Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Angela L. Slitt

Abstract

Drug transporters are membrane bound proteins, which are involved in facilitating both uptake and efflux of xenobiotics, endogenous compounds and their metabolites in various tissues such as liver, kidney, testis, and brain. Alteration in drug transporters may cause imbalances in endogenous compounds such as bile acids, hormones, and bilirubin. Xenobiotic metabolism is inefficient without drug transporters, and drug transporters are recognized as vital mediators for moving polar compounds across membranes. ATP binding cassette (Abc) transporters are a kind of drug transporters, which are ATP-dependent membrane bound proteins involved in transport of wide variety of compounds. Multiple hepatic conditions can alter drug transporter expression such as obesity, oxidative stress, cytokines, druginduced liver injury and environmental toxicants. Specifically, drug transporter expression is known to alter during several metabolic conditions. For example, both genetically modified and diet-induced obesity models of rodents displayed altered hepatic drug transporter expression. Previous studies shown not only during metabolic syndrome, but also exposure to xenobiotics such as estradiol and polybrominated diphenyls (PBDs), which are endocrine disrupting chemicals, alter drug transporter expression. The objective of this study is to determine whether certain liver conditions, such as development of fatty liver (i.e. steatosis) and developmental exposure to an environmental endocrine disruptor (i.e. Bisphenol A) alter hepatic Abc drug transporter expression in conjunction with nuclear receptor expression. This study also aims to show, changes in hepatic Phase II and Abc drug transporter expression during metabolic syndrome alters metabolism and disposition of endocrine disruptor such as Bisphenol A (BPA) This research will provide novel observation and mechanisms by which expression of drug transporters will be affected and regulated. To delineate these aspects whole thesis research was divided into three specific sub studies:

In the first study, which is presented as Manuscript I shows changes in hepatic Abc drug transporters, uptake transporters, Phase I enzyme expression, and factor involved in regulation of hepatic Abc transporter expression during development of obesity. This study also shows possible physiological and transcription factors role in regulation of hepatic Abc transporter expression during development of obesity. In this study, hepatic gene expression and physiological factors were analyzed in both C57BL/6 and ob/ob mice at different time points such as week-1, 3, 4 and 8 age to capture physiological and gene expression changes that occur during development of obesity. Correlation between physiological changes and gene expression was performed using canonical correlations. Significant correlations were observed between physiological changes, hormones, and gene expression changes during development of obesity. Correlation between metabolismrelated hormones and hepatic gene expression are sex-dependent. Correlation between physiological changes and gene expression indicated metabolism-related hormones might have a role in regulation of hepatic genes involved in drug metabolism and transport.

In second study, which is presented as Manuscript II shows changes in hepatic Abc transporter expression with developmental exposure to endocrine disruptor BPA and identifies mechanistic pathways by which BPA exposure causes these effects. In this study, female mice are exposed to either control or BPA or ethinyl estradiol (EE) or phytoestrogen enriched diet. Studies were performed in male pups to analyze gene expression and functional activities of respective genes. Developmental exposure to BPA and EE downregulated hepatic drug transporters and Phase II enzyme expression and activity that are involved in BPA metabolism and excretion, whereas genistein co-administration reversed these changes. Changes observed with developmental exposure of BPA were persistently observed even after cessation of BPA exposure to male pups. Decrease in nuclear factors mRNA expression and binding activity could be partially responsible for downregulation of hepatic Phase II enzymes and drug transporter expression. Further, increase in expression of histone deactylases (Hdac’s) upon BPA exposure could be responsible for decreased transcription factor expression and activity. Our data suggest that developmental BPA and EE exposure may work via similar pathways, and greatly affect the expression of key hepatic genes involved in BPA and hormone metabolism and clearance.

In third study, presented as Manuscript III shows changes in hepatic drug metabolism gene expression observed during obesity alters BPA, an endocrine disruptor disposition. This study aim to identify whether changes in Phase II and drug transporter expression alters BPA disposition, as increase in urinary total BPA levels and BPA exposure in humans are correlated to occurrence of obesity. BPA (10 mg/kg, i.v.) was administered; parent and its metabolites were analyzed in bile, plasma and urine sample of lean and obese rats. Along with in vivo BPA disposition studies, hepatic glucuronidation and sulfation enzymatic assays were performed to identify whether obesity altered hepatic metabolic processes. Changes in hepatic Phase II and III protein expression in obese rats resulted in altered BPA metabolism and disposition. In obese rats, BPA metabolites specifically BPA-glucuronide levels were increased in urine and decreased in bile compared to lean rats. This altered BPA metabolism and disposition during obesity suggests, in humans detailed evaluation of urinary BPA levels such as ratio of metabolite to parent compound, are needed to correlate BPA exposure to occurrence of obesity.

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