(98) Exposure to a Phthalate Metabolite Mixture Decreases Viability of Mouse Ovarian Endothelial Cells In Vitro: Mechanisms and Implications on Ovulatory Angiogenesis

Brittney A. Williams¹; Caroline V. Harper²; Madison M. Wilson²; Patrick R. Hannon^1,2

1. Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, Kentucky, United States

2. Department of Obstetrics and Gynecology, University of Kentucky, Lexington, Kentucky, United States

Abstract Text:

Ovulation is the cornerstone of fertility and relies on a cascade of events resulting in the release of the oocyte. One such event is the development of blood vessels which penetrate the previously avascular granulosa cell layer. This process of ovulatory angiogenesis involves endothelial cells proliferating, migrating, and sprouting in response to an angiogenic stimulus. Exposure to endocrine-disrupting chemicals, such as phthalates, could impair the ability of endothelial cells to respond to this stimulus, threatening angiogenesis and ovulation itself. People are ubiquitously exposed to phthalates, and these chemicals have been found within follicular fluid, indicating their ability to reach the ovary. Our previous work has demonstrated that phthalate exposure alters ovarian endothelial cell expression of angiogenic receptors. We hypothesized that phthalates may induce these changes by decreasing viability and increasing apoptosis via oxidative stress. Because people are exposed to a mixture of phthalates, we utilized a mixture of phthalate metabolites (MPTmix) that was derived using urinary phthalate levels in women. Mouse ovarian endothelial cells were treated ±MPTmix (1-500μg/mL; DMSO=vehicle control) and ±VEGFA, a potent angiogenic stimulus, for 24 or 48hr to investigate the impacts of phthalates on the cells in both a basal and angiogenic state. The cells were subjected to MTS assays for viability, qPCR for gene expression analyses, or TUNEL staining for apoptosis. The media were used to measure levels of reactive oxygen and nitrogen species (ROS/RNS) using OxiSelect in vitro ROS/RNS assays. The MTS assay (n=8; p≤0.05) indicated that exposure to MPTmix decreased endothelial cell viability under basal (100μg/mL; 24hr) and angiogenic (500μg/mL; 24hr) conditions. Cells exposed to MPTmix in basal conditions exhibited signs of apoptosis through changes in gene expression (n=7; p≤0.05), indicated by an increase in Bax at 100μg/mL and 500μg/mL (48hr), while anti-apoptotic Bcl2 was decreased at 500μg/mL (48hr). Consequently, MPTmix increased the Bax/Bcl2 ratio at 100μg/mL and 500μg/mL (48hr). There was a significant increase in TUNEL staining (n=4; p≤0.05) under angiogenic conditions (500μg/mL; 24hr), as well as a strong trend of increased positive TUNEL staining (n=3-4; p=0.08) under basal conditions at 10μg/mL and 500μg/mL (48hr) and under angiogenic conditions at 10μg/mL (24hr). This decreased viability and increased cell death could be attributed to oxidative stress, suggested by changes in oxidative stress-related gene expression (n=7; p≤0.05). Under basal conditions, MPTmix exposure decreased Sod1 at 1μg/mL and 100μg/mL (24hr); increased Sod1 at 100μg/mL and 500μg/mL (48hr); decreased Gpx1 at 1μg/mL (24hr) and 10μg/mL (48hr); increased Gpx2 at 500μg/mL (48hr); and increased Gclm at 100μg/mL and 500μg/mL (24hr). Under angiogenic conditions, MPTmix exposure decreased Cat at 10μg/mL (48hr) and increased Gclm at 500μg/mL (24, 48hr). Under basal conditions, there was a strong trend toward increased levels of ROS/RNS with 500μg/mL at 24hr (n=3; p=0.06). Ultimately, exposure to phthalates decreased endothelial cell viability under both basal and angiogenic conditions, an effect that could be due to apoptosis, as suggested by the increased positive TUNEL staining. This apoptotic response could be attributed to oxidative stress, as indicated by changes in oxidative stress-related gene expression. These signs of apoptosis by oxidative stress were exacerbated under basal conditions, reflected by the increased Bax/Bcl2 ratio and trend toward increased ROS/RNS. Given this, MPTmix exposure may threaten ovulatory angiogenesis by altering angiogenic receptor levels via increased oxidative stress and/or apoptosis in ovarian endothelial cells. Supported by R01ES033767, P30ES026529.