Chiharu Hattori

The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.

Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats.

Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis.

Multiple-dosing effects of benzo[a]pyrene in the mouse bone marrow micronucleus test

Multiple-dosing effects of benzo[a]pyrene (B[a]P) in the micronucleus test were studied using CD-1 male mice. Mice were treated orally once, twice or 3 times with 250, 500, 1000 or 2000 mg/kg, at 24-h intervals. Bone marrow cells were sampled 24 h after the last administration. The present study indicated that the incidence of polychromatic erythrocytes with micronuclei significantly increased more in the group of animals that received B[a]P twice than in those receiving it one or 3 times. The dose of 500 mg/kg B[a]P yielded the greatest response of any dose regimen.

Structural and numerical chromosome aberration inducers in liver micronucleus test in rats with partial hepatectomy.

The liver micronucleus test is an important method to detect pro-mutagens such as active metabolites not reaching bone marrow due to their short lifespan. We have already reported that dosing of the test compound after partial hepatectomy (PH) is essential to detect genotoxicity of numerical chromosome aberration inducers in mice [Mutat. Res. 632 (2007) 89-98]. In naive animals, the proportion of binucleated cells in rats is less than half of that in mice, which suggests a species difference in the response to chromosome aberration inducers. In the present study, we investigated the responses to structural and numerical chromosome aberration inducers in the rat liver micronucleus test. Two structural chromosome aberretion inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and two numerical chromosome aberration inducers (colchicine and carbendazim) were used in the present study. PH was performed a day before or after the dosing of the test compound in 8-week old male F344 rats and hepatocytes were isolated 4 days after the PH. As a result, diethylnitrosamine and 1,2-dimethylhydrazine, structural chromosome aberration inducers, exhibited significant increase in the incidence of micronucleated hepatocyte (MNH) when given either before and after PH. Colchicine and carbendazim, numerical chromosome aberration inducers, did not result in any toxicologically significant increase in MNH frequency when given before PH, while they exhibited MNH induction when given after PH. It is confirmed that dosing after PH is essential in order to detect genotoxicity of numerical chromosome aberration inducers in rats as well as in mice. Regarding the species difference, a different temporal response to colchicine was identified. Colchicine increased the incidence of MNH 4 days after PH in rats, although such induction in mice was observed 8-10 days after PH.

PIGRET assay can detect mutagenicity of ethyl methanesulfonate much earlier than RBC Pig-a assay.

The comparison between the original red blood cell (RBC) Pig-a assay, which measures Pig-a mutant RBCs, and the PIGRET assay, which uses reticulocytes, was conducted using in vivo mutagenesis by ethyl methanesulfonate (EMS) as a part of a collaborative study by the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen Society. Three dose levels of EMS (180, 360, and 720mg/kg) were administered once by oral gavage to 8-week-old male Crl:CD(SD) rats, and peripheral blood was sampled at 0 (1 day before dosing), 1, 2, and 4 weeks after dosing with EMS. As a result, a statistically significant increase in the mutant frequency of the Pig-a gene was observed from 2 weeks after dosing and a higher value was obtained on week 4 at the highest dose only in the RBC Pig-a assay. In the PIGRET assay, on the other hand, a statistically significant increase in Pig-a mutant frequency was obtained at the highest dose from 1 week after dosing, and it decreased on weeks 2 and 4 compared to the value at week 1. The Pig-a mutant frequency appeared to reach a plateau 1 week after dosing in the PIGRET assay and it might continue to increase even after week 4 in the RBC Pig-a assay. These results indicate that the PIGRET assay can detect Pig-a mutants much earlier than the original RBC Pig-a assay, and it can enable judgement of mutagenicity of EMS within 1 week after a single dosing.

Effect of buprenorphine on genotoxicity evaluation of chemicals by the rat liver micronucleus test with partial hepatectomy

In the view of animal welfare considerations, we investigated the suitability of modifying the rat liver micronucleus test with partial hepatectomy to include administration of an analgesic drug to minimize pain and distress as much as possible. The effects of the analgesic, buprenorphine, on the genotoxicity evaluation of structural chromosome aberration inducers (cyclophosphamide, diethylnitrosamine and 1,2-dimethylhydrazine) and numerical chromosome aberration inducers (colchicine and carbendazim) were examined. The genotoxicants were given orally to 8-week-old male F344 rats a day before or after partial hepatectomy and hepatocytes were isolated 4 days after the partial hepatectomy. Buprenorphine was injected subcutaneously twice a day with at least a 6-hr interval for 2 days from just after partial hepatectomy. As results, buprenorphine caused neither change in clinical signs (except for one animal death) nor increase in the incidence of micronucleated hepatocytes of vehicle treated animals. In the case of concomitant treatment of buprenorphine and a genotoxicant, one out of 8 animals died in each group given buprenorphine with cyclophosphamide, carbendazim or colchicine (lower dose level only). Slight changes in clinical signs were noted in the group given buprenorphine with cyclophosphamide or carbendazim. A statistically significant increase in the incidence of micronucleated hepatocytes was obtained in concomitant treatment of buprenorphine and genotoxicant compared with genotoxicant alone for 1,2-dimethylhydrazine, colchicine and carbendazim. It is concluded that use of buprenorphine as an analgesic drug to minimize pain and distress for rats that are given partial hepatectomy is not appropriate under the present experimental conditions, because it could enhance the general toxicity and genotoxicity of the test chemical.

Gender differences in the liver micronucleus test in rats with partial hepatectomy

The liver micronucleus test in rats with partial hepatectomy is a useful method to detect pro-clastogens such as diethylnitrosamine, the active metabolites of which do not reach the bone marrow due to their short lifespan. We have already reported that structural or numerical chromosome aberration inducers should be given before or after partial hepatectomy, respectively, to detect genotoxicity in the liver of rats. In the present study, we found that the percentage of binucleated cells in the liver from naive male rats is approximately 60% of that in female rats, which suggests a gender difference in the response to chromosome aberration inducers. Therefore, we investigated the responses to structural chromosome aberration inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and numerical chromosome aberration inducers (colchicine and carbendazim) in male and female rats. The chemicals were given to 8-week-old male and female F344 rats a day before or after partial hepatectomy and hepatocytes were isolated 4 days after the partial hepatectomy. As the results, diethylnitrosamine and 1,2-dimethylhydrazine produced a significant increase in the frequency of micronucleated hepatocytes in both genders and the responses were comparable. In the case of colchicine and carbendazim, higher frequencies in the micronucleated hepatocytes were obtained in males than in females. Taken together, the response to chromosome aberration inducers in male rats was equal to or stronger than that in female rats. It seems that the use of only male rats in the liver micronucleus test is sufficient, unless existing data indicate a toxicologically meaningful gender difference in rats.

Evaluation of in vivo mutagenicity of isopropyl methanesulfonate by RBC Pig-a and PIGRET assays

A comparison between the original red blood cell (RBC) Pig-a assay, which measures Pig-a mutant cells in RBCs, and the PIGRET assay, which uses reticulocytes, was conducted using the in vivo mutagenesis assay with isopropyl methanesulfonate (iPMS) as a part of a collaborative study by the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen Society. Three dose levels of iPMS (50, 100, and 200mg/kg) were administered once intraperitoneally to 8-week-old male Crl:CD(SD) rats, and peripheral blood was sampled at 0 (1 day before dosing), and 1, 2, and 4 weeks after dosing with iPMS. As a result, a time-dependent increase in the mutant frequency of Pig-a mutant RBCs was observed in the RBC Pig-a assay, and a statistically significant increase was observed from 2 weeks after dosing. In the PIGRET assay, on the other hand, a statistically significant increase in Pig-a mutant frequency was obtained from 1 week after dosing at all dose levels, and the Pig-a mutant frequency at the highest dose level had already reached a plateau on week 1. The maximum Pig-a mutant frequency induced by a single treatment with iPMS at 200mg/kg in the PIGRET assay was approximately two times higher than that in the RBC Pig-a assay. These results indicate that the PIGRET assay can detect Pig-a mutants much earlier and with a higher value in Pig-a mutant frequency compared with the original RBC Pig-a assay, and it can enable judgement of mutagenicity of iPMS within 1 week after a single dose.

Assessment of a twice dosing regimen both before and after partial hepatectomy in the rat liver micronucleus test

The liver micronucleus test is an important method to detect in vivo genotoxicants, especially those that require metabolic activation for their genotoxicity. We have already reported that structural or numerical chromosome aberration inducers have to be given before or after partial hepatectomy, respectively, to detect their genotoxicity in the liver of rats. In the present study, we assessed a twice dosing regimen, in which the genotoxicant is dosed both before and after partial hepatectomy, using the four chromosome aberration inducers used in the previous study. Two structural chromosome aberration inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and two numerical chromosome aberration inducers (colchicine and carbendazim) were used. The genotoxicant was administered to 8-week old male F344 rats one day before and again one day after the partial hepatectomy and hepatocytes were isolated 3 days after second dosing (4 days after the partial hepatectomy). As a result, all genotoxicants (structural or numerical chromosome aberration inducers) caused a dose-dependent statistically significant increase in the incidence of micronucleated hepatocytes when given both before and after partial hepatectomy. No marked difference was observed in general toxicity, relative liver weight and cell classification between single dosing regimens and twice dosing regimen of the genotoxicants. These results confirm that the twice dosing regimen, in which the test compound is dosed both before and after partial hepatectomy, can detect in vivo induction of micronucleated hepatocytes by structural or numerical chromosome aberration inducers qualitatively similar to their appropriate regimen in which the test compound is administered either before or after partial hepatectomy.

In vivo genotoxicity of 1,4-dioxane evaluated by liver and bone marrow micronucleus tests and Pig-a assay in rats

1,4-Dioxane, used widely as a solvent in the manufacture of chemicals and as a laboratory reagent, induced liver adenomas and carcinomas in mice and rats, and nasal tumors in rats in several long-term studies. 1,4-Dioxane has been reported to be non-genotoxic in vitro, and there is no clear conclusion concerning its in vivo genotoxicity in rodents. In the present study, we investigated the ability of 1,4-dioxane to induce micronuclei in the liver and bone marrow of rats. For the liver micronucleus test, we performed the juvenile animal method and two methods using partial hepatectomy (PH), dosing before PH or dosing after PH. We also evaluated the in vivo mutagenicity of 1,4-dioxane by Pig-a gene mutation assay using rat peripheral blood. As a result, all methods of liver micronucleus test showed an increase in the frequency of micronucleated hepatocytes by 1,4-dioxane. The dosing before PH, a suitable method for detecting structural chromosome aberration inducers, showed the clearest response for micronucleated hepatocytes induction among the three methods. This finding is consistent with a previous report that 1,4-dioxane induces mainly chromosome breakage in the liver. Negative results were obtained in the bone marrow micronucleus test and Pig-a gene mutation assay in our study. These results suggested that 1,4-dioxane is clastogenic in the liver but not genotoxic in the bone marrow of rats.