Numerous chemicals are associated with carcinogenesis through epigenetic alterations in cells. To detect global epigenetic changes induced by carcinogens, the housekeeping gene can serve as a reporter locus, offering a baseline for identifying shifts in epigenetic marks. To investigate this potential, we developed a simple, cost-effective, and quantitative reporter system to assess chemically induced epigenetic effects, utilizing the thymidine kinase (TK) gene mutation assay as a foundation. Using a standard genotoxicity test cell line, human lymphoblast TK6, we edited the CpG promoter loci of the endogenous TK gene using the CRISPR/dCas9-SunTag-DNMT3A system. This epi-genotoxicity assay, employing modified mTK6 cells, provides a simple method for quantifying chemically induced epigenetic effects. The assay successfully detects both increased TK reversion rates induced by DNMT inhibitors, such as 5-Aza-2'-deoxycytidine and GSK-3484862, and, for the first time, a significant reduction in TK revertant frequency caused by the non-genotoxic carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA). Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment led to a global decrease in H3K27Ac levels, likely driven by TPA-mediated inflammation. These results demonstrate the utility of the epi-genotoxicity assay as a valuable tool for evaluating dual-directional epigenetic changes triggered by chemical exposure.

Sodium azide is a strong mutagen that has been successfully employed in mutation breeding of crop plants. In biological systems, it is metabolically converted to the proximate mutagen azidoalanine, which requires further bioactivation to a putative ultimate mutagen that remains elusive. The nature of the DNA modifications induced by azides leading to mutations is also unknown. Other mutagenic organic azido compounds seem to share the same bioactivation pathway to the ultimate mutagenic species as they induce point mutations dependent on the same DNA repair pathways. We investigated mutations induced by the representative mutagen 3-azido-1,2-propanediol (azidoglycerol, AZG) in the human TK6 cell line. Until now, azides have been considered to be non-mutagens and non-carcinogens in mammals, including humans, as judged only by the conventional clastogenicity chromosomal aberration types of bioassays. Here, we show the potent mutagenicity of AZG in cultured human cells, comparable to alkylating agents such as methyl methanesulfonate at concentrations with similar lethality. The potent ability of an organic azide to induce base substitutions in a mammalian system raises an alert with respect to human exposure to organic and inorganic azido compounds.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoidTK6 cells lacking XRCC1 and XPA (XRCC1(-)(/-)/XPA(-/-)), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficientTK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1(-)(/-)/XPA(-/-) cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1(-)(/-)/XPA(-/-) cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

Abstract 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.

Background Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies). Results Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay. Conclusions The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

Background Gene mutation assays in transgenic rodents are useful tools to investigate in vivo mutagenicity in a target tissue. Using a lambda EG10 transgene containing reporter genes, gpt delta transgenic mice and rats have been developed to detect point mutations and deletions. The transgene is integrated in the genome and can be rescued through an in vitro packaging reaction. However, the packaging efficiency is lower in gpt delta rats than in mice, because of the transgene in gpt delta rats being heterozygous and in low copy number. To improve the packaging efficiency, we herein describe a newly developed homozygous gpt delta rat strain. Results The new gpt delta rat has a Wistar Hannover background and has been successfully maintained as homozygous for the transgene. The packaging efficiency in the liver was 4 to 8 times higher than that of existing heterozygous F344 gpt delta rats. The frequency of gpt point mutations significantly increased in the liver and bone marrow of N -nitroso- N -ethylurea (ENU)- and benzo[ a ]pyrene (BaP)-treated rats. Spi − deletion frequencies significantly increased in the liver and bone marrow of BaP-treated rats but not in ENU-treated rats. Whole genome sequencing analysis identified ≥ 30 copies of lambda EG10 transgenes integrated in rat chromosome 1. Conclusions The new homozygous gpt delta rat strain showed a higher packaging efficiency, and could be useful for in vivo gene mutation assays in rats.

The human genome is constantly exposed to numerous environmental genotoxicants. To prevent the detrimental consequences induced by the expansion of damaged cells, cellular protective systems such as nucleotide excision repair (NER) exist and serve as a primary pathway for repairing the various helix-distorting DNA adducts induced by genotoxic agents. NER is further divided into two sub-pathways, namely, global genomic NER (GG-NER) and transcription-coupled NER (TC-NER). Both NER sub-pathways are reportedly involved in the damage response elicited by exposure to genotoxins. However, how disruption of these sub-pathways impacts the toxicity of different types of environmental mutagens in human cells is not well understood. To evaluate the role of NER sub-pathways on the cytotoxic effects of mutagens, we disrupted XPC and CSB to selectively inactivate GG-NER and TC-NER, respectively, in human lymphoblastoid TK6 cells, a standard cell line used in genotoxicity studies. Using these cells, we then comparatively assessed their respective sensitivities to representative genotoxic agents, including ultraviolet C (UVC) light, benzo [a] pyrene (B(a)P), 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), γ-ray, and 2-acetylaminofluorene (2-AAF). CSB−/− cells exhibited a hyper-sensitivity to UVC, B(a)P, and MeIQx. On the other hand, XPC−/− cells were highly sensitive to UVC, but not to B(a)P and MeIQx, compared with wild-type cells. In contrast with other genotoxins, the sensitivity of XPC−/− cells against PhIP was significantly higher than CSB−/− cells. The toxicity of γ-ray and 2-AAF was not enhanced by disruption of either XPC or CSB in the cells. Based on our findings, genetically modified TK6 cells appear to be a useful tool for elucidating the detailed roles of the various repair factors that exist to combat genotoxic agents, and should contribute to the improved risk assessment of environmental chemical contaminants.

Introduction: It is known that fibrous particles of micrometer length, such as carbon nanotubes, which have same dimensions as asbestos, are carcinogenic. Carcinogenicity of nanomaterials is strongly related to inflammatory reactions; however, the genotoxicity mechanism(s) is unclear. Indeed, inconsistent results on genotoxicity of multi-walled carbon nanotubes (MWCNTs) have been shown in several reports. Therefore, we analyzed the in vivo genotoxicity induced by an intratracheal instillation of straight MWCNTs in rats using a different test system-the Pig-a gene mutation assay-that can reflect the genotoxicity occurring in the bone marrow. Since lungs were directly exposed to MWCNTs upon intratracheal instillation, we also performed the gpt assay using the lungs.Findings: We detected no significant differences in Pig-a mutant frequencies (MFs) between the MWCNT-treated and control rats. Additionally, we detected no significant differences in gpt MFs in the lung between the MWCNT-treated and control rats.Conclusions: Our findings indicated that a single intratracheal instillation of MWCNTs was non-mutagenic to both the bone marrow and lung of rats.

The recently introduced Pig-a in vivo gene mutation assay measures endogeneous mutations of Pig-a (human, PIG-A), an X-linked gene that is conserved across species from rodents to humans. Flow cytometric analysis enables the enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient erythrocytes, resulting from a mutation in Pig-a/PIG-A, in only a few microliters of peripheral blood. Pig-a/PIG-A mutations appear to function in a neutral manner, allowing evaluation of the accumulated genotoxic effects of repeated exposures. To date, most Pig-a studies have been conducted in rodents; only a few reports regarding human applications of the PIG-A assay have been published. We have conducted a PIG-A assay in the context of human genotoxicity monitoring. Peripheral blood was collected from healthy human donors and chemotherapy-treated cancer patients at Yamagata University Hospital. To investigate the PIG-A mutant frequency (MF) induced by chemotherapy, red blood cells were analyzed via flow cytometry following staining with allophycocyanin-conjugated anti-CD235ab (erythrocyte specific) and fluorescein isothiocyanate-conjugated anti-CD59 antibodies (GPI-anchored protein specific). Reticulocyte frequencies (%RET) were also analyzed using a phycoerythrin-conjugated anti-CD71 antibody to monitor bone marrow suppression and reticulocytosis. Two of 27 patients exhibited a significantly elevated frequency of PIG-A mutants. Although we observed either a reduced or an increased %RET in all patients, no association was observed between this factor and the PIG-A MF. Unfortunately, we could not analyze blood samples collected before treatment during therapeutic processes. Additionally, the sampling time point for some patients was too short to express the PIG-A mutant phenotypes. Therefore, the possibility of natively high PIG-A MFs prior to treatment must be considered. The human PIG-A assay shows promise as a human genotoxicity monitoring method.

The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1 × 106 RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol.

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.

The Pig-a gene mutation assay, a powerful tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells. (RBCs), which are deficient in glycosylphosphatidylinositol anchored proteins caused by mutation(s) in the Pig-a gene. Various approaches for measuring cells with mutated Pig-a gene have been developed. The Pig-a assay targeting concentrated reticulocytes - the PIGRET assay - has the potential to detect genotoxicity in early stages of the study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted a joint research with the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen Society. As part of this study, we evaluated the genotoxicity of a single oral administration of acrylamide (AA) at 25, 50, 100, 137.5, and 175 mg/kg using the PIGRET and Pig-a assays targeting RBCs (RBC Pig-a assay) at 7, 14, and 28 days after dosing. Toxic effects induced by AA, such as hind limb weak-paralysis, reduction of body weight gain, and reticulocytosis, were observed in AA-treated groups. However, we detected no significant increases in Pig-a mutant frequencies using either the PIGRET or RBC Pig-a assay. Therefore, we concluded that the genotoxicity of AA could not be detected by these assays under our experimental conditions. (C) 2015 Elsevier B.V. All rights reserved.

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.

The emerging Pig-a gene mutation assay, a powerful and promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs), which are deficient in glycosylphosphatidylinositol anchored protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly those focused on peripheral RBCs and reticulocytes (RETs). Previously, it had been reported that Pig-a and gpt mutant frequencies were relatively increased in N-ethyl-N-nitrosourea (ENU)- and benzo[a]pyrene (BP)-treated mice. The capacity and characteristics of the Pig-a assay relative to transgenic rodent (TGR) mutation assays, however, are unclear in rats. Here, using transgenic gpt delta rats, we compared the in vivo genotoxicity of single oral doses of ENU (40 mg/kg) in the gpt gene mutation assay in bone marrow and liver, and Pig-a gene mutation assays on RBCs and RETs in the same animals. The Pig-a gene mutation assays were conducted at 1, 2, and 4 weeks after treatment, whereas gpt assays were conducted on tissues collected at the 4-week terminal sacrifice. Consequently, we detected that Pig-a and gpt mutant frequencies were clearly increased in ENU-treated rats, indicating that both the Pig-a and TGR gene mutation assays can detect in vivo ENU genotoxicity equally.

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20-30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.

The recently developed Pig-a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient red blood cells caused by a forward mutation in the Pig-a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig-a assay could be integrated into repeat-dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig-a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N-ethyl-N-nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4-nitroquinoline-1-oxide (4NQO, 50 mg/kg) in the Pig-a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig-a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7-week terminal sacrifice. ENU increased both Pig-a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig-a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two- or threefold above control) were detected in the bone marrow in both the Pig-a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig-a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol. Mutagen. 54:747–754, 2013. © 2013 Wiley Periodicals, Inc.

The recently developed Pig-a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient red blood cells caused by a forward mutation in the Pig-a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig-a assay could be integrated into repeat-dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig-a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N -ethyl- N -nitrosourea (ENU, 40 mg/kg), benzo[ a ]pyrene (BP, 100 and 200 mg/kg), and 4-nitroquinoline-1-oxide (4NQO, 50 mg/kg) in the Pig-a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig-a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7-week terminal sacrifice. ENU increased both Pig-a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig-a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two- or threefold above control) were detected in the bone marrow in both the Pig-a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig-a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol. Mutagen. 54:747–754, 2013. © 2013 Wiley Periodicals, Inc.

The recently developed Pig‐a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor‐deficient red blood cells caused by a forward mutation in the Pig‐a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig‐a assay could be integrated into repeat‐dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig‐a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N‐ethyl‐N‐nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4‐nitroquinoline‐1‐oxide (4NQO, 50 mg/kg) in the Pig‐a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig‐a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7‐week terminal sacrifice. ENU increased both Pig‐a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig‐a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two‐ or threefold above control) were detected in the bone marrow in both the Pig‐a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig‐a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol. Mutagen. 54:747–754, 2013. © 2013 Wiley Periodicals, Inc.