Evaluation of the novel liver micronucleus assay using formalin-fixed tissues.

BACKGROUND: The repeated-dose liver micronucleus (RDLMN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly for those that require metabolic activation to show genotoxicity. In a collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS), micronucleus induction of 22 chemicals with the RDLMN assay employing the collagenase digestion method was examined and reported on. Recently, we have developed a method which enables retrospective evaluation of micronucleus induction in formalin-fixed liver tissues (the formalin-fixed method) obtained in general toxicity studies completed in the past. Using this method, we were able to easily evaluate clastogenic potential of chemicals from the formalin-fixed tissues obtained in the general toxicity studies.In this study, to evaluate the usefulness of the formalin-fixed method, we have conducted a liver micronucleus assay using the formalin-fixed liver samples obtained from the above collaborative study (18 of 22 test chemicals) and carried out a comparison with the results obtained by the collagenase digestion method. RESULTS: Comparison of the collagenase digestion and formalin-fixed methods was conducted using the results of the micronucleus assays with a total of 18 test chemicals which included 12 genotoxic hepatocarcinogens (Group A), 4 genotoxic carcinogens but not liver targeted (Group B), and 2 nongenotoxic hepatocarcinogens (Group C). The formalin-fixed method obtained the similar results as the collagenase digestion method in 10 out of the 12 chemicals of Group A, and all chemicals of Group B and Group C. Although the results were statistically contradictive due to different levels of concurrent negative control, the 2 other chemicals of Group A showed comparable responses between the two methods. CONCLUSION: The present study shows that the formalin-fixed method is capable of detecting liver carcinogens with sensitivity equal to or higher than that of the collagenase digestion method. We recommend use of the formalin-fixed method because of its capability of enabling retrospective evaluation of micronucleus induction in the formalin-fixed liver tissues obtained in general toxicity studies completed in the past.

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.

Detection of micronuclei in hepatocytes isolated from young adult rats repeatedly treated with N-nitrosodi-n-propylamine.

To assess the effectiveness of the multiple dose liver micronucleus (MN) assay, the induction of micronuclei by N-nitrosodi-n-propylamine (NDPA), a genotoxic rodent carcinogen, was compared in hepatocytes (HEPs) and bone marrow (BM) cells. Young adult male rats were treated orally with NDPA at 6 weeks of age for 14 days using daily doses of 10, 20 and 40mg/kg. Samples of the liver and BM tissues were harvested from each animal one day following the last treatment with NDPA and were evaluated for the frequencies of micronucleated cells. Repeated doses with 40mg/kg/day of NDPA caused systemic and hepatic toxicity, including suppressed body weight gains and histopathological hepatic lesions. The frequencies of micronucleated HEPs were significantly increased in all the NDPA-treated groups in a dose-dependent manner. In contrast, the induction of micronuclei in the BM was undetectable, even at the high dose level of 40mg/kg, for which the inhibition of hematopoiesis was observed. For the detection of micronucleated HEPs induced by NDPA treatment, a 14-day administration period is adequate. The liver MN assay using naive young adult rats may be integrated into general repeated-dose toxicity studies including histopathological examinations. Our results suggest that the liver MN assay using multiple doses is more efficient and sensitive than the BM MN assay in detecting the in vivo genotoxic potential of NDPA.

Formation and development of maxillary first molars with delayed eruption.

Cases of congenitally missing and delayed eruption of the maxillary first molar are rare. However, in recent years, we have experienced cases of suspected delayed eruption of or congenitally missing first molars. The purpose of this study was to analyze the formation of delayed erupted maxillary first molars (M1) (>2 standard deviations), which play important roles in occlusion, and normal eruption of the maxillary first molars (U6). The frequency of M1 among patients born between 1974 and 1994 in one institution with a clear total patient number and personal oral histories was 1.55 % [80 % bilateral eruption in 8 of 806 male patients (0.99 %) and 23 of 1195 female patients (1.92 %)]. To evaluate the formation and eruption of M1 according to Moorrees's tooth formation stages, panoramic X-ray films were obtained every year for 73 patients with M1 from 3 institutions (20 male and 53 female patients, total 131 M1s) without systematic histories or genetic disorders. The development/growth curve of M1 was fitted to both the logistic curve and U6 curve. The M1 development/growth curve was started behind with U6 curve; however, the straight part of the M1 curve exhibited steep inclination compared with the straight part of the U6 curve. The curve of the eruption pathway of M1 also exhibited a sigmoid S shape. These results indicate that the development and migration speed of M1 are faster than that of U6, excluding the delayed start point. These results may help orthodontists in treatment planning for patients with M1.

Effects of male sexual maturity of reproductive endpoints relevant to DART studies in Wistar Hannover rats.

Wistar Hannover rats have been utilized as one of major strains in regulatory toxicology studies. This study was performed to verify the appropriate age of male sexual maturity in the development and reproductive toxicity (DART) study in Wistar Hannover rats (RccHan:WIST) by comparing reproductive endpoints between 8, 10 and 12 weeks of ages. Although fertility showed a tendency toward decrease in 8-week-old males, copulation index was not different among three ages. Testis weights reached a plateau at 10 weeks of age, whereas weights of other reproductive organs developed until 12 weeks of age. Indices of spermatogenesis (sperm motility, number of sperm in the epididymis and testis and contents of morphologically abnormal sperm) showed age-related progress and did not fully develop except for 12-week-old. For histology, epididymal tubules in 8-week-old animals showed immaturity with tall epithelium. At cesarean section, dams mated with 8-week-old males showed high incidence of preimplantation loss and the number of live fetuses was less than 10. In conclusion, although reproductive performance attained maturity by age of 10 weeks, spermatogenesis was not fully established at 10-week-old, which could result in a low fertility index. Therefore, we recommend that Wistar Hannover male rats at 12-week-old or older are used to conduct DART study properly and evaluate any adverse effects on dams and embryo-fetal development.

Microbiologically influenced corrosion of orthodontic metallic appliances.

Biocorrosion (microbiologically influenced corrosion; MIC) occur in aquatic habitats varying in nutrient content, temperature, stress and pH. The oral environment of organisms, including humans, should be one of the most hospitable for MIC. Corrosion of metallic appliances in the oral region is one cause of metal allergy in patients. In this study, an inductively coupled plasma-optical emission spectrometer revealed elution of Fe, Cr and Ni from stainless steel (SUS) appliances incubated with oral bacteria. Three-dimensional laser confocal microscopy also revealed that oral bacterial culture promoted increased surface roughness and corrosion pits in SUS appliances. The pH of the supernatant was lowered after co-culture of appliances and oral bacteria in any combinations, but not reached at the level of depassivation pH of their metallic materials. This study showed that Streptococcus mutans and Streptococcus sanguinis which easily created biofilm on the surfaces of teeth and appliances, did corrode orthodontic SUS appliances.

Magnetic fields from electric toothbrushes promote corrosion in orthodontic stainless steel appliances but not in titanium appliances.

Electric toothbrushes are widely used, and their electric motors have been reported to produce low-frequency electromagnetic fields that induced electric currents in metallic objects worn by the users. In this study, we showed that electric toothbrushes generated low-frequency magnetic fields (MFs) and induced electric currents in orthodontic appliances in artificial saliva (AS), which accelerated corrosion in stainless steel (SUS) appliances, but not in titanium (Ti) appliances; the corrosion was evaluated by using an inductively coupled plasma-optical emission spectrometer and a three-dimensional laser confocal microscope. The pH of AS used for appliance immersion did not change during or after MF exposure. These results suggested that MF-induced currents from electric toothbrushes could erode SUS appliances, but not Ti appliances, because of their high corrosion potentials. Further studies are required to clarify the mechanisms of metallic corrosion by induced currents in dental fields, which may trigger metal allergies in patients.

Evaluation of a liver micronucleus assay in young rats (III): a study using nine hepatotoxicants by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

We have been investigating a liver micronucleus assay to detect genotoxic chemicals using young rats for several years, and had established its advantages with respect to using autonomous proliferation of young rat hepatocytes. Nine chemicals known to induce hepatotoxic effects such as necrosis (2,6-dinitrotolune, bromobenzene, isoniazid, phenacetin, allyl alcohol and thioacetamide), cholestasis (chlorpromazine hydrochloride and alpha-naphthyl isothiocyanate) and oxidative stress (clofibrate) were selected for this study. A liver micronucleus assay was conducted in 4-week-old male F344 rats using two or three dose levels of test chemicals given orally by gavage to evaluate the compound's ability to induce micronucleated hepatocytes. Several of these test chemicals were additionally examined in a peripheral blood micronucleus assay conducted concurrently and in the same animals. The genotoxic rodent hepatocarcinogen, 2,6-dinitrotoluene showed a positive result in the liver micronucleus assay, but the nongenotoxic hepatocarcinogens, clofibrate and thioacetamide gave negative responses. Bromobenzene, known to produce DNA adducts but is noncarcinogenic in rodent liver, was judged equivocal in this assay. alpha-Naphthyl isothiocyanate is noncarcinogenic and showed negative response in the liver. The other four chemicals, known to be either noncarcinogenic or carcinogenic in other non-liver target organs, showed negative results in the liver micronucleus assay. Based on the results in the present study and previous report described above, it was concluded that this technique is able to effectively predict genotoxic rodent hepatocarcinogenicity, and does not give false positives due to hepatotoxicity.

Evaluation of a liver micronucleus assay in young rats (IV): a study using a double-dosing/single-sampling method by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

A collaborative study was conducted to evaluate whether a liver micronucleus assay using four-week-old male F344 rats can be used to detect genotoxic rat hepatocarcinogens using double-dosing with a single-sampling 4 days after the second dose. The assay methods were thoroughly validated by the seven laboratories involved in the study. Seven chemicals, 2,4-diaminotoluene, diethyl nitrosamine, p-dimethylaminoazobenzene, 1,2-dimethylhydrazine dihydrochloride, 2,4-dinitrotolunene, 2,6-dinitrotoluene and mitomycin C, known to produce positive responses in the single-dosing/triple-sampling method were selected for use in the present study, and each chemical was examined in two laboratories with the exception of 2,4-dinitrotolunene. Although several of the compounds were examined at lower doses for reasons of toxicity than in the single-dosing/triple-sampling method, all chemicals tested in the present study induced micronuclei in liver cells indicating a positive result. These findings suggest that the liver micronucleus assay can be used in young rats to detect genotoxic rat hepatocarcinogens using a double-dosing/single-sampling procedure. Further, the number of animals used in the liver micronucleus assay can be reduced by one-third to a half by using the double-dosing/single-sampling method. This reduction in animal numbers also has significant savings in time and resource for liver perfusion and hepatocyte isolation.

Collaborative work on evaluation of ovarian toxicity. 2) Two- or four-week repeated dose studies and fertility study of mifepristone in female rats.

In order to assess ovarian pathological changes and their relationship to changes in female fertility parameters, mifepristone, a progesterone receptor antagonist, was selected as the test article and was administered orally to female rats at dose levels of 0, 0.8, 4, 20 and 100 mg/kg for 2 or 4 weeks in repeated dose-toxicity studies and in a female fertility study at dose levels of 0, 0.8, 4 and 20 mg/kg from > 2 weeks before copulation to postcoital day 7. In the repeated dose toxicity studies, persistent estrus was seen in the vaginal smears, and multiple cysts in the ovaries at necropsy, increases in luteinized cysts and hypertrophy of previously formed corpora lutea were observed in the histopathological examination of ovaries in rats receiving 20 mg/kg or more for 2 or 4 weeks. In female fertility studies, persistent vaginal cornification was also observed at 20 mg/kg and the precoital interval was significantly shortened. All of the animals were completely infertile when dosed with 20 mg/kg during the post-coital period. An increase in pre-implantation losses was observed in the animals treated with 20 mg/kg during the pre-coital phase, while treatment with 4 mg/kg mifepristone during the post-coital phase induced an increase in post-implantation losses. These results suggested that a 2-week administration period would be sufficient to detect the ovarian toxicity of mifepristone in repeated dose toxicity study and the pathological findings in the ovaries would reflect the alterations in female reproductive endpoints in the female fertility study.

Evaluation of a liver micronucleus assay with 12 chemicals using young rats (II): a study by the Collaborative Study Group for the Micronucleus Test/Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group.

The partial hepatectomy method, co-treatment method with mitogens and an in vivo/in vitro assay method have been reported as in vivo liver micronucleus (MN) assays. These methods have disadvantages with respect to widespread use as an in vivo assay, i.e. they are time consuming, labour intensive and there is the possibility of interaction with the mitogens used. Therefore, we have attempted to develop a new method to overcome these disadvantages. The assay as described herein utilises the autonomous proliferation of hepatocytes of young rats. Nine chemicals have been evaluated using this method thus far. We have also assessed the sensitivity and detectability according to the following methods. A liver MN assay was performed in two strains of young rats using one or two doses of 12 chemicals to investigate the inducibility of micronucleated hepatocytes. For some of the chemicals, a peripheral blood MN assay was performed concurrently in the same animals. The following chemicals were used: diethylnitrosamine (DEN), 2-acetylaminofluorene (2AAF), 2,4-diaminotoluene (2,4-DAT), quinoline, p-dimethylaminoazobenzene (DAB), dimethylnitrosamine (DMN), ethylmethanesulphonate, 5-fluorouracil, mitomycin C (MMC), 1,2-dimethylhydrazine.2HCl, cyclophosphamide and 2,4-dinitrotoluene (2,4-DNT). The rodent hepatocarcinogens, quinoline, DAB and DMN showed positive responses in previous assays. The results of the present assay revealed new positive responses for single doses of 2AAF, 2,4-DAT, MMC, 1,2-dimethylhydrazine.2HCl and 2,4-DNT. These chemicals are known rodent hepatocarcinogens, whose clastogenicity is believed to be related to the formation of reactive metabolites generated through enzymatic activation, or the chemicals act directly. Two doses of 2AAF and DMN appeared to be more effective than a single dose in terms of MN induction. Although there were quantitative differences in the incidences of MNs, both strains of rat (F344 and SD) responded positively after treatment with DEN, DMN, 2,4-DAT, DAB, quinoline and 2AAF, suggesting that both strains are appropriate for the assay. Based on these results, it is concluded that this technique could be effective for detecting chemical clastogenicity in hepatocytes in vivo.

Evaluation of liver and peripheral blood micronucleus assays with 9 chemicals using young rats. A study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

We conducted simultaneous liver and peripheral blood micronucleus assays in young rats with seven rodent hepatocarcinogens-4,4'-methylenedianiline (MDA), quinoline, o-toluidine, 4-chloro-o-phenylenediamine (CPDA), dimethylnitrosamine (DMN), p-dimethylaminoazobenzene (DAB), and di(2-ethylhexyl)phthalate (DEHP)-and two mutagenic chemicals-kojic acid and methylmethanesulfonate (MMS). Quinoline, DMN, and DAB were positive in the liver assay, while o-toluidine, kojic acid, DAB, and MMS were positive in the peripheral blood assay. o-Toluidine, kojic acid, and DAB are reportedly negative in mouse bone marrow micronucleus assays, indicating a species difference. Our results revealed a correlation between micronucleus induction in hepatocytes and hepatocarcinogenicity. This technique can be useful for the detection of micronucleus-inducing chemicals that require metabolic activation, and it enables simultaneous comparison of the micronucleus-inducing potential of chemicals in the liver and peripheral blood in the same individual.