Yoshifumi Uno

Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody.

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay.

The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals

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.

Application of the DNA adductome approach to assess the DNA-damaging capability of in vitro micronucleus test-positive compounds

The in vitro micronucleus (MN) test is widely used for screening genotoxic compounds, but it often produces false-positive results. To consider the significance of positive results, it is important to know whether DNA adducts are formed in the cells treated with the test compound. Recently, Matsuda et al. developed the DNA adductome approach to detect DNA adducts comprehensively ([4] Kanaly, et al., Antioxid. Redox Signal., 2006, 8, 993-1001). We applied this method to assess the DNA-damaging capability of in vitro MN test-positive compounds. CHL/IU cells were treated with compounds from three categories: (1) carcinogens causing DNA alkylation, ethyl methanesulfonate and N-methyl-N-nitro-N-nitrosoguanidine; (2) carcinogens producing DNA bulky adducts, 2-amino-6-phenyl-1-methylimidazo[4,5-b]pyrene, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and 4-nitroquinoline-1-oxide, and (3) non-carcinogens, caffeine, maltol, and sodium chloride, with or without metabolic activation. With the conditions in which all test compounds gave positive results in the MN tests, DNA was extracted from the cells and hydrolyzed to deoxyribonucleosides, which were subsequently subjected to LC/ESI-MS/MS analysis. All carcinogens (categories 1 and 2) produced various DNA adduct peaks, and some of the m/z peak values corresponded to known adducts. No non-carcinogens produced DNA adducts, indicating that these compounds produced MN through different mechanisms from the adduct formation. These results indicate that the adductome approach is useful to demonstrate DNA damage formation of MN test-positive compounds and to understand their mechanisms of action.

Chromosomal damage and micronucleus induction by MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor: Evidence for a non-DNA-reactive mode of action

MP-124, a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that competes with the binding of the PARP substrate nicotinamide adenine dinucleotide (NAD), is being developed as a neuroprotective agent against acute ischemic stroke. MP-124 increased structural chromosomal aberration in CHL/IU cells, but showed negative results in the bacterial reverse mutation test, and the rat bone marrow micronucleus (MN) and the rat liver unscheduled DNA synthesis tests after the intravenous bolus injection. Thus, MP-124 did not appear to be direct-acting mutagen. Since, PARP-1 is a key enzyme in DNA repair, the effect of continuous PARP-1 inhibition by MP-124 was further examined in the rat MN test under 24-h intravenous infusion, and an increase in micronucleated immature erythrocytes (MNIE) was observed. The increase was clearly reduced by co-treatment with nicotinic acid, which resulted in increased intracellular NAD levels. This is consistent with the established activity of MP-124 as a competitive inhibitor of PARP and provides strong evidence that the DNA-damaging effect that leads to the increase in MNIE is a secondary effect of PARP-1 inhibition. This mechanism is expected to result in a threshold for the induction of MNIE by MP-124, and allows for the establishment of a safe margin of exposure for the therapeutic use of MP-124.

Evaluation of the mutagenicity of alkylating agents, methylnitrosourea and temozolomide, using the rat Pig-a assay with total red blood cells or reticulocytes

A collaborative study of the endogenous phosphatidylinositol glycan class A (Pig-a) gene mutation assay was conducted by the Japanese Environmental Mutagen Society/Mammalian Mutagenicity Study Group with a single-dosing regimen of test chemicals administered to male rats. As a part of the study, two DNA alkylating agents, methylnitrosourea (MNU) and temozolomide (TMZ), were dosed by single oral gavage at 25, 50, and 100mg/kg body weight. Pig-a mutant analysis of total red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay) was performed on Days 8, 15 and 29 after the administration. Both chemicals increased Pig-a mutants among RBCs and RETs with dose dependency on all days examined. The mutant frequencies were higher among RETs compared with RBCs, indicating that the PIGRET assay could detect mutagenicity more sensitively than the RBC Pig-a assay after a single dose of test chemicals.

Contribution to regulatory science and a next challenge of the Japanese Environmental Mutagen Society (JEMS)

Many members of The Japanese Environmental Mutagen Society (JEMS) have significantly contributed to guidelines on chemical genotoxicity. The guidelines have been useful for the hazard identification and risk assessment of genotoxic chemicals. However, risk assessors and developers of drugs and other commercial products might eliminate beneficial chemicals from further development simply based on positive results of genotoxicity testing. Experts in the field of genotoxicity should better characterize the biological significance of genotoxicants and more correctly assess human risk. I hope that one of the next challenges undertaken by JEMS will be to assess the human risk of genotoxic chemicals more correctly based on the precise analysis of their mechanisms of action.

Assessment of methyl methanesulfonate using the repeated-dose liver micronucleus assay in young adult rats.

A repeated-dose liver micronucleus assay using young adult rats was conducted with methyl methanesulfonate (MMS) as a part of a collaborative study supported by the Collaborative Study Group for the Micronucleus Test/the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. MMS is a classical DNA-reactive carcinogen, but it is not a liver carcinogen. In the first experiment (14-day study), MMS was administered per os to 6-week-old male Crl:CD (SD) rats every day for 14 days at a dose of 12.5, 25, or 50mg/kg/day. In the second experiment (28-day study), 6-week-old male SD rats were treated with MMS at 7.5, 15, or 30mg/kg/day for 28 days, because the highest dose used in the 14-day study (50mg/kg/day) caused mortality. Hepatocyte and bone marrow cell specimens were prepared on the day after the final dose. The frequency of micronucleated hepatocytes (MNHEPs) in the liver and that of micronucleated immature erythrocytes (MNIMEs) in the bone marrow were evaluated. Exposure to 50mg/kg/day MMS for 14 days resulted in an increased frequency of MNHEPs, but MMS had no effect on the frequency of MNHEPs in the rats exposed to the chemical for 28 days at doses up to 30mg/kg/day. MMS induced MNIMEs production at doses of 25 and 50mg/kg/day in the 14-day study and at doses of 15 and 30mg/kg/day in the 28-day study. Overall, the effect of MMS on the frequency of MNHEPs was considered to be equivocal.

Correlation between the results of in vitro and in vivo chromosomal damage tests in consideration of exposure levels of test chemicals

IntroductionWe examined the correlation between the results of in vitro and in vivo chromosomal damage tests by using in-house data of 18 pharmaceutical candidates that showed positive results in the in vitro chromosomal aberration or micronucleus test using CHL/IU cells, and quantitatively analyzed them especially in regard to exposure levels of the compounds.FindingsEight compounds showed that the exposure levels [maximum plasma concentration (Cmax) and AUC0-24h] were comparable with or higher than the in vitro exposure levels [the lowest effective (positive) concentration (LEC) and AUCvitrou2009=u2009LEC (μg/mL)u2009×u2009treatment time (h)]. Among them, 3 compounds were positive in the in vivo rodent micronucleus assays using bone marrow cells. For 2 compounds, cytotoxicity might produce false-positive results in the in vitro tests. One compound showed in vitro positive results only in the condition with S9 mix which indicated sufficient concentration of unidentified active metabolite(s) might not reach the bone marrow to induce micronuclei.ConclusionThese facts suggested that the in vivo exposure levels being equal to or higher than the in vitro exposure levels might be an important factor to detect in vivo chromosomal damage induced by test chemicals.