griseofulvin and Leukemia-L5178

In this report, results are presented from an international study of the in vitro micronucleus assay using mouse lymphoma L5178Y cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, 5-fluorouracil, colchicine and griseofulvin. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in mouse lymphoma L5178Y cells. Mannitol was the only exception, being tested in only one laboratory. Mannitol was negative, while bleomycin induced a concentration-dependent increase in micronucleated cells. Equivocal results were obtained for 5-fluorouracil, colchicine and griseofulvin. High levels of cytotoxicity interfered with the assessment of aneuploidy for colchicine and griseofulvin, preventing the ability to obtain clear results in all the treatment schedules. Experiments with 5-fluorouracil, colchicine and griseofulvin showed that both short and long treatment times are required as each compound was detected using one or more treatment protocol. No clear differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. It was also found that a recovery period may help to detect compounds which induce a genotoxicity associated to a reduction in cell number or cell proliferation. Overall, the results of the present study show that mouse lymphoma L5178Y cells are suitable for the in vitro micronucleus assay.

Topics: Aneugens; Animals; Bleomycin; Colchicine; Fluorouracil; Griseofulvin; In Vitro Techniques; International Cooperation; Leukemia L5178; Mannitol; Mice; Micronucleus Tests; Mutagens

This study, coordinated by the SFTG (French branch of European Environmental Mutagen Society), included 38 participants from Europe, Japan and America. Clastogens (bleomycin, urethane), including base and nucleoside analogs (5-fluorouracil and cytosine arabinoside), aneugens and/or polyploidy inducers (colchicine, diethylstilboestrol, griseofulvin and thiabendazole), as well as non-genotoxic compounds (mannitol and clofibrate), were tested. Four cell types were used, i.e. human lymphocytes in the presence of cytochalasin B and CHO, CHL and L5178Y cell lines, in the presence or absence of cytochalasin B, with various treatment-recovery schedules. Mitomycin C was used as a positive control for all cell types. Mannitol and clofibrate were consistently negative in all cell types and with all treatment-recovery conditions. Urethane, known to induce questionable clastogenicity, was not found as positive. Bleomycin and mitomycin C were found positive in all treatment-recovery conditions. The base and nucleoside analogs were less easy to detect, especially 5-fluorouracil due to the interference with cytotoxicity, while cytosine arabinoside was detected in all cell types depending on the treatment-recovery schedule. Aneugens (colchicine, diethylstilboestrol and griseofulvin) were all detected in all cell types. In this study, the optimal detection was ensured when a short treatment followed by a long recovery was associated with a long continuous treatment without recovery. There was no impact of the presence or absence of cytochalasin B on the detection of micronucleated cells on cell lines. Scoring micronucleated cells in both mononucleated and binucleated cells when using cytochalasin B was confirmed to be useful for the detection and the identification of aneugens. In conclusion, these results, together with previously published validation studies, provide a useful contribution to the optimisation of a study protocol for the detection of both clastogens and aneugens in the in vitro micronucleus test.

Topics: Aneugens; Animals; Bleomycin; Cell Line; CHO Cells; Clofibrate; Colchicine; Cricetinae; Cytarabine; Cytochalasin B; Diethylstilbestrol; Fluorouracil; Griseofulvin; Humans; In Vitro Techniques; International Cooperation; Leukemia L5178; Lymphocytes; Mannitol; Mice; Micronucleus Tests; Mitomycin; Mutagens; Thiabendazole

This study was designed to investigate a previously unidentified potential mechanism for mutation induction as well as to clarify a biological consequence of micronuclei with mutation induction as measured by trifluorothymidine (TFT) resistance in mouse L5178Y cells using four aneugens: colcemid, diethylstilbestrol, griseofulvin and vinblastine. All four compounds induced micronuclei which appeared in the first cell cycle after treatment. More than 85% of the micronuclei induced by each compound stained positive for the presence of kinetochores implying that the micronuclei contained whole chromosomes. However, these same compounds were unable to induce TFT resistance under three different treatment regimes. We concluded that these compounds, under conditions where they induce primarily kinetochore positive micronuclei, were not able to induce mutations. Thus, the induction of micronuclei containing whole chromosomes harboring a selectable gene is not an early event leading to mutations in these cells.

Topics: Animals; Demecolcine; Diethylstilbestrol; Drug Resistance; Griseofulvin; Kinetochores; Leukemia L5178; Mice; Micronuclei, Chromosome-Defective; Micronucleus Tests; Mutagenesis; Mutagens; Thymidine Kinase; Trifluridine; Tumor Cells, Cultured; Vinblastine