Detection of germ cell mutagenicity of trophosphamide by the spermatid micronucleus test in the rat

The effects of the antineoplastic drug trophosphamide (TP) on male rat germ cells were examined with the spermatid micronucleus test (SMNT). We used the microdissection technique in order to isolate stage I of the seminiferous epithelial cycle, where the cells that have just passed the meiotic divisions can be found. Micronuclei (MN) were scored at different time-points after TP treatment at dose levels of 25 and 50 mg/kg. An induction of MN was detected in cells exposed at preleptotene (18 and 19 days) and late pachytene (3 days), as well as at the diplotene-diakinesis stage (1 day). The dose-response for MN induction was linear at all time intervals studied, except for 18 days time point. The highest frequency of MN (5.20 ± 0.57/1000 spermatids) could be found with the lower TP dose at 18 days, corresponding to exposed preleptotene spermatocytes and reflecting S-dependent clastogenicity. While a significant increase in MN could only be detected in exposed preleptotene spermatocytes with the lower TP dose, the higher dose level also induced MN significantly in late pachytene and the diplotene-diakinesis stage. DNA flow cytometry at 18 days showed cytotoxicity of TP to exposed primary spermatocytes at pachytene, but no cytotoxicity to the preleptotene sypermatocytes that exhibited a significant MN induction. The results show that the SMNT using the stage-I-specific examination of the rat seminiferous epithelium can detect the germ cell mutagenicity of TP and gives further evidence of the usefulness of this technique in the testing of chemicals for genotoxic effects in male germ cells.