Etoposide (VP‐16) is a potent inducer of micronuclei in male rat meiosis: Spermatid micronucleus test and DNA flow cytometry after etoposide treatment

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.

Chromosome analysis of mouse one-cell androgenones derived from a sperm nucleus exposed to topoisomerase II inhibitors at pre- and post-fertilization stages

“…Unless sperm chromatin was adequately and timely exposed to ooplasmic topo II at the M phase, the torsional stress on DNA during chromatin remodeling would not be relieved completely, leading to strand breaks. This may explain why structural aberrations were significantly induced in paternal chromosomes when sperm nuclear development was delayed behind egg development in cross-fertilization 17 between Chinese hamster spermatozoa and Syrian hamster oocytes [42,43], and it may explain the significant increase of structural aberrations in paternal chromosomes of mouse embryos produced by delayed sperm injection into parthenogenetic eggs [44].…”

“…ICRF-193 was also clastogenic and aneugenic to mouse secondary oocytes [12]. Etoposide's ability to induce structural chromosome aberrations and aneuploidy in male meiosis has been reported in mice [13−16] and rats [17]. Furthermore, merbarone has been shown to be aneugenic to mouse spermatocytes [16,18].…”

Effects of Male Germ-Cell Stage on the Frequency, Nature, and Spectrum of Induced Specific-Locus Mutations in the Mouse

“…The topoisomerase-II inhibitor etoposide now fills this slot . While etoposide is also effective during diplotene, when it induces breaks near centromeres that result in micronucleated spermatids (La¨hdetie et al, 1994), its mutagenicity during a portion of pachytene is of particular interest because these are the stages during which events associated with recombination are taking place. Indeed, crossingover in the p-c segment of Chromosome 7 was found to be significantly decreased in offspring conceived 25-32 days after injection with etoposide (Russell et al, 2000a), corresponding to midand early pachytene, the same interval during which deletion mutations could be induced by this chemical.…”

Etoposide and merbarone are clastogenic and aneugenic in the mouse bone marrow micronucleus test complemented by fluorescence in situ hybridization with the mouse minor satellite DNA probe

“…When this analysis was conducted on bone marrow erythrocytes of mice treated with MER, the frequencies of CREST-negative MN were increased dose-dependently, indicating only a clastogenic effect [Wang and Eastmond, 2002]. In rodent spermatocytes, VP-16 and MER (80 mg/ kg) induced meiotic MN in vitro and in vivo which were analyzed in the subsequent stage of early spermatids Laehdetie, 1996, 1997;Laehdetie et al, 1994;Sjoblom et al, 1994]. Further analyses of meiotic MN induced in vivo by VP-16 (10 and 20 mg/kg) with CREST-staining and with FISH using the minor probe revealed that 58.8 -74.4% were signal-positive, depending on the treated stage of meiosis Laedetie, 1996, 1997].…”