DNA Repair Gene Expression Up-Regulation in Response to Phosphoramide Mustard Exposure in Cultured Rat Ovaries.

Abstract

Cyclophosphamide (CPA) is used as an anti-neoplastic and immunosuppressant therapy in females. Phosphoramide Mustard (PM) is the ovotoxic metabolite of CPA, and destroys rapidly dividing cells by binding covalently to DNA, inducing DNA-DNA and DNA-protein crosslinks, and DNA double-strand breaks (DSB). Previous studies have shown that PM induces destruction of primordial and small primary follicles in a dose-dependent manner in cultured rat ovaries. Interestingly, PM induced follicle destruction is preceded by the appearance of DSBs in oocytes. This study was designed to explore the expression of genes involved in DNA repair at time points prior to the appearance of PM-induced DSBs. Fisher 344 postnatal day 4 rat ovaries were cultured in medium containing vehicle control (DMSO) ± PM (30 µM) for 2 or 4 days. Three biological replicates were used to perform a real-time RT-PCR array designed to identify genes involved in DNA repair. The data were analyzed using mixed models, correcting for multiple testing by lme4 and q value packages in R software. Relative to control treated ovaries, 18 and 3 repair genes were up-regulated (q < 0.1) on days 2 and 4, respectively, in response to PM exposure. A single gene, DNA polymerase delta 3 (Pold3) was increased at both time points. These results support that the ovary up-regulates genes involved in DNA repair in response to a DNA damaging xenobiotic exposure. It is likely that repeated ovotoxic chemical exposure eventually overwhelms this protective response, resulting in loss of oocytes containing imperfect DNA. (Supported by ES016818 to AFK). (poster)