An assay to detect chemically induced DNA repair in rat spermatocytes

Abstract

An in vivo/in vitro DNA repair assay has been developed to quantitate chemically induced unscheduled DNA synthesis (UDS) in rat spermatocytes utilizing autoradiography. Male Fischer-344 rats were treated by i.p. injection or gavage with a variety of genotoxic agents dissolved in dimethyl sulfoxide, corn oil, or water. At selected times after treatment, spermatocytes were isolated by trypsin digestion of testes and cultured for 24 hr in the presence of ³H-thymidine. The direct-acting genotoxicants methyl methanesulfonate (MMS) and ethyl methanesulfonate and the chemotherapeutic agent cyclophosphamide (CPA) produced positive UDS responses in spermatocytes isolated 1 hr after i.p. injection. The UDS response evoked by either CPA or MMS was maximal within 1 hr after injection and declined rapidly thereafter to control levels. Other known genotoxicants—including dimethylnitrosamine, aflatoxin B₁, 2-acetylaminofluorene, 2,6-dinitrotoluene, and 1,6-dinitropyrene—failed to induce UDS, even with routes of administration and at times of exposure known to produce a positive response in hepatocytes. This negative response is consistent with these genotoxicants lack of mutagenic effect in rodent germ cells. These results demonstrate that the in vivo/in vitro spermatocyte DNA repair assay may be useful as a predictive screen for germ cell mutagens. Moreover, by its compatibility with similar assays which utilize other tissues from the same treated animal, this assay permits assessment of the organ specificity of the genotoxic response.