Micronucleus test using formalin-fixed rat glandular stomach and colon.

BACKGROUND: Genotoxicity in tissues other than hematopoietic tissues, such as the liver and gastrointestinal (GI) tract, is an important focus in the risk assessment of chemicals in humans. We previously developed a rat micronucleus test for the GI tract, which is the first contact tissue where chemicals are introduced into the body through oral exposure. Target cells were obtained from fresh tissue samples by ethylenediaminetetraacetic acid disodium salt (EDTA) treatment. As an improvement to this method, we have used formalin-fixed tissues instead of fresh tissues; this approach can be used for tissues that are sampled from other toxicological tests and that are archived for several years. This new method can be used for examining micronucleus induction retrospectively when needed. In the present study, we compared the performance of the EDTA method and the new method with formalin-fixed tissues (formalin-fixation method). RESULTS: Histological examination showed that both the EDTA and formalin-fixation methods could be used for collecting cells located in or above the proliferative zone of the GI tract tissues of rats. In addition, the collected cells were similar in shape. We conducted micronucleus tests with rat GI tract tissues by the two methods using model chemicals, which were used as positive control chemicals (a combination of diethylnitrosamine, 1,2-dimethylhydrazine dihydrochloride, and potassium bromate). The two methods showed similar results. We additionally evaluated the aging effect of tissues stored in formalin fixative. The results showed that 1 year of storage did not affect the frequency of micronucleated cells. CONCLUSION: The equivalence of the EDTA and formalin-fixation methods was confirmed, and micronucleus analysis was possible up to at least 1 year after formalin fixation of the GI tract, indicating that the formalin-fixation method is valuable for the rat GI tract micronucleus test.

Evaluation of a 4-day repeated-dose micronucleus test in rat glandular stomach and colon using aneugens and non-genotoxic non-carcinogens.

BACKGROUND: We previously developed a rodent gastrointestinal (GI) tract micronucleus (MN) test using the glandular stomach and/or colon, and evaluated this test method using several genotoxic carcinogens (clastogens) and genotoxic non-carcinogens; we demonstrated that this test method could detect genotoxic stomach and/or colon carcinogens with target organ specificity. In the present study, we further evaluated the sensitivity and specificity of the MN test for the rat glandular stomach and colon using three aneugens (colchicine, vinblastine sulfate, and docetaxel hydrate) and two non-genotoxic non-carcinogens (sodium chloride and sucrose). RESULTS: Male Crl:CD (SD) rats were administered test compounds through clinical administration route (orally or intravenously) for four consecutive days and then examined for the micronucleated cell frequencies in the glandular stomach and colon. We observed that all three aneugens significantly and dose-dependently increased the micronucleated cell frequencies in the stomach and colon. In contrast, neither of the two non-genotoxic non-carcinogens increased the micronucleated cell frequency in these tissues. Notably, an increase in cell proliferation was observed in the glandular stomach of rats administered a stomach toxicant, sodium chloride, but this increase did not affect the induction of micronuclei in the gastric cells. CONCLUSIONS: In the present study, it was demonstrated that the glandular stomach and colon MN tests could detect aneugens as positive and could adequately evaluate non-genotoxic non-carcinogens as negative, including a chemical that enhances cell proliferation. These results provide important evidence supporting good performance of the rat glandular stomach and colon MN tests with a 4-day treatment regimen.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS.

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.

Evaluation of a 28-day repeated-dose micronucleus test in rat glandular stomach, colon, and liver using gastrointestinal tract-targeted genotoxic-carcinogens and non-carcinogens.

The usefulness of the rat repeated-dose liver and gastrointestinal (GI) tract micronucleus (MN) tests to detect site-specific carcinogens has been shown previously using 22 chemicals in a study conducted by the Mammalian Mutagenicity Study group in the Japanese Environmental Mutagen Society. However, in the 6th International Workshop on Genotoxicity Testing, the need for further data to identify the sensitivity and specificity of the GI tract MN test and the specificity of the liver MN test, for the purpose of regulatory use, was mentioned. In the present study, we conducted additional studies to validate the performance of the 28-day repeated-dose GI tract and liver MN tests using genotoxic stomach carcinogens, N-nitroso-N-methylurea (MNU) and N-methyl-N-nitrosourethane (NMUT); genotoxic colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), and non-carcinogens, sodium chloride, sucrose, and amaranth. Male Crl:CD (SD) rats were administered with each chemical by oral gavage for 28 days and the micronucleated cell frequencies in the glandular stomach, colon, and liver were monitored. MNU and NMUT showed positive results in the glandular stomach, and PhIP did so in the colon. These carcinogens showed negative results in the liver, which is not a target organ for these chemicals. Negative results were obtained for all three non-carcinogens in the glandular stomach, colon, and liver. Therefore, it was shown that the glandular stomach and colon MN tests with 28-day repeated-dose regimen have a good sensitivity for detecting genotoxic GI tract carcinogens as positive and have a good specificity to determined non-carcinogens as negative. The negative results with these six chemicals in the liver provide additional evidence supporting the good specificity of the 28-day repeated-dose liver MN test.

Detection of Pig-a gene mutants in rat peripheral blood following a single urethane treatment: A comparison of the RBC Pig-a and PIGRET assays.

The rat red blood cell (RBC) Pig-a assay has been recommended by an expert working group of the International Workshop on Genotoxicity Testing as a potential new method to evaluate in vivo gene mutations in regulatory genotoxicity risk assessments. In a collaborative study in Japan, an improved Pig-a assay using reticulocytes (PIGRET assay) with magnetic enrichment of CD71-positive cells was evaluated, and it was revealed that this assay could detect the mutagenicity of chemicals earlier than the RBC Pig-a assay could. To verify further the suitability of the PIGRET assay for an in vivo short-term genotoxicity screening test, a joint research study was conducted by the Japanese Environmental Mutagen Society, and 24 compounds were evaluated. One of the compounds evaluated in this study was urethane, a multi-organ rodent carcinogen. Urethane (250, 500, and 1000mg/kg body weight) was orally administered once to 8-week-old male Crl:CD (SD) rats. Blood samples were collected at 1, 2, and 4 weeks after the administration and processed for the RBC Pig-a and PIGRET assays. In the PIGRET assay, the Pig-a mutant frequency (MF) significantly increased at both 2 and 4 weeks after the treatment of 1000mg/kg of urethane. However, in the RBC Pig-a assay, a significant increase in the Pig-a MF was observed only at 1 week after the treatment with 500mg/kg, but the MF value was within our historical control range; therefore, it was judged to be negative. These results suggest that the PIGRET assay might be useful for evaluating the in vivo mutagenicity more clearly than the RBC Pig-a assay after a single treatment of test compounds.

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.

Measuring reproducibility of dose response data for the Pig-a assay using covariate benchmark dose analysis.

The reproducibility of the in vivo Pig-a gene mutation test system was assessed across 13 different Japanese laboratories. In each laboratory rats were exposed to the same dosing regimen of N-nitroso-N-ethylurea (ENU), and red blood cells (RBCs) and reticulocytes (RETs) were collected for mutant phenotypic analysis using flow cytometry. Mutant frequency dose response data were analysed using the PROAST benchmark dose (BMD) statistical package. Laboratory was used as a covariate during the analysis to allow all dose responses to be analysed at the same time, with conserved shape parameters. This approach has recently been shown to increase the precision of the BMD analysis, as well as providing a measure of equipotency. This measure of equipotency was used here to demonstrate a reasonable level of interlaboratory reproducibility. Increased reproducibility could have been achieved by increasing the number of cells scored, as this would reduce the number of zero values within the mutant frequency data. Overall, the interlaboratory trial was successful, and these findings support the transferability of the in vivo Pig-a gene mutation assay.

Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone.

Repeated-dose liver and gastrointestinal tract micronucleus assays for quinoline in rats.

Repeated-dose liver, bone marrow, and gastrointestinal tract micronucleus assays that use young adult rats were evaluated in a collaborative study that was organized by the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. A genotoxic hepatocarcinogen quinoline was orally administered to independent groups of five Crl:CD (SD) male rats at doses of 30, 60 and 120mg/kg for 14 days and at doses of 15, 30 and 60mg/kg for 28 days. After treatment, the livers were harvested and hepatocytes were isolated by collagenase treatment. The frequency of micronucleated hepatocytes (MNHEPs) increased significantly in both the 14- and 28-day repeated dose studies. However, the frequency of micronucleated cells did not increase in the bone marrow, stomach or colon cells, which were not quinoline-induced carcinogenic target organs in the rats. These results indicate that a repeated-dose liver micronucleus (RDLMN) assay using young adult rats is capable of detecting the genotoxicity of quinoline at the target organ of carcinogenicity. The protocol may also permit the integration of the genotoxic endpoint into general repeated-dose toxicity studies. Furthermore, we elucidated that conducting the micronucleus assay in multiple organs could potentially assess organ specificity.

Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS).

The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.

Evaluation of a repeated dose liver micronucleus assay in rats treated with two genotoxic hepatocarcinogens, dimethylnitrosamine and 2-acetylaminofluorene: the possibility of integrating micronucleus tests with multiple tissues into a repeated dose general toxicity study.

As part of a collaborative study by the Collaborative Study Group for Micronucleus Test (CSGMT) of the Mammalian Mutagenicity Study Group (MMS) in the Japanese Environmental Mutagen Society (JEMS), the present study evaluated the effectiveness of the repeated dose liver micronucleus (RDLMN) assay. Two genotoxic hepatocarcinogens, dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2-AAF), were administered orally to male rats (6 weeks old at the initial dosing) once daily for 14 and 28 days to evaluate the micronucleus (MN) inducibility in the liver. In addition, these chemicals were evaluated for MN inducibility in the bone marrow (BM) and gastrointestinal (GI) tract, i.e. glandular stomach and colon of the same animals used in the RDLMN assay. As a result, both chemicals produced positive results in the liver, although a weak positive response was given by 2-AAF. DMN gave negative results in the tissues other than the liver. 2-AAF produced positive responses in the BM and glandular stomach, and a prominent response was particularly observed in the glandular stomach, which is directly exposed to the test chemicals by gavage. The present results suggest that the RDLMN assay is a useful method for detecting genotoxic hepatocarcinogens, and that it is especially effective for evaluating test chemicals, such as DMN, undetectable by the BM and GI tract MN assay. Moreover, the results in this investigation indicate that the use of multiple tissues in the study integrating the MN tests is more effective than using a single tissue, for detection of the MN induction produced by chemical exposure to rats, and helps to determine the characteristics of the test chemicals.

Evaluation of repeated dose micronucleus assays of the liver and gastrointestinal tract using potassium bromate: a report of the collaborative study by CSGMT/JEMS.MMS.

The food additive potassium bromate (KBrO3) is known as a renal carcinogen and causes chromosomal aberrations in vitro without metabolic activation and in vivo in hematopoietic and renal cells. As a part of a collaborative study by the Mammalian Mutagenicity Study group, which is a subgroup of the Japanese Environmental Mutagen Society, we administered KBrO3 to rats orally for 4, 14, and 28 days and examined the micronucleated (MNed) cell frequency in the liver, glandular stomach, colon, and bone marrow to confirm whether the genotoxic carcinogen targeting other than liver and gastrointestinal (GI) tract was detected by the repeated dose liver and GI tract micronucleus (MN) assays. In our study, animals treated with KBrO3 showed some signs of toxicity in the kidney and/or stomach. KBrO3 did not increase the frequency of MNed cells in the liver and colon in any of the repeated dose studies. However, KBrO3 increased the frequency of MNed cells in the glandular stomach and bone marrow. Additionally, the MNed cell frequency in the glandular stomach was not significantly affected by the difference in the length of the administration period. These results suggest that performing the MN assay using the glandular stomach, which is the first tissue to contact agents after oral ingestion, is useful for evaluating the genotoxic potential of chemicals and that the glandular stomach MN assay could be integrated into general toxicity studies.

A four-day oral treatment regimen for simultaneous micronucleus analyses in the glandular stomach, colon, and bone marrow of rats.

Our aim was to develop a multi-tissue micronucleus (MN) test method for the simultaneous analysis of rat glandular stomach, colon, and bone marrow. We have evaluated the multi-tissue MN test method with a regimen in which rats were administered chemicals orally once per day for four days and the cells of each tissue were collected 24 h after the final dose. The following compounds were studied: N-nitroso-N-methylurea (MNU), 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourethane (NMUT), 1,2-dimethylhydrazine 2HCl (DMH), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine HCl (PhIP), KBrO(3), amaranth (AM), and quercetin (QN). The gastrointestinal tract carcinogens increased the frequencies of micronucleated (MNed) cells in target tissue in a dose-dependent manner: MNU in gastric- and colonic-cells; 4NQO, MNNG, and NMUT in gastric cells; DMH and PhIP in colonic cells. In immature erythrocytes, MNU, 4NQO, DMH, and PhIP increased the frequency of MNed cells but MNNG and NMUT did not. The food additive KBrO(3), which is known to be a renal carcinogen, increased the frequencies of MNed cells in the glandular stomach and bone marrow. The food additive AM and the plant flavonoid QN, which are non-carcinogenic in most studies, did not cause increased MNed cells in any of the three tissues. Our results indicate that this multi-tissue MN test method is useful for the comprehensive evaluation of the genotoxicity of orally administered compounds.

In vivo rat glandular stomach and colon micronucleus tests: Kinetics of micronucleated cells, apoptosis, and cell proliferation in the target tissues after a single oral administration of stomach- or colon-carcinogens.

We have developed in vivo micronucleus (MN) tests by using an epithelial cell suspension isolated from the glandular stomach and colon of rodents. In the present study, our aim was to demonstrate the characteristics of the glandular stomach and colon MN tests by analyzing time-related changes in MN frequencies, apoptosis and cell proliferation in the target tissues of male CD (SD) rats that were orally administered a single dose of a stomach- or colon-targeted carcinogen, i.e., N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for the stomach and 1,2-dimethylhydrazine dihydrochloride (DMH) for the colon. After treatment, the MN frequencies significantly increased in the respective target tissues, peaking at 48-96h and decreasing afterwards. The time-response pattern could be explained by the epithelial cell turnover confirmed with a labeling experiment using the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU). In the study with MNU and DMH, we also prepared paraffin sections of the respective target tissues for the immunohistochemical evaluation of apoptosis and cell proliferation. The incidence of apoptosis increased in the early phase (6 and/or 24h) after treatment, and then decreased. Cell proliferation was depressed when a high incidence of apoptosis was observed, and then it recovered until 72h. MN frequencies increased with the recovery of cell proliferation occurring later than the peak apoptosis response. These results indicated that micronuclei were induced in the glandular stomach and colon epithelial cells by administration of the model chemicals. On the other hand, MNU induced significant increases of MNed cells in both the glandular stomach and bone marrow in the same rats, while MNNG did only in the glandular stomach when administered orally up to 1/4 of the LD50. These results suggest that the glandular stomach- and colon-MN tests would be useful for evaluating the genotoxicity of agents in the gastrointestinal tract.

Persistence and accumulation of micronucleated hepatocytes in liver of rats after repeated administration of diethylnitrosamine.

A repeat-dose micronucleus assay in adult rat liver was recently developed [Mutat. Res. 747 (2012) 234-239]. This assay demonstrated a high detectability of hepatocarcinogens at relatively low doses, as indicated by dose-dependent micronucleus induction. Because the adult rat liver is known to have a long life-span, this desirable property of the assay will be an advantage in detecting micronucleated hepatocytes (MNHEPs) that have persisted for long periods in the liver following repeated dosing. However, no data directly supporting the underlying mechanisms have been published to date. In the present study, we verified the mechanisms by means of pulse-labeling of micronucleated hepatocytes with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). The rodent hepatocarcinogen diethylnitrosamine (DEN) was repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old) for up to 2 weeks, and EdU was injected intraperitoneally on days 1, 7, or 14. Hepatocytes were isolated by use of a non-perfusion technique at 24h, 1 week, or 2 weeks after EdU injection and analyzed for EdU incorporation and micronucleus formation. The results of our study confirmed that MNHEPs labeled with EdU on the first day of DEN administration persisted until 2 weeks post-administration in the rat livers. However, the frequency of MHNEPs among EdU-labeled hepatocytes decreased over time. In addition, the number of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive cells in the liver tissue increased, suggesting selective removal of micronucleated cells. Theoretical calculation of the cumulative MNHEP frequency on each of the days on which DEN was administered, taking into account the rate of loss, came out closer to the actual value observed in the liver micronucleus test. Taken together, these results indicate that although micronucleated cells induced in rat livers by administration of the genotoxic hepatocarcinogen DEN undergo selective removal, they persist for a long time in a certain proportion, and repeated administration results in their accumulation and increased frequency.

Development of a repeated-dose liver micronucleus assay using adult rats (II): further investigation of 1,2-dimethylhydrazine and 2,6-diaminotoluene.

Detecting genotoxicity in the liver is considered an effective approach for predicting hepatocarcinogenicity, as many genotoxic chemicals in vivo may act as hepatocarcinogens in rodents. Here, a genotoxic rodent hepatocarcinogen, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), and a genotoxic (Ames positive) noncarcinogen, 2,6-diaminotolunene (2,6-DAT), were administered orally to rats for up to 28 days, and liver samples were then examined in a repeated-dose liver micronucleus (MN) assay, and additionally tested in the bone marrow (BM) MN assay concurrently. We recently established a simple method to isolate hepatocytes without in situ liver perfusion procedures, and applied this method in the liver MN assay. As a result, 1,2-DMH increased the proportion of micronucleated hepatocytes in both a dose- and duration-dependent manner at relatively low-dose levels that are routinely used in repeated-dose toxicity studies. In contrast to 1,2-DMH, 2,6-DAT did not have a detectable effect. In addition to these two chemicals, two genotoxic rodent hepatocarcinogens, diethylnitrosamine and 2,4-diaminotoluene, which gave positive responses in the liver MN assay in our previous investigation [Narumi et al., Mutat. Res. 747 (2012) 234-239], were subjected to the BM MN assay and histopathological evaluation. All four test chemicals gave negative responses in the BM MN assay. Furthermore, the three hepatocarcinogens displayed hepatotoxicity, including hepatocellular hypertrophy and anisokaryosis, but no abnormal findings were observed in the liver of rats treated with 2,6-DAT. Taken together, the present results indicate that the liver MN assay is effective for predicting hepatocarcinogenicity and may be integrated into repeated-dose toxicity studies without disturbing routine examinations, such as histopathology. Furthermore, with repeat-dose treatment protocols, our findings indicate that the liver MN assay is superior to the BM MN assay for detecting genotoxic or carcinogenic chemicals in rats.

Regulation of fowl sperm motility: evidence for the indirect, but not direct, involvement of dynein-ATPase activity on the reversible temperature-dependent immobilization.

Potential mechanisms of the reversible temperature-dependent immobilization of fowl sperm were investigated. At 30 °C, motility of demembranated fowl sperm was inhibited by adding 2 mM ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), but restored immediately after the subsequent addition of 2 mM CaCl(2), whereas at 40 °C, such additions did not appreciably affect motility (which remained almost negligible). With intact sperm, 10(-9) to 10(-3) M Ca(2+) had no effect on motility at 30 °C, which remained high. In contrast, intact sperm at 40 °C were almost immotile below 10(-5) M Ca(2+), and then gradually recovered motility at higher Ca(2+) concentrations. The negligible motility of demembranated sperm at 40 °C, and at 30 °C in the presence of EGTA, was stimulated by addition of 100 nM of the protein phosphatase inhibitor calyculin A. Dynein-ATPase activities of sperm at 40 °C in the presence of 2 mM EGTA, 2 µM CaCl(2), 2 mM CaCl(2,) or 100 nM calyculin A were higher than those at 30 °C. Therefore, stimulation of fowl sperm motility by temperature, Ca(2+), and phosphatase inhibition was not simply associated with an increase of flagellar dynein-ATPase activity. Furthermore, Ca(2+) was essential, at the axonemal level, for initiation of the 'intrinsic' motility of fowl sperm at 30 °C, but this Ca(2+)-dependent mechanism might be different from that involved in restoration of motility of intact sperm at 40 °C. In addition, perhaps inhibition of protein phosphatase activity was involved in initiation of sperm motility, but acting at a location different from Ca(2+) on the axoneme.

Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene.

Various liver micronucleus assay methods, such as those involving partial hepatectomy, treatment with mitogens, and the use of juvenile animals, have been developed. These assays have been proven to be of high sensitivity and specificity to predict hepatocarcinogenicity of compounds that cannot be detected by bone marrow micronucleus assays. On the contrary, the existing assays have only been evaluated for their use in detecting micronucleus induction in the settings of relatively short-term cell proliferation. However, the integration of in vivo genotoxicity endpoints into routine toxicity studies is increasingly desired from the viewpoint of animal welfare to reduce the number of animals used. In the present study, the rodent hepatocarcinogens diethylnitrosamine (DEN) and 2,4-diaminotoluene (2,4-DAT) were repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old at the beginning of administration) for 5, 14, and 28 days, and changes in the frequency of hepatocytes with micronuclei in liver tissues that had undergone no artificial treatment to accelerate cell proliferation were evaluated. At the same time, a new method of hepatocyte isolation involving the treatment of a portion of the liver with collagenase in a centrifuge tube, without the use of in situ perfusion, was established. The induction of micronucleated hepatocytes was achieved after the repeated administration of DEN for 5 days or longer and of 2,4-DAT for 14 days or longer. Micronucleus frequencies were increased depending on the number of administrations, indicating that micronucleated hepatocytes had possibly remained for a long period of time and accumulated additively. It therefore appears that even in adult rat liver with low mitotic activity, a repeated-dose of a chemical substance for 14 days or longer enables the detection of micronucleus induction. In addition, the establishment of a method to isolate hepatocytes without perfusion using only a part of the liver enables the integration of liver micronucleus assays into general toxicity studies.

Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR.

The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.

Temperature-dependent regulation of sperm motility of Ijima's copper pheasants (Syrmaticus soemmerringii ijimae), one of 'near threatened' species.

In order to conserve the copper pheasants, one of the Japanese 'near threatened' species, the knowledge of the sperm characteristics is the inevitable issue. Therefore, temperature-dependent regulation of copper pheasant sperm motility was investigated in comparison with that of domestic fowl spermatozoa. Motility of intact spermatozoa from both species was markedly affected by temperature. During incubation at 30 degrees C, copper pheasant spermatozoa showed around 60-70% motility, but became almost immotile when the temperature was raised to 40 degrees C. Then, when the temperature of the sperm suspension was subsequently cooled to 30 degrees C, the spermatozoa regained their motility. The motility of domestic fowl spermatozoa showed a similar pattern. Temperature also affected the motility of both demembranated copper pheasant and domestic fowl spermatozoa in the same way. The motility of intact copper pheasant and domestic fowl spermatozoa at 30 degrees C was unaffected following the addition of 2 mM CaCl(2), 100 nM calyculin A, an inhibitor of protein phosphatase-type 1 (PP1), or 4 mM diB-cAMP, respectively, compared with those with no effectors. However, the presence of 10 microM ML-7, a selective inhibitor of myosin light chain kinase (MLCK), inhibited motility of spermatozoa from both species. At 40 degrees C, the presence of CaCl(2) or calyculin A restored the motility of spermatozoa from both species, but the addition of diB-cAMP or ML-7 could not prevent the immobilization of spermatozoa. At 30 degrees C in the presence of ATP, the motility of demembranated copper pheasant spermatozoa was over 60% but was inhibited following the addition of 10 microM ML-7; a similar pattern was found with demembranated domestic fowl sperm motility. The motility of demembranated spermatozoa from both species was inhibited following the addition of 2mM EGTA to the reactivation medium at 30 degrees C, but restored by the subsequent addition of 4 mM CaCl(2). These results suggest that copper pheasant sperm motility might be regulated by similar mechanisms to that of domestic fowl spermatozoa: i.e., the balance of Ca(2+)/MLCK or an MLCK-like protein-dependent phosphorylation and PP1-dependent dephosphorylation. The similarity in physiological regulation of spermatozoa from both species shows that extensive technology developed for artificial breeding of the domestic fowl might be applicable to captive breeding of copper pheasants.