Amifostine-doxorubicin association causes long-term prepubertal spermatogonia DNA damage and early developmental arrest.

ABSTRACT

BACKGROUND: In a previous study, we found that amifostine provides some protection to the seminiferous epithelium of prepubertal doxorubicin-treated male rats but does not improve their fertility status as adults. Based on these results, a long-term study was undertaken to evaluate the DNA damage caused to spermatogonia and the consequences for embryo development. METHODS: Twenty-four male prepubertal rats (30-day-old) were divided into four equal groups and treated with doxorubicin (D--5 mg/kg), amifostine (A--400 mg/kg), amifostine/doxorubicin (AD--amifostine 15 min before doxorubicin) and control (C--0.9% saline solution). Sixty-four days after the treatment, animals were euthanized and the testes and epididymides were excised. The testes were fixed in Bouin's solution and historesin-embedded for histopathological analysis. Spermatozoa from the cauda epididymides were collected for chromatin structure analyses (Comet Assay and SCSA™). Adult rats (100-day-old) were mated with fertile females for embryo analyses on 2.5, 4.5 and 20 days post-coitum (d.p.c.). RESULTS: The seminiferous epithelium histopathology of AD group was better preserved compared with the D group. On the other hand, rats from the D and AD groups presented an increased percentage of sperm DNA strand breaks, as assessed by the comet assay, as well as an increased level of sperm chromatin denaturation, as assessed by the SCSA™ assay. In amifostine-treated groups (A and AD) there was a significant increase in the number of arrested embryos, as observed by the number of oocytes/zygotes on 2.5 d.p.c., when compared with control and doxorubicin groups; however, this number was increased when the AD group was compared with the A group. CONCLUSIONS: These results raise a concern about the effects of the association of these two drugs on the germ cell genome. Amifostine-doxorubicin-exposed rat spermatogonia produced long-term damage on sperm DNA, compromised conceptus development and reduced pregnancy outcome.