Comparison of the frequencies of ENU-induced point mutations in male germ cells and inherited germline mutations in their offspring.

BACKGROUND: Gene mutations induced in germ cells may be transmitted to the next generation and cause adverse effects such as genetic diseases. Certain mutations may result in infertility or death in early development. Thus, the mutations may not be inheritable. However, the extent to which point mutations in male germ cells are transmitted to the next generation or eliminated during transmission is largely unknown. This study compared mutation frequencies (MFs) in sperm of N-ethyl-N-nitrosourea (ENU)-treated gpt delta mice and de novo MFs in the whole exome/genome of their offspring. RESULTS: Male gpt delta mice were treated with 10, 30, and 85 mg/kg of ENU (i.p., weekly × 2) and mated with untreated females to generate offspring. We previously reported a dose-dependent increase in de novo MFs in the offspring estimated by whole exome sequencing (WES) (Mutat. Res., 810, 30-39, 2016). In this study, gpt MFs in the sperm of ENU-treated mice were estimated, and the MFs per reporter gene were converted to MFs per base pair. The inherited de novo MFs in the offspring (9, 26 and 133 × 10- 8/bp for 10, 30, and 85 mg/kg ENU-treated groups, respectively) were comparable to those of the converted gpt MFs in the sperm of ENU-treated fathers (6, 16, and 69 × 10- 8/bp). It indicated that the gpt MFs in the ENU-treated father's sperm were comparable to the inherited de novo MFs in the offspring as estimated by WES. In addition, de novo MFs in the offspring of 10 mg/kg ENU-treated and control fathers were estimated by whole genome sequencing (WGS), because WES was not sufficiently sensitive to detect low background MF. The de novo MF in the offspring of the ENU-treated fathers was 6 × 10- 8/bp and significantly higher than that of the control (2 × 10- 8/bp). There were no significant differences in de novo MFs between gene-coding and non-coding regions. WGS analysis was able to detect ENU-induced characteristic de novo base substitutions at a low dose group. CONCLUSIONS: Despite a difference between exome/genome and exogenous reporter genes, the results indicated that ENU-induced point mutations in male germ cells could be transmitted to the next generation without severe selection.

Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo.

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.

Transgenic rat models for mutagenesis and carcinogenesis.

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.

Dose-dependent de novo germline mutations detected by whole-exome sequencing in progeny of ENU-treated male gpt delta mice.

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.

Estimation of the frequency of inherited germline mutations by whole exome sequencing in ethyl nitrosourea-treated and untreated gpt delta mice.

BACKGROUND: Germline mutations are heritable and may cause health disadvantages in the next generation. To investigate trans-generational mutations, we treated male gpt delta mice with N-ethyl-N-nitrosourea (ENU) (85 mg/kg intraperitoneally, weekly on two occasions). The mice were mated with untreated female mice and offspring were obtained. Whole exome sequencing analyses were performed to identify de novo mutations in the offspring. RESULTS: At 20 weeks after the treatment, the gpt mutant frequencies in the sperm of ENU-treated mice were 21-fold higher than those in the untreated control. Liver DNA was extracted from six mice, including the father, mother, and four offspring from each family of the ENU-treated or untreated mice. In total, 12 DNA samples were subjected to whole exome sequencing analyses. We identified de novo mutations in the offspring by comparing single nucleotide variations in the parents and offspring. In the ENU-treated group, we detected 148 mutation candidates in four offspring and 123 (82 %) were confirmed as true mutations by Sanger sequencing. In the control group, we detected 12 candidate mutations, of which, three (25 %) were confirmed. The frequency of inherited mutations in the offspring from the ENU-treated family was 184 × 10(-8) per base, which was 17-fold higher than that in the control family (11 × 10(-8) per base). The de novo mutation spectrum in the next generation exhibited characteristic ENU-induced somatic mutations, such as base substitutions at A:T bp. CONCLUSIONS: These results suggest that direct sequencing analyses can be a useful tool for investigating inherited germline mutations and that the germ cells could be a good endpoint for evaluating germline mutations, which are transmitted to offspring as inherited mutations.

Organ specific Gst-pi expression of the metastatic androgen independent prostate cancer cells in nude mice.

BACKGROUND: Elucidating the mechanisms of metastasis in prostate cancer, particularly to the bone, is a major issue for treatment of this malignancy. We previously reported that an androgen-independent variant had higher expression of glutathione S-transferase pi (Gst-pi) compared with a parent androgen-dependent transplantable rat prostate carcinoma which was established from the transgenic rat for adenocarcinoma of the prostate (TRAP). METHODS: A new cell line, PCai1, was established from the androgen-independent tumor and investigated its metastatic potential in nude mice. The tumorigenesis of PCai1 cells in vivo was studied by subcutaneous transplantations into nude mice. The growth in the microenvironment of the prostate was studied by orthotopic transplantation of PCai1 cells into nude mice. The metastatic potential of PCai1 cells was studied by tail vein injections. Effects of Gst-pi knocked down were analysis in PCai1 cells. RESULTS: PCai1 frequently formed metastatic lesions in the lung and lymph nodes after orthotopic implantation in the prostate. Intravenous injections of PCai1, metastasis to lung and bone were obvious. PCai1 had strong expression for Gst-pi, therefore we tried knocked down Gst-pi. Gst-pi-siRNA in vitro significantly suppressed cell proliferation rate. In addition, high levels of intracellular reactive oxygen species (ROS) were recognized in the Gst-pi knockout. CONCLUSIONS: Gst-pi expression of the prostate cancers are dependent on metastatic site, and that Gst-pi has an important role in adapting prostate cancer for growth and metastasis involving an alteration of ROS signals.

Chemopreventive effects of silymarin against 1,2-dimethylhydrazine plus dextran sodium sulfate-induced inflammation-associated carcinogenicity and genotoxicity in the colon of gpt delta rats.

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.

Integration of in vivo genotoxicity and short-term carcinogenicity assays using F344 gpt delta transgenic rats: in vivo mutagenicity of 2,4-diaminotoluene and 2,6-diaminotoluene structural isomers.

An important trend in current toxicology is the replacement, reduction, and refinement of the use of experimental animals (the 3R principle). We propose a model in which in vivo genotoxicity and short-term carcinogenicity assays are integrated with F344 gpt delta transgenic rats. Using this model, the genotoxicity of chemicals can be identified in target organs using a shuttle vector lambda EG10 that carries reporter genes for mutations; short-term carcinogenicity is determined by the formation of glutathione S-transferase placenta form (GST-P) foci in the liver. To begin validating this system, we examined the genotoxicity and hepatotoxicity of structural isomers of 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT). Although both compounds are genotoxic in the Ames/Salmonella assay, only 2,4-DAT induces tumors in rat livers. Male F344 gpt delta rats were fed diet containing 2,4-DAT at doses of 125, 250, or 500 ppm for 13 weeks or 2,6-DAT at a dose of 500 ppm for the same period. The mutation frequencies of base substitutions, mainly at G:C base pairs, were significantly increased in the livers of 2,4-DAT-treated rats at all three doses. In contrast, virtually no induction of genotoxicity was identified in the kidneys of 2,4-DAT-treated rats or in the livers of 2,6-DAT-treated rats. GST-P-positive foci were detected in the livers of rats treated with 2,4-DAT at a dose of 500 ppm but not in those treated with 2,6-DAT. Integrated genotoxicity and short-term carcinogenicity assays may be useful for early identifying genotoxic and nongenotoxic carcinogens in a reduced number of experimental animals.