COMBINED CYTOKINESIS BLOCK MICRONUCLEUS AND CHROMOSOMAL ABERRATION ASSAY FOR THE EVALUATION OF RADIOSENSITIZERS AT LOW RADIATION-DOSES

Purpose: Several methods have been tried for evaluating the efficacy o f hypoxic cell radiosensitizers at clinically relevant low radiation d oses (1-4 Gy). The cytokinesis-block micronucleus assay is known to be useful for both the in vitro and in vivo evaluation of radiosensitize rs, while the chromosomal aberration assay has been commonly used to a ssess the mutagenicity of various agents. In the present study, the ch romosomal aberration assay and the cytokinesis-block micronucleus assa y mere performed simultaneously to assess the radiosensitizing effect of etanidazole and KU-2285 at low radiation doses. The correlation bet ween the two assays was also evaluated. Methods and Materials: In vitr o 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 suspensi ons were then obtained in both studies and were used for the cytokines is-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 cell s was evaluated in the latter assay. Results: In vitro study: the sens itizer enhancement ratios of etanidazole and KU-2285 were 1.73 and 2.2 1, respectively, in the micronucleus assay, and 1.41 and 1.79 in the c hromosomal aberration assay. In vivo-in vitro study: the sensitizer en hancement ratios of etanidazole and KU-2285 were 1.18 and 1.31, respec tively, in the micronucleus assay, and 1.16 and 1.42 in the chromosoma l aberration assay. In both studies, a linear correlation was observed between the micronucleus frequency and the chromosomal aberration fre quency. The background (i.e., the frequency at 0 Gy) of the latter ass ay 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 radi osensitizers. However, a combination of the cytokinesis-block micronuc leus assay and the chromosomal aberration assay seems to be useful for assessing radiosensitizers at low radiation doses for the following r easons: a) the chromosomal aberration assay is as sensitive as the mic ronucleus assay and possibly more specific, because chromosomal aberra tions can be observed before cells pass through the first metaphase af ter irradiation and, thus, reflect quite acute damage, even though the y reflect only a part of the total damage; b) the combined assay provi des relatively more information for the time and labor required.