Phase III biotransformation enzyme involvement in the ovarian response to ovotoxic environmental exposures

• Main Author: Thomas, Porsha Q
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01.01.2016
• Subjects: Animal sciences; Surgery
• ISBN: 1339845334; 9781339845333

The mammalian ovary is comprised of follicles at various stages of growth and is responsible for gamete and steroid hormone production. Conservation of ovarian function is vital for female reproductive and general health, and disruption can lead to infertility and/or ovarian senescence. Stressors affect the ovary by inflicting damage to the primordial oocyte and/or inducing follicular activation thereby depleting the follicular reserve and disrupting biological pathways or defense mechanisms necessary for maintaining proper ovarian function. Cyclophosphamide (CPA) is a chemotherapy drug that biotransforms into an ovotoxic metabolite phosphoramide mustard (PM) via hepatic biotransformation. Our previous findings demonstrated that PM (60 µM) exposure depleted primordial follicles as well as all follicle types in cultured postnatal day (PND) 4 rat ovaries. With the advancement of science and medicine, there has been a decline in mortality rates of females who receive chemotherapy. The viability and health of the ovary post-chemotherapy treatment is of major concern for female cancer survivors due to the harsh effects of anti-neoplastic agents. An ovarian defensive mechanism would challenge the adverse effects of PM-induced ovotoxicity. We hypothesized that the ovarian defense response to PM-induced damage involves both the ABCB1 and ABCC1 proteins to excrete PM from the ovary and that their regulation involved the PI3K pathway. ABCB1 and ABCC1 are involved phase III chemical biotransformation, having enzymatic activity that requires ATP derived energy for the transportation of endogenous and exogenous compounds. Briefly, PND4 rat ovaries were cultured in PM (60 µM), LY294002 (20 µM), or vehicle control (1% DMSO) followed by protein isolation and western blotting. Adult (PFTN) gilts included to eliminate bias of feed intake followed by protein isolation, RNA isolation, western blotting, and qRT-PCR. Additionally, post-pubertal gilts were synchronized in their follicular phase with Matrix feeding and exposed to TN (20.3°C) or HS (26–32°C) conditions for 5 d followed by protein isolation, RNA isolation, western blotting, and qRT-PCR. We found that chronic HS (35 d) of pre-pubertal gilts increased both ABCC1 and SULT1E1 protein abundance without having an effect at the transcriptional level. HS during the follicular phase in post-pubertal estrous synchronized gilts resulted in reduction of ABCC1 mRNA levels and increased levels of SULT1E1 protein, but no impact on ABCC1 protein was observed. The data generated from these studies suggest that PI3K plays a critical role in ovarian phase III biotransformation enzymes providing evidence that the ovary has the ability to conduct phase III drug metabolism and that SULT1E1 and ABCC1 are active in the ovary and may alter 17β- estradiol metabolism during HS.