The application of an in vitro micronucleus test in mouse fibroblast L929 cells

Abstract

The MN^a (in vitro the micronucleus assay) is recommended for studying genotoxicity of chemicals. However, no protocol is currently available for experiments with mouse fibroblast L929 cells. The aim of this study was to improve the scope of CBMN^b (cytokinesis-block micronucleus) test. Optimization consisted of: selection of a non-cytotoxic concentration of cytokinesis blocker - cytoB^c (cytochalasin B) and type and definition of the positive controls, verification of the efficacy of phenobarbital/5,6-benzoflavone as an S9 enzyme inducer as well as the identification of an optimal staining method. The compounds were tested in three exposure regimens: 6 h exposure with S9 activation followed by a 24 h recovery period, 6 h exposure followed by a 24 h recovery without metabolic activation of S9 and 30 h continuous exposure without S9. Different parameters, such as internal and interlaboratory reproducibility were investigated and criteria for test correctness were proposed. Higher MN rates were achieved using 1 μg/mL cytoB^c as a cytokinesis blocker, and MMS^d (methyl methanesulfonate), (250 μM), Col^e (colchicine), (0.5 μM) and CP^f (cyclophosphamide), (30 μM) as positive controls. In regard to the recommended S9 inducer, phenobarbital/5,6-benzoflavone was more effective as Aroclor 1254. Giemsa and acridine orange stains were optimal for the evaluation of MN formation. The protocol described in this study with L929 cells produced the reliable results and is suitable for performing the CBMN^b experiments according to the current OECD Guideline #487.

Keywords: Cytochalasin B; Cytokinesis block; Micronuclei assay; Mouse fibroblasts L929; Phenobarbital/5,6-benzoflavone.