Investigating mechanisms of ovotoxicity of Perfluorooctanoic acid and Zearalenone exposure and the influence of altered metabolic status on ovarian function

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González-Alvarez, María Estefanía
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Keating, Aileen F
Koziel, Jacek
Munkvold, Gary
Leandro, Leonor
Bradbury, Steven
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Animal Science
Exposure to xenobiotics termed ovotoxicants can alter the quality and maintenance of oocytes and synthesis and secretion of hormones, which are the major roles of the ovary, leading to premature ovarian failure and infertility. Obesity has several negative reproductive effects in women, and in female mice, mechanistic studies have discovered that obesity decreases the number of primordial follicles, alters steroidogenesis and folliculogenesis, altering the estrous cycle, and blunts the ovarian response to toxicant exposure. In this dissertation, the hypothesis that altered physiological status such as obesity or heat stress would enhance ovarian sensitivity to the ovotoxicants perfluorooctanoic acid (PFOA) and zearalenone (ZEN) was investigated. The rationale for this hypothesis was based upon similar metabolic alterations including hyperinsulinemia between obesity and heat stress in females. A study to evaluate obesity-induced alterations to the ovarian proteome in obese rats was conducted and potential species differences in obesity-induced altered ovarian function related to ovotoxicant exposure were identified (Chapter 2). While no impacts of obesity on several markers of DNA damage were noted, LC-MS/MS analysis provided mechanistic insight into the impact of obesity in a rat model on ovarian endpoints related to genotoxicant response. Per- and polyfluoroalkylated substances (PFAS) including PFOA are characterized by having strong bonds between carbon and fluorine groups and are persistent in the environment. They are extensively used in consumer goods including flame retardant coatings, non-stick cookware, food packaging, fire-fighting foam and cosmetics. In females, exposure to PFOA has been linked to delayed puberty, early menopause, endocrine disruption, reduced fertility, and premature ovarian insufficiency. Mechanisms of PFOA-induced ovarian toxicity including alterations to the levels of E2 and P4, changes to the ovarian abundance of proteins with documented involvement in reproduction, DNA damage sensing and repair, and chemical metabolism and changes in serum metabolome were investigated (Chapter 3). Additionally, hepatic abundance of genes involved in chemical metabolism were to understand mechanisms of action in which PFOA can cause non-alcoholic fatty liver disease and the potential subsequent ovarian effects (Chapter 4). Additionally, an obese group of mice were studied to ascertain if obesity would modulate the ovarian impacts of PFOA exposure (Chapters 3 and 4). We found alterations in ovarian abundance of proteins involved in DNA damage sensing and repair, chemical metabolism and reproduction. The alterations in the ovarian abundance of the proteins involved in DNA damage sensing and repair could suggest an increased risk for ovarian cancer. In addition, even though PFAS are not biotransformed in the body, the alterations in the ovarian abundance of proteins involved in chemical biotransformation could lead to changes in the capacity of the ovary to respond to other toxicants or can lead to alterations in how endogenous chemicals are biotransformed in the body. We also investigated if the metabolic status could impact how the ovary can respond to PFOA exposure and we found altered 17-estradiol levels, differences in the ovarian proteome, and in the serum metabolome. These differences might suggest that the livers of obese females exposed to PFOA function less efficiently than the liver of lean females. The second ovotoxicant investigated herein was ZEN, a non-steroidal mycotoxin with estrogenic activity that causes adverse effects on the female reproductive system, including altered folliculogenesis, decreased fertility, anovulation, vulvovaginitis, and altered follicle-stimulating hormone, progesterone, and 17-estradiol. Swine are the most sensitive species to ZEN exposure. Similar to obesity, heat stress (HS) causes hyperinsulinemia and impairs reproduction. It was hypothesized that HS would alter ovarian abundance of proteins involved in chemical metabolism, that ZEN exposure would alter abundance of ovarian chemical biotransformation proteins, and that an additive impact of HS and ZEN exposure would be observed. The levels of ovarian EPHX1, CYP2E1 and GSTP1 proteins were evaluated by western blots of protein isolated from prepubertal pigs held in a thermal-neutral environment with ad libitum access to a standard swine diet, a thermal-neutral environment with a restricted access to a standard swine diet, and a heat-stressed environment with the same plane of nutrition as thermal neutral feed restricted pigs. (Chapter 5). We found that HS itself alters the basal levels of EPHX1, CYP2E1 and GSTP1 in the ovary and identified that EPHX1 and GSTP1 are involved in the ovarian response to ZEN exposure. In addition, the ovarian abundance of EPHX1 was decreased in pigs who were feed restricted, probably indicating a response to reduced nutrient intake. These findings suggest that HS influences the ability of the ovary to respond to environmental stress possible leading to negative reproductive effects. This dissertation supports the hypothesis that obesity in rats alters the ovarian proteome, that PFOA can induce DNA damage thereby activating DNA damage sensing and repair proteins and alters ovarian abundance of proteins involved in chemical metabolism and reproduction. Taken together this research supports the generation of the hypothesis by which PFOA may cause ovotoxicity and generates additional hypothesis-based questions for future research. Additionally, the work described in this dissertation demonstrates that PFOA exposure in females causes hepatic enlargement and alters hepatic abundance of genes involved in chemical biotransformation, observations that could be informative to ovarian function. It was also discovered that there are differences in the toxicity induced by PFOA in obese females as well as species difference between mice and rats in obesity-induced ovarian alterations. Finally, this work identifies ZEN-induced ovarian biotransformation in pigs and demonstrated an impact of hyperthermia on chemical biotransformation and a differential impact of ZEN exposure in HS females. In conclusion, this work provides evidence of a physiological influence of altered systemic metabolism on ovotoxicity outcomes.
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