No abstract

The genotoxic and cytotoxic effects of etoposide (VP-16), a topoisomerase II inhibitor, on male rat spermatogenic cells were studied by analysing induction of micronuclei during meiosis. Micronuclei (MN) were scored in early spermatids after different time intervals corresponding to exposure of different stages of meiotic prophase. Etoposide had a strong effect on diplotene-diakinesis I cells harvested 1 day after exposure, and a significant effect also on late pachytene cells harvested 3 days after exposure. The effect at 18 days corresponding to exposure of preleptotene stage of meiosis (S-phase) was weaker but also statistically significant. Adriamycin was used as a positive control in this study. The results indicate a different mechanism of action of etoposide compared with adriamycin and other chemicals studied previously with the spermatid micronucleus test. DNA flow cytometry was carried out to assess cytotoxic damage at the same time intervals (1, 3, and 18 days after treatment) at stages I and VII of the seminiferous epithelial cycle allowing a study of cytotoxicity to different spermatogenic cell stages. Damage of differentiating spermatogonia was observed by a decrease in the cell numbers of the 2C peak 1 and 3 days after treatment and by a reduction of the number of 4C cells (primary spermatocytes) 18 d after etoposide treatment. Adriamycin also killed differentiating spermatogonia. Since the cell population which showed a high induction of MN by etoposide was not reduced in number, the genotoxic effect is remarkable. We conclude that etoposide is a potent inducer of genotoxicity and patients treated with this agent during cancer chemotherapy are at a risk of genetic damage.

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 spermatocytes 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.