Akihiro Wakata

Report from the In Vitro Micronucleus Assay Working Group.

At the Washington International Workshop on Genotoxicity Test Procedures (March 25–26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA‐specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one‐third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis‐block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 μg/ml [lymphocytes cultured from whole blood cells] and 3–6 μg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages. Environ. Mol. Mutagen. 35:167–172, 2000

Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (Groups 1, 2A and 2B): The summary report of the 6th collaborative study by CSGMT/JEMS·MMS

To assess the correlation between micronucleus induction and human carcinogenicity, the rodent micronucleus assay was performed on known and potential human carcinogens in the 6th MMS/CSGMT collaborative study. Approximately 100 commercially available chemicals and chemical groups on which there was little or no micronucleus assay data were selected from IARC (International Agency for Research on Cancer) Groups 1 (human carcinogen), 2A (probable human carcinogen) and 2B (possible human carcinogen). As minimum requirements for the collaborative study, 5 male mice were treated by intraperitoneal injection or oral gavage once or twice with each chemical at three dose levels, and bone marrow and/or peripheral blood was analyzed. Five positives and 2 inconclusives out of 13 Group 1 chemicals, 7 positives and 5 inconclusives of 23 Group 2A chemicals, and 26 positives and 6 inconclusives of 67 Group 2B chemicals were found. Such low positive rates were not surprising because of a test chemical selection bias, and we excluded well-known micronucleus inducers. The overall evaluation of the rodent micronucleus assay was based on the present data combined with published data on the IARC carcinogens. After merging, the positive rates for Groups 1, 2A and 2B were 68.6, 54.5 and 45.6%, respectively. Structure-activity relationship analysis suggested that the micronucleus assay is more sensitive to the genetic toxicity of some classes of chemicals. Those to which it is sensitive consist of (1) aziridines and bis(2-chloroethyl) compounds; (2) alkyl sulfonate and sulfates; (3) acyl-type N-nitroso compounds; (4) hydrazines; (5) aminobiphenyl and benzidine derivatives; and (6) azo compounds. Those to which it is less sensitive consist of (1) dialkyl type N-nitroso compounds; (2) silica and metals and their compounds; (3) aromatic amines without other functional groups; (4) halogenated compounds; and (5) steroids and other hormones. After incorporation of structure-activity relationship information, the positive rates of the rodent micronucleus assay became 90.5, 65.2 and 60.0% for IARC Groups 1, 2A and 2B, respectively. Noteworthy was the tendency of the test to be more sensitive to those carcinogens with stronger evidence human carcinogenicity.

Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS·MMS

The mouse has traditionally been used for the micronucleus test, with bone marrow the usual target organ. The aim of the 9th collaborative study by CSGMT was to evaluate the suitability of the rat for the micronucleus test, with bone marrow and peripheral blood as the target organ. Since the rat spleen eliminates circulating micronucleated erythrocytes, a rat peripheral blood micronucleus assay might not be feasible.

Induction of lacZ mutation by 7,12-dimethylbenz[a]anthracene in various tissues of transgenic mice.

The induction of gene mutations was examined in MutaMouse after an intraperitoneal injection of 7, 8-dimethylbenz[a]anthracene (DMBA) at 20 mg/kg in a collaborative study participated by four laboratories. Although the DMBA dose used was lower than the level that has been reported to induce micronucleated erythrocytes maximally in several mouse strains, a killing effect appeared after day 9 of the post-treatment interval. Mutations in lacZ transgene were detected by the positive selection assay following in vitro packaging of phage lambda from the genomic DNA of the transgenic animals that survived. The mutant induction was evaluated in the bone marrow, liver, skin, colon, kidney, thymus, and testis 7 to 28 days after the treatment. In the bone marrow, the mutant frequency reached a maximum, approximately a 30-fold increase, 14 days after the treatment and the increased frequency persisted at least up to day 28 of the post-treatment. Induction of mutants was detected in the liver, colon, thymus, and skin to lesser extents. Marginal responses were obtained in the kidney and testis. The slight increases in the mutant frequencies in the kidney and testis observed in some laboratories were within laboratory-to-laboratory or animal-to-animal variations. In contrast to the gene mutation induction in the bone marrow, the frequency of micronucleated reticulocytes increased transiently 3 days after the treatment and returned to a control level before day 8 of the post-treatment. It was suggested that DMBA induced gene mutation is fixed in stem cells depending on cell proliferation while DNA damages responsible for chromosome breakage are not transmitted to progeny cells.

Evaluation of the sensitivity and specificity of in vivo erythrocyte micronucleus and transgenic rodent gene mutation tests to detect rodent carcinogens

Sensitivity and/or specificity of the in vivo erythrocyte micronucleus (MN) and transgenic rodent mutation (TGR) tests to detect rodent carcinogens and non-carcinogens were investigated. The Carcinogenicity and Genotoxicity eXperience (CGX) dataset created by Kirkland et al. was used for the carcinogenicity and in vitro genotoxicity data, i.e., Ames and chromosome aberration (CA) tests. Broad literature surveys were conducted to gather in vivo MN or TGR test data to add to the CGX dataset. Genotoxicity data in vitro were also updated slightly. Data on 379 chemicals (293 carcinogens and 86 non-carcinogens) were available for the in vivo MN test; sensitivity, specificity or concordances were calculated as 41.0%, 60.5% or 45.4%, respectively. For the TGR test, data on 80 chemicals (76 carcinogens and 4 non-carcinogens) were available; sensitivity was calculated as 72.4%. Based on the recent guidance on genotoxicity testing strategies, performance (sensitivity/specificity) of the following combinations was calculated; Ames+in vivo MN (68.7%/45.3%), Ames+TGR (83.8%/not calculated (nc)), Ames+in vitro CA+in vivo MN (80.8%/21.3%), Ames+in vitro CA+TGR (89.1%/nc), Ames+in vivo MN+TGR (87.5%/nc), Ames+in vitro CA+in vivo MN+TGR (89.3%/nc). Relatively good balance in performance was shown by the Ames+in vivo MN in comparison with Ames+in vitro CA (74.3%/37.5%). Ames+TGR and Ames+in vivo MN+TGR gave even higher sensitivity, but the specificity could not be calculated (too few TGR data on non-carcinogens). This indicates that in vivo MN and TGR tests are both useful as in vivo tests to detect rodent carcinogens.

Induction of a whole chromosome loss by colcemid in human cells elucidated by discrimination between FISH signal overlap and chromosome loss.

Aneuploidy is a change in the number of chromosomes and an essential component in tumorigenesis. Therefore, accurate and sensitive detection of aneuploidy is important in screening for carcinogens. In vitro micronucleus (MN) assay has been adopted in the recently revised International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S2 guideline and can be employed to predict both clastogenic and aneugenic chromosomal aberrations in interphase cells. However, distinguishing clastogens and aneugens is not possible using this assay. The Organization for Economic Co-operation and Development (OECD) guideline TG487 therefore recommends the use of centromere/kinetochore staining in micronuclei to differentiate clastogens from aneugens. Here, we analyzed numerical changes of a specific chromosome in cytokinesis-blocked binucleated cells by fluorescence in situ hybridization (FISH) using the specific centromere probe in human lymphoblastoid TK6 cells treated with aneugens (colcemid and vincristine) or clastogens (methyl methanesulfonate [MMS] and 4-nitroquinoline-1-oxide [4-NQO]). Colcemid and vincristine significantly increased the frequencies of nondisjunction and loss of FISH signals, while MMS and 4-NQO slightly increased only the frequency of loss of FISH signals. The loss of FISH signals of a specific chromosome from two to one per nucleus implies either a loss of a whole chromosome or an overlap of two signals. To distinguish a chromosome loss from signal overlap, we investigated the number of FISH signals and the fluorescent intensity of each signal per nucleus using a probe specific for whole chromosome 2 in binucleated TK6 cells and primary human lymphocytes treated with colcemid and MMS. By discriminating between chromosome loss and FISH signal overlap, we revealed that colcemid, but not MMS, induced a loss of a whole chromosome in primary lymphocytes and TK6 cells.

Repeated-dose liver micronucleus assay of mitomycin C in young adult rats.

The repeated-dose liver micronucleus (RDLMN) assay has been previously reported to be effective for the detection of hepatocarcinogens and suitable for general toxicology studies. A collaborative study was conducted to evaluate whether this RDLMN assay using young adult rats without collagenase perfusion of the liver can be used to detect genotoxic carcinogens. In this study, we performed the RDLMN assay in young adult rats that received intraperitoneal injections of 0.25, 0.5 or 1.0mg/kg/day of mitomycin C (MMC) for 14 and 28 days. The micronucleus induction in the bone marrow was concurrently measured, and a histopathological examination of the liver was conducted. The results revealed that the frequency of micronucleated hepatocytes (MNHEPs) was significantly increased in all of the treatment groups. However, the highest occurrence of MNHEPs was observed in the low-dose treatment group in both the 14- and the 28-day study periods. In addition, histopathological changes indicating hepatotoxicity were not observed even in the group that received the highest dose of MMC. There was no change in the frequency of metaphase hepatocytes in any of the treatment groups compared with our facilitys background data. However, the frequency of proliferating hepatocytes, as assessed by Ki-67 positivity, was decreased at the highest dose, as was the frequency of MNHEPs. Therefore, the decreased induction of MNHEPs in the high-dose groups might be explained by suppression of hepatocyte cell division. In contrast, the frequency of micronucleated immature erythrocytes in the bone marrow significantly increased in a dose-dependent manner in all of the treatment groups in both study periods. Repeated treatment of MMC induced micronuclei in the liver. These results suggest that the novel RDLMN assay can be used to detect MMC genotoxicity in the liver.

Chromosome loss caused by DNA fragmentation induced in main nuclei and micronuclei of human lymphoblastoid cells treated with colcemid.

Aneuploidy, a change in the number of chromosomes, plays an essential role in tumorigenesis. Our previous study demonstrated that a loss of a whole chromosome is induced in human lymphocytes by colcemid, a well-known aneugen. Here, to clarify the mechanism for colcemid-induced chromosome loss, we investigated the relationship between chromosome loss and DNA fragmentation in human lymphoblastoid cells treated with colcemid (an aneugen) compared with methyl methanesulfonate (MMS; a clastogen). We analyzed the number of fluorescence in situ hybridization (FISH) signals targeted for a whole chromosome 2 in cytokinesis-blocked binucleated TK6 cells and WTK-1 cells treated with colcemid and MMS, and concurrently detected DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results revealed that DNA fragmentation occurred in 60% of all binucleated TK6 cells harboring colcemid-induced chromosome loss (30% of micronuclei and 30% of main nuclei). DNA fragmentation was observed in colcemid-induced micronuclei containing a whole chromosome but not in MMS-induced micronuclei containing chromosome fragments. In contrast, colcemid-induced nondisjunction had no effect on induction of DNA fragmentation, suggesting that DNA fragmentation was triggered by micronuclei containing a whole chromosome but not by micronuclei containing chromosome fragments or nondisjunction. In addition, the frequency of binucleated cells harboring chromosome loss with DNA fragmentation in micronuclei or main nuclei was higher in wild-type p53 TK6 cells than in mutated-p53 WTK-1 cells treated with colcemid. Taken together, these present and previous results suggest that colcemid-induced chromosome loss is caused by DNA fragmentation, which is triggered by a micronucleus with a whole chromosome and controlled by the p53-dependent pathway.

Evaluation of in vivo gene mutation with etoposide using Pig-a and PIGRET assays

The Pig-a assay detects the expression of the endogenous phosphatidylinositol glycan anchor biosynthesis, class A gene (Pig-a) as a reporter of mutations and shows promise for detecting mutations in vivo. This assay requires two to four weeks to detect mutations in erythrocytes after animals are administered a single dose of test compound. In contrast, the more recently developed PIGRET assay using reticulocytes detects mutation sensitively one week after dosing, which is an advantage for conducting short-term genotoxicity studies in vivo. As part of the Japanese Environmental Mutagen Society/Mammalian Mutagenicity Study Group collaborative study, we conducted Pig-a and PIGRET assays to detect Pig-a mutations in rats treated with etoposide. The DNA topoisomerase II-inhibitor, etoposide, causes DNA cleavage and subsequent protein-linked DNA double-strand breaks, induces chromosomal aberrations and micronuclei, and is classified as 2A (probably carcinogenic to humans) by IARC. Etoposide was intravenously given once to 7-week old SD rats at doses of 0, 5, 10, and 20mg/kg. Severe myelosuppression was induced 2days after dosing in all dosing groups. We conducted Pig-a mutant analysis in Pig-a and PIGRET assays 1, 2, and 4 weeks after dosing. Neither Pig-a nor PIGRET assays showed any statistically significant increases in Pig-a mutant frequency in any of the dose groups at any of the sampling times. It was concluded that etoposide was judged negative in Pig-a and PIGRET assays of bone marrow cells, and the results of the two assays showed hardly any differences.

Prolonged rest period enables the detection of micronucleated hepatocytes in the liver of young adult rats after a single dose of diethylnitrosamine or mitomycin C.

A repeated-dose micronucleus assay utilizing young adult rat hepatocytes was recently developed to evaluate the genotoxicity. In this assay, accumulation of micronucleated hepatocytes (MNHEPs) induced by repeated dosing of genotoxic chemicals is considered to be a key factor in the detection of micronuclei induction. Then, we hypothesized that the period following chemical exposure enable the detection of MNHEP induction in young adult rats, namely that MNHEPs can be generated from chromosomally damaged cells and accumulate following initiation of chemical exposure until sampling. We therefore measured MNHEP induction at 2 or 4 weeks after a single oral administration of 12.5, 50, or 100mg/kg of diethylnitrosamine (DEN) or an intraperitoneal administration of 0.5, 1.0, or 2.0mg/kg of mitomycin C (MMC) to young adult rats. Results showed a statistically significant, dose-dependent increase in the numbers of MNHEPs in DEN- or MMC-treated rats, indicating that prolonged rest period following a single dose of a genotoxic chemical enables the detection of MNHEP induction in the liver of young adult rats. From these results, a single oral administration of 50mg/kg of DEN with a 2- or 4- week rest period can be used as a positive control in repeated-dose liver micronucleus assays. This procedure is superior in terms of labor saving and animal welfare to repeated dosing of DEN.