title: Ovarian xenobiotic biotransformation enzymes are altered during phosphoramide mustard-induced ovotoxicity.
abstract: The anti-neoplastic prodrug, cyclophosphamide, requires biotransformation to phosphoramide mustard (PM), which partitions to volatile chloroethylaziridine (CEZ). PM and CEZ are ovotoxicants, however their ovarian biotransformation remains ill-defined. This study investigated PM and CEZ metabolism mechanisms through the utilization of cultured postnatal day 4 (PND4) Fisher 344 (F344) rat ovaries exposed to vehicle control (1% dimethyl sulfoxide (DMSO)) or PM (60μM) for 2 or 4 days. Quantification of mRNA levels via an RT(2) profiler PCR array and target-specific RT-PCR along with Western blotting found increased mRNA and protein levels of xenobiotic metabolism genes including microsomal epoxide hydrolase (Ephx1) and glutathione S-transferase isoform pi (Gstp). PND4 ovaries were treated with 1% DMSO, PM (60μM), cyclohexene oxide to inhibit EPHX1 (CHO; 2mM), or PM + CHO for 4 days. Lack of functional EPHX1 increased PM-induced ovotoxicity, suggesting a detoxification role for EPHX1. PND4 ovaries were also treated with 1% DMSO, PM (60μM), BSO (Glutathione (GSH) depletion; 100μM), GEE (GSH supplementation; 2.5mM), PM ± BSO, or PM ± GEE for 4 days. GSH supplementation prevented PM-induced follicle loss, whereas no impact of GSH depletion was observed. Lastly, the effect of ovarian GSH on CEZ liberation and ovotoxicity was evaluated. Both untreated and GEE-treated PND4 ovaries were plated adjacent to ovaries receiving PM + GEE or PM + BSO treatments. Less CEZ-induced ovotoxicity was observed with both GEE and BSO treatments indicating reduced CEZ liberation from PM. Collectively, this study supports ovarian biotransformation of PM, thereby influencing the ovotoxicity that ensues.
authors: Department of Animal Science, Iowa State University, Ames, Iowa 50011.
authors: Jill A
authors: JA
authors: Madden
authors: Department of Animal Science, Iowa State University, Ames, Iowa 50011 akeating@iastate.edu.
authors: Aileen F
authors: AF
authors: Keating
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity*; D001711:Biotransformation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005978:Glutathione / Q000378:metabolism; D010053:Ovary / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D010752:Phosphoramide Mustards / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism; D011916:Rats, Inbred F344; D013997:Time Factors; D046509:Tissue Culture Techniques; D015262:Xenobiotics / Q000378:metabolism*
chemical_list: D018906:Antineoplastic Agents, Alkylating; D004791:Enzyme Inhibitors; D010752:Phosphoramide Mustards; D012333:RNA, Messenger; D015262:Xenobiotics; C030090:phosphoramide mustard; D004851:Epoxide Hydrolases; D005978:Glutathione
keywords: chemotherapy; glutathione; metabolism; ovary