exposed to a variety of chemical agents as well as radiation, health effects of radiation should be evaluated in combination with chemicals. To explore combined genotoxic effects of radiation and chemicals, we examined modulating effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting methylating agent, against genotoxicity of γ-radiation. Human lymphoblastoid TK6 cells and its mismatch-deficient derivative, i.e., MT1 cells, were treated with MNNG for 24h before they were exposed to γ-irradiation at a dose of 1.0 Gy, and the resulting genotoxicity was examined. In TK6 cells, the pretreatments with MNNG at low doses suppressed frequencies of the thymidine kinase (TK) gene mutation and micronucleus (MN) formation induced by γ-irradiation and thus the dose responses of TK and MN assays were U-shaped along with the pretreatment doses of MNNG. In contrast, the genotoxic effects of MNNG and γ-irradiation were additive in MT1 cells and the frequencies of TK mutations and MN induction increased along with the doses of MNNG. Apoptosis induced by γ-radiation was suppressed by the pretreatments in TK6 cells, but not in MT1 cells. The expression of p53 was induced and cell cycle was delayed at G2/M phase in TK6, but not in MT1 cells, by the treatments with MNNG. These results suggest that pretreatments of MNNG at low doses suppress genotoxicity of γ-radiation in human cells and also that mismatch repair proteins are involved in the apparent adaptive responses.

Silymarin, a natural flavonoid from the seeds of milk thistle, is used for chemoprevention against various cancers in clinical settings and in experimental models. To examine the chemopreventive mechanisms of silymarin against colon cancer, we investigated suppressive effects of silymarin against carcinogenicity and genotoxicity induced by 1,2-dimethylhydrazine (DMH) plus dextran sodium sulfate (DSS) in the colon of F344 gpt delta transgenic rats. Male gpt delta rats were given a single subcutaneous injection of 40 mg/kg DMH and followed by 1.5% DSS in drinking water for a week. They were fed diets containing silymarin for 4 weeks, starting 1 week before DMH injection and samples were collected at 4, 20 and 32 weeks after the DMH treatment. Silymarin at doses of 100 and 500 p.p.m. suppressed the tumor formation in a dose-dependent manner and the reduction was statistically significant. In the mutation assays, DMH plus DSS enhanced the gpt mutant frequency (MF) in the colon, and the silymarin treatments reduced the MFs by 20%. Silymarin also reduced the genotoxicity of DMH in a dose-dependent manner in bacterial mutation assay with Salmonella typhimurium YG7108, a sensitive strain to alkylating agents, and the maximum reduction was >80%. These results suggest that silymarin is chemopreventive against DMH/DSS-induced inflammation-associated colon carcinogenesis and silymarin might act as an antigenotoxic agent, in part.

Ochratoxin A (OTA) can induce renal tumors that originate from the S3 segment of the proximal tubules in rodents, but the results of conventional mutagenicity tests have caused controversy regarding the role of genotoxic mechanisms in the carcinogenesis. Human exposure to OTA from various foods is unavoidable. Therefore, an understanding of OTA-induced renal carcinogenesis is necessary for accurate estimates of the human risk hazard. In the present study, a 13-week exposure of gpt delta rats to OTA at a carcinogenic dose induced karyomegaly and apoptosis at the outer stripe of the outer medulla (OM) of the kidney but failed to affect the reporter gene mutations in DNA extracted from whole kidneys. This site specificity resulting from the kinetics of specific transporters might be responsible for the negative outcome of in vivo mutagenicity. The kidney was then macroscopically divided, based on anatomical characteristics, into the cortex, the OM, and the inner medulla, each of which was histopathologically confirmed. Spi⁻ mutant frequencies (MFs) but not gpt MFs in the OM after a 4-week exposure to OTA were significantly higher than in controls despite the absence of cortical changes. There were also no changes in 8-hydroxydeoxyguanosine levels in kidney DNA. These results strongly suggest the involvement of a genotoxic mechanism, with the exception of oxidative DNA damage in OTA-induced renal carcinogenesis. In addition, the reporter gene mutation assay using DNA from target sites could be a more powerful tool to investigate in vivo genotoxicities.

The recent discovery that the potent carcinogen acrylamide (AA) is present in a variety of fried and baked foods raises health concerns, particularly for children, because AA is relatively high in child-favoured foods such as potato chips and French fries. To compare the susceptibility to AA-induced genotoxicity of young versus adult animals, we treated 3- and 11-week-old male gpt delta transgenic F344 rats with 0, 20, 40 or 80 p.p.m. AA via drinking water for 4 weeks and then examined genotoxicity in the bone marrow, liver and testis. We also analysed the level of N7-(2-carbamoyl-2-hydroxyethyl)-guanine (N7-GA-Gua), the major DNA adduct induced by AA, in the liver, testis and mammary gland. At 40 and 80 p.p.m., both age groups yield similar results in the comet assay in liver; but at 80 p.p.m., the bone marrow micronucleus frequency and the gpt-mutant frequency in testis increased significantly only in the young rats, and N7-GA-Gua adducts in the testis was significantly higher in the young rats. These results imply that young rats are more susceptible than adult rats to AA-induced testicular genotoxicity.

Two UV-sensitive syndrome patients who have mild photosensitivity without detectable somatic abnormalities lack detectable Cockayne syndrome group B (CSB) protein because of a homozygous null mutation in the CSB gene. In contrast, mutant CSB proteins are produced in CS-B patients with the severe somatic abnormalities of Cockayne syndrome and photosensitivity. It is known that the piggyBac transposable element derived 3 is integrated within the CSB intron 5, and that CSB-piggyBac transposable element derived 3 fusion (CPFP) mRNA is produced by alternative splicing. We found that CPFP or truncated CSB protein derived from CPFP mRNA was stably produced in CS-B patients, and that wild-type CSB, CPFP, and truncated CSB protein interacted with DNA topoisomerase I. We also found that CPFP inhibited repair of a camptothecin-induced topoisomerase I-DNA covalent complex. The inhibition was suppressed by the presence of wild-type CSB, consistent with the autosomal recessive inheritance of Cockayne syndrome. These results suggested that reduced repair of a DNA topoisomerase I-DNA covalent complex because of truncated CSB proteins is involved in the pathogenesis of CS-B.