Combined cytokinesis-block micronucleus and chromosomal aberration assay for the evaluation of radiosensitizers at low radiation doses

Abstract

Purpose : Several methods have been tried for evaluating the efficacy of hypoxic cell radiosensitizers at clinically relevant low radiation doses (1–4 Gy). The cytokinesis-block micronucleus assay is known to be useful for both the in vitro and in vivo evaluation of radiosensitizers, while the chromosomal aberration assay has been commonly used to assess the mutagenicity of various agents. In the present study, the chromosomal aberration assay and the cytokinesis-block micronucleus assay were performed simultaneously to assess the radiosensitizing effect of etanidazole and KU-2285 at low radiation doses. The correlation between the two assays was also evaluated. Methods and Materials : In vitro study: EMT-6 cells were irradiated at a dose of 1–3 Gy under hypoxic conditions with or without the drugs at 1 mM. In vivo-in vitro study: EMT-6 tumor-bearing BALB/c mice received 2–4 Gy of radiation with or without administration of the drugs at 200 mg/kg. Single-cell suspensions were then obtained in both studies and were used for the cytokinesis-block micronucleus assay and the chromosomal aberration assay. The micronucleus frequency in binucleate cells was evaluated in the former assay, and the frequency of chromosomal aberrations in metaphase cells was evaluated in the latter assay. Results : In vitro study: the sensitizer enhancement ratios of etanidazole and KU-2285 were 1.73 and 2.21, respectively, in the micronucleus assay, and 1.41 and 1.79 in the chromosomal aberration assay. In vivo-in vitro study: the sensitizer enhancement ratios of etanidazole and KU-2285 were 1.18 and 1.31, respectively, in the micronucleus assay, and 1.16 and 1.42 in the chromosomal aberration assay. In both studies, a linear correlation was observed between the micronucleus frequency and the chromosomal aberration frequency. The background (i.e., the frequency at 0 Gy) of the latter assay was considerably lower than that of the former assay, especially in the in vivo study. Conclusions : The chromosomal aberration assay has not yet been established as a method for evaluating the effect of radiosensitizers. However, a combination of the cytokinesis-block micronucleus assay and the chromosomal aberration assay seems to be useful for assessing radiosensitizers at low radiation doses for the following reasons: a) the chromosomal aberration assay is as sensitive as the micronucleus assay and possibly more specific, because chromosomal aberrations can be observed before cells pass through the first metaphase after irradiation and, thus, reflect quite acute damage, even though they reflect only a part of the total damage; b) the combined assay provides relatively more information for the time and labor required.