Effect of Metabolic Disease and Xenobiotic Exposure on Hepatic ATP-Binding Cassette (ABC) Drug Transporter Expression

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.

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. xi

LIST OF TABLES
MANUSCRIPT-I  Table - conditions, such as cholestasis, hyperbilirubinemia, drug-induced liver injury (Faber et al., 2003;Geier et al., 2003;Lecureux et al., 2009  In liver, ABC drug transporters are important in maintaining the bile acid pool, fatty acids, and cholesterol transport and efflux of xenobiotic metabolites. Drug transporters in liver are regulated by several nuclear receptors and transcription factors such as nuclear factor-E2-related factor 2 (Nrf2), aryl 3 hydrocarbon receptor (Ahr), constitutive androstane receptor (Car), estrogen receptor (Er), and Peroxisome proliferator-activated receptor alpha (Ppar-α) (Faber et al., 2003;Geier et al., 2003;Maher et al., 2005a). Activation of nuclear hormones via pharmacological means or genetic manipulation often upregulates drug Abcc transporter expression in liver (Faber et al., 2003;Maher et al., 2005a). Drug transporters and Phase-II enzyme expression in liver differs from neonates to adults in rodent and humans (Coughtrie et al., 1988;Cui et al., 2010). In rodents, Abcc transporter expression change with age (Maher et al., 2005b;Cheng et al., 2007). In neonates and children, Phase II enzymes such as UGTs expression is low compared to adults (Coughtrie et al., 1988).
Similarly drug transporters such as Abcc family transporters, have low hepatic expression in neonates compared to adult mice (Maher et al., 2005b). In rodents, gender dependent Abc transporter expression is observed in liver.

4
For example, Abcc4 is highly expressed in females compared to males (Maher et al., 2005b). In rats, age dependent differences in Phase-II and drug transporter expression in neonates are taught to be responsible for cardiac glycoside induced toxicity (Guo et al., 2002;Maher et al., 2005b). Low expression of hepatic Abcc2 was taught to be one of the reasons for occurrence of neonatal jaundice (Maher et al., 2005b).

Obesity:
Obesity is metabolic disease characterized with increased body mass index (BMI≥30) (2010). Obesity is a hallmark disease of metabolic syndrome and widely considered as a complex condition. There is growing concerns of obesity affecting people worldwide and approximately above 30% of US population was effected by obesity of which 5% are considered as morbidly obese. During obesity, fat accumulation in liver (steatosis) was observed which represents non-alcoholic fatty liver (Wanless and Lentz, 1990). It is well established that during obesity and other metabolic disorders several genes expression changes take place particularly in liver. Few studies documented the coordinate regulation of drug transporter and nuclear receptor expression in steatosis. Altered nuclear hormone expression in fatty liver, was taught to be one of the factors responsible for change in metabolic genes (Cheng et al., 2008;. Obesity and other metabolic syndrome components are also characterized with altered levels of metabolism-related hormones such as insulin, resistin, glucagon and amylin levels (Azuma et al., 2003;Reinehr et al., 2007). Hyperinsulinemia was observed during obese and diabetic conditions. No studies have shown relation between metabolismrelated hormones role in regulating drug transporters. Recently, drugs that target these metabolism-related hormones are developed for treating obesity and diabetes (Schmitz et al., 2004). There is a need to identify whether these metabolism-related hormones have role in regulating gene expression changes particularly drug transporter expression during development of obesity.

Leptin and rodent models of obesity:
There are of several models of obesity in rodents such as diet induced obesity or genetically modified obseity. Ob/ob mice are genetically modified obese model, these mice have a spontaneous mutation in Ob gene (leptin). Leptin a hormone produced by adipose tissue both in human and rodent plays crucial role in food consumption (Frederich et al., 1995;Kennedy et al., 1997). Leptin regulates food consumption by acting on the central nervous system as a negative feedback system for body fat accumulation (Elmquist et al., 1998;Schwartz et al., 2000). Decreases in fat and body weight were observed with leptin supplements in normal and mice lacking Ob gene (Muzzin et al., 1996;Elmquist et al., 1998;Friedman and Halaas, 1998;Ahima and Flier, 2000;Schwartz et al., 2000). Leptin levels in body correspond to amounts of fat tissue. Leptin acts through membrane receptors (Ob-R), there are several sub types of OB-R receptors they differs by length, location and functionality (Kastin et al., 1999;Hileman et al., 2000;Martin et al., 2008). Obese Zucker rat have mutated leptin receptor (fa, Ob-R gene), these rats display similar 6 conditions as ob/ob mice. Both in rodent and humans, there is an increase in circulating leptin levels proportional to increase in adiposity in diet induced obesity (Li et al., 1997) . Ob-Re is a soluble leptin receptor isoform and several other Serum Leptin Interacting Proteins (SLIPs) play crucial roles in regulating leptin activity both centrally and peripherally (Lammert et al., 2001;Zastrow et al., 2003;Chen et al., 2006). Leptin has no central action in neonatal mice despite fact that there are high levels of circulating levels of leptin. In neonates, high expression of OB-Re leptin receptor, which act as leptin inhibitory protein was thought to be responsible for lack of leptin action over CNS during that age (Ahima et al., 1998;Pan et al., 2008). These studies suggest that leptin has no or minimal regulation on food consumption in neonates, which is contrary to leptin action in adults.
Adult ob/ob mice have well characterized models for obesity diabetes and steatosis that is part of non-alcoholic fatty liver disease. Ob/ob mice have characteristic features such as hyperphagia, hyperglycemia, glucose intolerance, elevated plasma insulin, subfertility, hypometabolism and hypothermia (Lindstrom, 2007). All these physiological changes were predominantly observed in ob/ob mice from four weeks of age (Dubuc, 1976).
Previous studies show that leptin has minimal or no central action until mice are two weeks old; moreover ob/ob mice did not show any significant physiological changes until they reach adolescence. This suggests neonatal Ob mice should have normal physiological and gene expression changes as normal mice. Drug transporter expression is known to alter during several 7 metabolic conditions. Both genetically modified and diet-induced obesity alters drug transporter expression. Analyzing ontogeny of Ob mice hepatic drug transporter expression will provide an insight how lack of leptin action and increased levels of serum hormone levels can affect drug transporters expression.

Bispheonl A:
Bisphenol A (BPA) is a monomer used in plastic manufacturing. According to National Health and Nutrition Examination Survey, 92.6% of 2500 participants have BPA in urine samples . BPA exposure during gestation and lactation periods resulted in dysregulation of several aspects in pups. The negative effects of BPA exposure are considered to be higher in children compared to adults may be due to low expression of elimination pathway. Adverse effects of BPA exposure includes increased body weights in females, early onset of puberty, alterations in mammary glands and reproductive glands, changes metabolic features, causes insulin resistance, increases adipogenesis and predisposes to metabolic syndrome in pups (Alonso-Magdalena et al., 2006;Wei et al., 2011). All these adverse effects shown by BPA exposure involve multiple mechanisms, such as epigenetic modulation and endocrine disruption. In mice, methyl donor supplementation along with BPA treatment has muted epigenetic modulation shown by BPA (Dolinoy et al., 2007). BPA acts as an endocrine disruptor by mimicking estrogen and binds to estrogen receptors (ER) such as ER-alpha and beta. BPA is also known to act as an antagonist for thyroid 8 hormone receptors. BPA developmental exposure in A vy mice has resulted in change in coat color of pups through epigenetic modulation which was muted by methyl donor supplementation along with BPA exposure (Dolinoy et al., 2007). BPA developmental exposure in these A vy mice altered sexual dimorphic gene expression and sexual traits expressed in adult mice (Jasarevic et al.; Mao et al.).
BPA is highly metabolized in gut and liver, and eliminated by urinary and fecal excretion . Drug transporters play key role in elimination of BPA from body, as they are involved in elimination of BPA metabolites. BPA-glucuronide and BPA-sulfate are the major metabolites formed in humans and mice ). The Ugt2b family and Sult1a1 enzymes are major biotransformation enzymes involved in BPA conjugation . Early developmental toxicity caused by BPA in neonates may be due to low expression of several Phase II enzymes compared to adults (Hines, 2008).
Decrease in these Phase-II and drug transporter expression in liver may cause accumulation of BPA in body. BPA metabolite elimination differs from rodent to human and non-human primates. In rodents, BPA primarily is excreted by the biliary route whereas in human BPA undergoes urinary excretion

ABSTRACT
Purpose: Obesity is a predominant risk factor for metabolic syndrome, which is defined as a cluster of risk factors that occur simultaneously and increase the likelihood of coronary artery disease, stroke, and type-2 diabetes. One manifestation of metabolic syndrome is the development of hepatic steatosis in conjunction with insulin resistance. In recent years, the occurrence of obesity in the adult population has significantly increased, which warrants the need for better drug efficacy and toxicity prediction. The purpose of this study was to determine whether clinical biomarkers and hormones could correlate with nuclear receptor and transporter-related pathways at different ages, in order to better predict altered drug metabolism or disposition.
Methods: Livers from male and female C57BL/6 and ob/ob mice littermates at 1, 3, 4 and 8 weeks of age were collected. Serum hormone and mRNA levels were analyzed using a Luminex platform. Correlation between physiological changes and gene expression was performed using canonical correlations.
Results: Significant ontogenic changes in both C57BL/6 and ob/ob mice were observed during and post-weaning. In males and females, the ontogenic pattern started to differ from week 3, but significant changes were observed at week 4 and 8 in ob/ob mice compared C57BL/6 mice. Significant correlations were observed between physiological changes, hormones, and gene expression changes during development of obesity. 19 Conclusion: The correlation between physiological changes and gene expression indicate metabolism-related hormones can regulate or be coregulated with genes involved in drug metabolism and transport. Specifically, in males correlations indicate serum resistin and glucagon can regulate hepatic Abc transporter expression.

INTRODUCTION
Obesity is a metabolic disease characterized by an increased body mass index (BMI≥30). Obesity is a predominant risk factor for metabolic syndrome (MetS), which encompasses increase in body weight, adipose tissue mass, insulin resistance, and serum hormone levels (Grundy, 2004). One of the manifestations of metabolic syndrome is the development of hepatic lipid accumulation (e.g. steatosis), which represents non-alcoholic fatty liver disease (NAFLD) in conjunction with insulin resistance (Wanless and Lentz, 1990). There is a growing concern about absorption, distribution, metabolism, and excretion (ADME) in the obese population, as studies reveal an altered ADME in people affected with obesity and metabolic diseases . Ob/ob mice have a mutation in the Ob gene that encodes for the leptin, resulting in a phenotype that has many characteristics common to Mets.
Due to lack of leptin, ob/ob mice exhibit hyperphagia, profound weight gain, hyperglycemia, glucose intolerance, elevated plasma insulin and severe hepatic steatosis (Lindstrom, 2007). Most of these changes are observed predominantly in ob/ob mice from four weeks of age (Dubuc, 1976 Multiple hepatic conditions can alter drug transporter expression, such as obesity, oxidative stress, inflammation, drug-induced liver injury, and environmental toxicants (Geier et al., 2003;Cheng et al., 2008). Previous studies document alterations in drug transporter and drug metabolizing enzyme expression (DME's) in obese and diabetic conditions (Cheng et al., 2008;. In liver, transcription factors, such as pregnane X receptor (Pxr, Nr1i2), constitutive androstane receptor (Car, Nr 1i3), farnesoid X receptor (Fxr, Nr1h4) and nuclear factor E2-related factor 2 (Nrf2, Nfe2l2) regulate the basal and inducible expression of biotransformation enzymes and Abc transporters (Klaassen and Slitt, 2005). For example, Pxr and Car upregulate Cyp3a11 and Cyp2b10 expression, whereas Nrf2 upregulates Nqo1 gene transcription and expression (Aleksunes et al., 2006). With regard to transporters, hepatic Abcc2-4 induction by microsomal enzyme inducers is observed to be Nrf2dependent (Maher et al., 2005a). Prototypical Pxr activators upregulate hepatic Abcc2, 3, Slc10a1 and Slco1a4 expression (Cheng et al., 2005b;Maher et al., 2005a;Cheng et al., 2007), whereas Car activators upregulate Abcc2-6 in liver (Cheng et al., 2005b;Maher et al., 2005a). However, the mechanisms by which transcription factors regulate transporter expression during development of fatty liver disease are largely not well described. An increase in Pxr, Car and Nrf2 mRNA expression with no changes in binding to consensus sequences was observed in livers of 9 week old ob/ob mice 22 compared to C57BL/6 mice (Xu et al., 2012), suggesting other mechanisms may be involved in the coordinate regulation of drug transporter and transcription factor expression in steatosis.
Obesity alters levels of metabolic hormones, such as resistin, glucagon, insulin and incretins (Starke et al., 1984;Azuma et al., 2003;Reinehr et al., 2007), which could help explain the observed changes in gene expression. Known consequence of obesity is insulin resistance accompanied by hyperglycemia (Kahn et al., 2006). Although increased incretin such as glucagon like peptide-1 (GLP-1) levels are observed in obese people, GLP-1 activity associated with insulin secretion is decreased compared to lean individuals (Laferrere et al., 2007). Several therapies that target these hormones have been identified for treatment of obesity and other metabolic diseases (Schmitz et al., 2004). During obesity correlations have been identified between changes in serum resistin, leptin and insulin levels (Pantsulaia et al., 2007).
As more incretins based therapies are being utilized to manage diabetes a component of Mets, it is critical to understand whether incretin hormones could modulate drug disposition.
Ob/ob mice are commonly used to model MetS and fatty liver disease (Lindstrom, 2007). This study aims to correlate typical clinical endpoints and metabolic hormones with hepatic transcription factoes, prototypical drug metabolizing enzyme (DME), and transporter mRNA expression. The findings of this study provide potential insight into possible measures and serum 23 biomarkers that can be used to predict potential ADME changes in obese patients.

Animals and husbandry.
Heterozygous mice were mated and offspring were genotyped for sex and mutation of the leptin gene. Tissues from male and female wild type (C57BL/6J) and homozygous (ob/ob) were collected at age of week 1, 3, 4, and 8 (n=4-5 per group). Blood was collected and serum was obtained after centrifugation at 5,000 rpm for 5 minutes at 4°C. Livers were collected, snap frozen in liquid nitrogen, and stored at -80°C for future analysis. All animal experiments were approved by University of Rhode Island Institutional Animal Care and Use Committee (IACUC).
Hematoxylin and eosin staining. After collection, a small section of liver from the central lobe was fixed in formaldehyde for 24 h and then transferred to 75% ethanol prior to paraffin embedding. Paraffin-embedded tissues were cut to approximately 5 micron sections, and stained with hematoxylin and eosin by standard histology protocols (AML Laboratories, Rockland, MD).

RNA extraction.
Total RNA from livers was isolated by phenol-chloroform extraction method using RNA-Bee reagent (Tel-Test Inc., Friendswood, TX), according to the manufacturer's protocol. RNA concentration was quantified by absorbance at 260 nm using a Nanodrop ND1000 (Thermo Fisher Scientific, Waltham, MA) and the samples were diluted to 1 µg/µL.
Formaldehyde-agarose gel electrophoresis followed by UV illumination was used to visualize RNA and confirm integrity.

Branched DNA amplification (bDNA) assay.
Relative bile salt-export pump (Abcb11, Bsep) and Na + -taurocholate cotransporting polypeptide (Slc10a1, Ntcp) mRNA levels were quantified using bDNA assay using previously described probesets (Cheng et al., 2007). All reagents for analysis including lysis buffer, amplifier/label probe diluent and substrate solution were supplied in the QuantiGene 1.0 assay kit (Panomics, Fremont, CA). Briefly, the probe set stocks containing capture extenders, label extenders, and blockers were diluted 1:100 in lysis buffer before use. On day one, total RNA samples (10 µg) were added to wells containing 50 µL of capture hybridization buffer and 50 µL of diluted probe set. The RNA was allowed to hybridize overnight with the probe set at 53°C. On day two, subsequent hybridization steps were followed as detailed in the manufacturer's protocol, and fluorescence was measured with a GloRunner TM microplate luminometer interfaced with GloRunner DXL Software (Turner Biosystems, Sunnywale, CA). The fluorescence for each well was reported as relative light units (RLU) per 10 µg of total RNA (Donepudi et al., 2012). Data was normalized with the average of week 1 expression in C57BL/6 mice and expressed as arbitrary units (AU).

Serum metabolism-related hormone levels. Serum metabolism-related
hormones were quantified using a Millipore 10-plex kit (MMHMAG-44K) on a Bioplex® multiple array system. A custom Millipore-plex kit containing different targets such as insulin, glucagon, resistin, glucagon like peptide-1 (GLP-1), amylin and leptin was used and analyzed according to manufacturers protocol. Fluorescence was detected on a Bioplex® multiple array reader system (BioRad, Hercules, CA). Data was collected by Bioplex® manager 5.0 software and plotted as average concentration (µg/mL).

Correlation analysis.
Correlations between the mRNA levels of genes related to drug metabolism and transcription factors were performed using either Statistica 9.1 software (Stat Soft, Inc., Tulsa, OK) or canonical correlation analysis (CCA). Briefly, the canonical correlation analysis data generated 27 was log transformed and distributed in 3 blocks such that block 1 contained gene expression of drug transporters and phase I enzymes, block 2 contained transcription factor expression, and block3 contained physiological dataserum hormone and, glucose levels, body and, liver weights. Cross block pairwise bivariate correlations were performed between each block and heat maps were generated. Hierarchical clustering was performed using the same data with pearson correlation. Data presented as heat maps or with r value, p≤0.05 is considered as a statistically significant correlation.

Statistical analysis.
The statistical significance between groups was determined by factorial ANOVA followed by a Duncan's Multiple-range post hoc test, using Statistica 9.1 software (Stat Soft, Inc., Tulsa, OK). Data are presented as mean ± SE, with p ≤0.05 considered statistically significant. Figure 1A illustrates the body weights of male and female C57BL/6 and ob/ob mice. At week 1 of age, body weight was similar between all groups. At weeks 4 and 8, ob/ob mice had significantly higher body weight compared to C57BL/6 mice, as anticipated. Figure 1B illustrates serum glucose levels each group of mice. A 2.3-fold age-dependent increase in serum glucose levels was observed at 8 weeks compared to week1 C57BL/6 mice. In male ob/ob mice, serum glucose levels increased with age at week 4 and 8 by 2.1 and 4.5-fold respectively, compared to week 1 ob/ob male mice. In female ob/ob mice, serum glucose levels increased with age at week-3, 4 and 8 by 1.7, 1.9 and 3.8-fold respectively compared to week 1 female ob/ob mice. Serum glucose levels in both male and female ob/ob mice were significantly increased at

Tissue and body weights, blood glucose levels:
week 8 compared to C57Bl/6 counterparts by 1.3-fold, these changes in serum glucose levels between ob/ob mice and C57BL/6 mice were not observed at early ages. Figure 1C illustrates serum hormone changes observed in both male and female C57BL/6 and ob/ob mice from weeks 1, 3, 4 and 8. Serum hormone levels were similar between C57BL/6 and ob/ob mice at one week of age. In male C57BL/6 mice, glucagon, resistin and GLP-1 decreased with age, but insulin and amylin did not. In male C57BL/6 mice, serum glucagon, resistin, and GLP-1 levels were decreased by ~78% from week 3 compared to week 1.

Ontogeny of serum hormones in C57BL/6 and ob/ob mice.
However, in male ob/ob mice, serum insulin and amylin increased with age after week 4 by 4-fold. Also, serum insulin, glucagon, resistin, GLP-1 and amylin levels were significantly elevated in male ob/ob mice compared to male C57BL/6 mice from 4 weeks of age.
In female C57BL/6 mice serum glucagon, resistin and GLP-1 levels decreased with age. However, serum insulin and amylin levels were similar at all weeks assessed. In female ob/ob mice, insulin and amylin increased after four weeks of age while glucagon, resistin, and GLP-1 levels decreased by 57%, 31% and 40% respectively, compared to 1 week old female ob/ob mice.
Similar to observations in males, all serum hormone levels were increased at 8 week time point in females ob/ob mice compared to corresponding C57BL/6 females. Figure 2 illustrates hepatic efflux drug transporter expression observed in both male and female C57BL/6 and ob/ob mice. In C57BL/6 and ob/ob mice ontogenic changes were observed in Abc transporter expression for Abcc1, 3, 4, 5 and Abcb11 in both males and females. Similar to physiological changes, no significant changes were observed in Abc transporters expression between one-week old male and female C57BL/6 and ob/ob mice. In male ob/ob mice, Abcc3, 4 and Abcg2 mRNA expression was increased significantly compared to C57BL/6 mice by 1.8, 7 and 2.3-fold respectively. A similar increase was observed in female ob/ob mice, with

Transporter and prototypical metabolizing enzyme expression livers of C57BL/6 and ob/ob mice.
Abcc3, 4 and Abcg2 mRNA expression compared to female C57BL/6 mice. In 30 contrast to males, female ob/ob mice have significant decrease in Abcb11 mRNA expression by 49% compared to female C57BL/6 mice. Most of these significant changes in both male and female ob/ob mice were observed from 4 weeks of age. week old male and female ob/ob mice, Slco1a1 expression was significantly decreased by 97 and 98% respectively, compared to their C57BL/6 counterparts. In male and female, C57Bl/6 and ob/ob mice, ontogenic changes in Slco1a4 were observed only at 3 and 4 weeks of their age but not in week 8 old mice compared to week 1. Figure 4 illustrates hepatic DME expression observed in both male and female C57BL/6 and ob/ob mice. In male and female ob/ob mice Cyp2b20 and 4a14 mRNA expression significantly increased with age compared to week 1 whereas no ontogenic changes were observed in C57BL/6 mice. Changes in mRNA expression with age in Cyp3a11 were observed from 3 weeks of age whereas changes in Cyp2b20 and 4a14 mRNA expression were observed from 4 weeks of age. Male ob/ob mice also showed a significant increase in cyp2b20 and 4a14 expression from 4 weeks of age compared to their C57BL/6 counterparts by 4.3 and 2.2-fold respectively. Female ob/ob mice 31 had significant increase in expression of Cyp3a11 (1.3 fold) and Cyp2b20 (1.8 fold) expression compared to C57BL/6 mice at only week-4. Cyp4a14 mRNA expression in female ob/ob mice significantly greater at both 4 and 8 weeks by 2.5 and 3.1-fold respectively compared to female C57BL/6 mice.
Hepatic transcription factor expression of C57BL/6 and ob/ob mice. Figure 5 illustrates the hepatic transcription factor mRNA expression levels in livers of male and female C57BL/6 and ob/ob mice. In male C57BL/6 and ob/ob mice, there were no significant ontogenic changes in transcription factor mRNA expression, except Nrf2 and Ppar-α. In male ob/ob mice, Nrf2 mRNA expression increased at week-8 by 2 fold compared to week-1. At week-8, Car, Fxr and Nrf2 mRNA expression significantly increased by 2-3 fold, in male ob/ob compare to male C57BL/6 mice. In female C57BL/6 mice, Pxr mRNA expression decreased with age by 51% compared to week-1, however this is not observed in female ob/ob mice. In female ob/ob mice, only Fxr mRNA levels significantly increased by 2.5 fold at 3 and 4 weeks of their age compared to week-1. In females no significant changes were observed in transcription factors expression between ob/ob and C57BL/6 mice.

Correlation analysis between gene expression changes and
phenotypical changes. Role of gender and leptin in ontogenic changes in hepatic gene expression pattern is depicted pictorially using heat maps ( Figure   6 & 7). Gene expression values were log transformed to generate heat maps using R-language. These heat maps strongly indicate leptin and gender plays a huge role in the regulation of hepatic genes involved in metabolism and 32 disposition. Difference in ontogenic changes in ob/ob mice and C57BL/6 mice in both male and female illustrates influence of leptin over peripheral tissues apart its regulation of satiety at central nervous system. Cyp3a11, 2b20 and 2e1 expression. In males, serum glucose concentrations correlated to Abcc1, 3, 5, 6, Abcb1a, Abcb11, Cyp3a11 and 2b20 expression whereas it is correlated only to Abcc3-5, Cyp3a11 and 2b20 in females. Both body and liver weights correlated to Abcc1, 3, 5, 6, Abcb1a, abcb11, Slco1a1, Cyp3a11 and 2b20 expression in male mice, whereas in females they correlated with Abcc1, 3-6, Cyp3a11 and 2b20 expression.

DISCUSSION
Obesity and the subsequent MetS are major concerns in the United States (Grundy, 2004). Non-alcoholic fatty liver disease (NAFLD) is a manifestation of MetS. About 15-39% of the US population is affected with NAFLD, and about 95% of the morbidly obese are diagnosed with NAFLD (Younossi et al., 2002;. Ob/ob mice are used to model MetS and 35 NAFLD because they have multiple markers that are elevated in a manner similar to humans with uncontrolled MetS -morbid obesity, markedly elevated glucose levels, insulin resistance and dysregulation of metabolic hormones, dyslipidemia, increased markers of inflammation, and hepatic steatosis (Lindstrom, 2007). Studies have shown alteration in drug transporter and drug metabolizing enzyme expression during NAFLD, which resulted in altered drug elimination (Barshop et al.;Lickteig et al., 2007;Cheng et al., 2008;Hardwick et al., 2012). Moreover, changes in hepatic uptake and efflux transporter expression in ob/ob mice is somewhat similar to changes observed in dietinduced obese mice and human steatotic livers (Cheng et al., 2008;.
Previous studies have shown that expression of several DMEs and drug transporters in C57BL/6 mice change with age (Cheng et al., 2005a;Maher et al., 2005b;Cui et al., 2010). In this study we characterized ontogeny of transporters along with phase-I enzymes in coordination with transcription factors in both C57BL/6 and ob/ob mice. We selected four different ages (e.g. week-1, 3, 4 and 8) to capture different times during the progression of MetS.
Ontogeny of drug transporters in both male and female C57BL/6 mice we observed are similar to published studies (Cheng et al., 2005a;Maher et al., 2005b). Changes observed in transporters, phase-I enzymes, and transcription factors expression in ob/ob mice compared to C57BL/6 mice were similar to published studies (Cheng et al., 2008;Xu et al., 2012).
As mentioned earlier ob/ob mice have characteristic physiological changes. At week-1 body and liver weights do not show any significant difference from their lean counterparts. However, increased body and liver weight were observed with development of obesity in ob/ob mice irrespective of gender ( Figure 1A and 11). These observations with body and liver weights are consistent with previously published studies (Dubuc, 1976). In ob/ob mice, similar to body and liver weight changes, hyperglycemia developed with development of obesity. Liver histology was similar at week-1, steatosis was observed at week-3, but prominent changes in histology presented at week-4 Abc transporters comprise the majority of hepatic efflux pumps, which efflux compounds from hepatocytes into bile or blood (Faber et al., 2003). In adult mice livers, relative Abcc1 and 4 are low, whereas Abcc3 expression is moderate, and Abcc6 is high (Maher et al., 2005b). In both C57BL/6 and ob/ob mice, Abcc1 and 5 are expressed highly at week1 and decreased with age. This pattern of high expression at initial stages and decrease at later age period in expression of Abcc1 is also observed in liver regeneration after undergoing 90% hepatectomy, indicating Abcc1 to have a lesser role in adult liver (Kimura et al., 2012). Abcc3 is highly inducible basolateral efflux transporter that can cause altered vectorial disposition of xenobiotics (Slitt et al., 2003). Abcc3 and 4 play key role in efflux of several xenobiotics and endogenous compounds such as estrogen and bile acid conjugates. Abcc 4 protects hepatocyte from bile acid toxicity during cholestatic conditions (Mennone et al., 2006). In both male and female C57BL/6 mice there is a slight increase although not statistical difference was observed in Abcc3 and 4 expressions with age. In both males and females obesity increased Abcc3 and 4 mRNA levels with age as well as compared to their lean littermates.
Apical efflux transporters such as Abcc2, Abcg2, Abcb1a and Abcb11 plays major role in excretion of xenobiotics and endogenous substances from liver to bile. In both male and female mice, Abcc2 and Abcb1a mRNA levels are unaltered with age and obesity. Previous studies indicated no change in mRNA levels and increase in protein levels of Abcc2 in ob/ob mice, which is consistent with our results (Cheng et al., 2008). Abcc2 expression during obesity is species specific; in obese zucker rats Abcc2 expression decreases whereas it increases in ob/ob mice, but in humans there is no change with 39 obesity (Pizarro et al., 2004;Cheng et al., 2008;. In male ob/ob mice Abcb11 expression did not alter compared to their lean littermates whereas in females Abcb11 expression decreased with progression of obesity. Gender specific difference in Abcb11 expression pattern may be due to gender specific difference in growth hormone responsiveness in obesity (Cocchi et al., 1993) which is known to regulate Abcb11 expression (Cheng et al., 2007). Previous studies showed that Abc transporter expression is regulated by several transcription factors (Maher et al., 2005a). Interestingly,  (Table 3 and 4).
Hepatic uptake transporter Slco1a1 mRNA levels increased with age in C57BL/6 mice whereas in ob/ob mice it remained unchanged. In both male and female ob/ob mice Slco1a1 mRNA levels decreased significantly at week-8. Previous studies shown Slco1a1 mRNA expression is androgen dependent and negatively regulated by microsomal enzyme inducers that activate transcription factors Pxr, Car, Ppar-α and Nrf2 (Lu et al., 1996;Cheng et al., 2005b). Ob/ob mice have decreased androgen levels (Swerdloff et al., 1976) and increase in transcription factors expression (Xu et al., 2012), which explain decrease in Slco1a1 expression compared to C57BL/6 counterparts as 40 they become old. Moreover in males, at week-8 age Slco1a1 expression was negatively correlated with all transcription factors that are analyazed, although these correlations are not significant (Table 3). Obesity did not affect the ontogeny of Slco1a4, 1b2 and Slc10a1, which is contrary to previously published studies. Differences in our results are may be due to age, previously published studies showed altered hepatic Slco1a4, 1b2 and Slc10a1 expression in week-11 old mice (Cheng et al., 2008), moreover hepatic uptake transporters are shown to change their expression pattern with age (Fu et al., 2012).
Several studies have shown correlations and alteration in serum hormone levels during metabolic disorders. Studies also showed hormones such as estrogen, progesterone and androgens could alter hepatic uptake and efflux transporter expression (Geier et al., 2003;Kalabis et al., 2007). Phase-I DMEs such as Cyp3a11 and 2e1 were not altered with obesity.
Cyp2e1 expression during obesity and diabetes is species-specific. In humans, Cyp2e1 is increased during obesity and diabetes whereas in mice it either remained unchanged or decreased (Enriquez et al., 1999;Wang et al., 2003;Cheng et al., 2008). In both male and female ob/ob mice Cyp4a14 expression increased with development of obesity. Cyp4a plays a key role in fatty acid metabolism and is shown to be upregulated in adult male ob/ob mice 42 (Cheng et al., 2008). In males, Cyp2b20 mRNA levels increased with development of obesity compared to C57BL/6 mice whereas in female Cyp2b20 mRNA levels remained unchanged. These gender specific changes in Cyp2b20 are observed in other model such as treatment with phenobarbital, which is an inducer of Cyp2b family (Larsen et al., 1994;Cheng et al., 2008).
Insulin treatment altered Cyp3a, 2e1 and 4a expression in hepatocytes (Kim et al., 2003), In summary, ob/ob mice are indistinguishable from their lean littermates at week-1. Interestingly, during week1 in both male and females there are no significant changes in transporter, DME's and transcription factors mRNA levels. Not only mRNA levels, even physiological changes such as steatosis, metabolism-related hormone levels, body and liver weight are similar between ob/ob and C5BL/6 mice at week-1. In both males and females, although ontogeny pattern started to differ from week-3 in ob/ob mice compared C57Bl/6 mice, significant changes were observed in week-4 and 8.
IConsistent with previous studies, significant changes in physiological factors like hyperglycemia and insulin resistance were observed after weaning 43 (Dubuc, 1976;Lindstrom, 2007).      Asterisks (*) represent a statistical difference between wild type and ob/ob of same age group and pound (#) represent a statistical difference with respect to week-1 mice (p≤0.05).  week-1, 3, 4, and 8, C57BL/6 and ob/ob mice are log transformed and heat maps were generated using g-plots in R language. Hierarchical clustering was performed using canonical correlations.

Figure 11. Effect of age and leptin deficiency on liver weights in C57Bl/6
and ob/ob mice. Liver weight of both male and female, C57Bl/6 and ob/ob mice at week1, 3, 4 and 8. Asterisks (*) represent a statistical difference 56 between wild type and ob/ob of same age group and pound (#) represent a statistical difference with respect to week-1 mice.

Figure 12. Effect of age and leptin deficiency on liver pathology. A)
Representative liver pathology in male C57Bl/6 and ob/ob mice at week 1, 3, 4, and 8 (n=1 for each group). B) Representative liver pathology in female C57Bl/6 and ob/ob mice at week 1, 3, 4, and 8 (n=1 for each group).
Representative photomicrographs of Hematoxylin and Eosin stains of liver sections (200x).

Table 3. Correlation of hepatic transcription factors expression with hepatic drug transporters and DME's expression in adult (week-8 old)
male mice. Data presented as r values, * value indicates significant correlation (p≤0.05).  Transporters are membrane proteins, which facilitate chemical transport into and out of cells . In liver, the ATP-binding cassette (Abc) superfamily of transporters are involved in excretion of endogenous and xenobiotic compounds from the body, as well as enterohepatic recirculation of bile acids. Membrane transporter proteins are crucial in facilitating the uptake and biliary excretion of endogenous chemicals (e.g. conjugated hormones, bile acids, and conjugated bilirubin), and xenobiotics (e.g. environmental chemicals and drugs) (Faber et al., 2003).

Male
Changes in certain transporters can cause imbalance in endogenous chemicals, such as bile acids, endocrine hormones, and bilirubin (Lecureux et al., 2009). Moreover, xenobiotic metabolism is considered to be less efficient without drug transporters (Faber et al., 2003). Impairment of certain drug transporters function, such as Multidrug resistance-associated protein 2 (Mrp2, Abcc2) and Bile salt-export pump (Bsep, Abcb11), cause hyperbilirubinemia and cholestasis respectively (Faber et al., 2003). During different pathological and physiological conditions, such as cholestasis or acetaminophen-induced liver injury, Abc transporter expression in liver is altered .
Bisphenol A (BPA) is a monomer used in plastic manufacturing. According to National Health and Nutrition Examination Survey, 92.6% of 2500 participants have BPA in urine samples . Perinatal BPA exposure to A vy mice disrupted sexual dimorphic gene expression and sexual trait expression in adult mice (Mao et al., 2010;. In utero BPA exposure increased body weights in females altered mammary and reproductive glands, changed metabolic features, caused insulin resistance, increased adipogenesis, and predisposed to metabolic syndrome in pups (Alonso-Magdalena et al., 2006;Wei et al., 2011). BPA exposure is emerging as a well-studied example of the "fetal basis of disease".  Kundakovic and Champagne, 2011 BPA also acts as an endocrine disruptor by mimicking estrogen and binds to estrogen receptors (ER, such as ER-alpha and beta. BPA is also known to act as a thyroid hormone antagonist (Moriyama et al., 2002).
BPA is highly metabolized in liver and eliminated by urinary and fecal excretion . BPA glucuronide (BPA-Gluc) and -sulfate (BPA-S) are the major metabolites formed in humans and mice . The UGT2B family and SULT1A1 enzymes are major biotransformation enzymes involved in BPA conjugation Hanioka et al., 2008). In rodents, Abcc2 is the predominant transporter, which mediates BPA-Gluc excretion from liver into bile . Maternal exposure to other endocrine disruptors, such as polybrominated diphenyls (PBDEs) and BDE47 alters expression of metabolic enzymes and ABC transporters (Richardson et al., 2008;Szabo et al., 2009).
This study aimed to identify whether developmental BPA exposure could affect hepatic clearance processes, such as metabolism and disposition, in adulthood. In this study two different doses of BPA were administered; 50 µg/kg diet and 50 mg/kg diet (equivalent to 6.5 µg/kg body weight and 6.5 mg/kg bodyweight respectively) (Rosenfeld et al., 2013). Along with BPA 80 exposure, dams were also exposed to ethinyl estradiol as a positive control or co-administrated genestein with BPA as previously described (Dolinoy et al., 2007a;Rosenfeld et al., 2013). Overall, the results herein detail that maternal exposure to BPA during gestation and lactation decreased hepatic transporter expression in male offsprings that were 135 days or older, which was in association with decreased recruitment of Hdac to the Abbc2 and 3 promoter.

Animals and treatments: All animal experiments including breeding and
dietary exposure were conducted by Dr. Cheryl Rosenfeld's laboratory at University of Missouri Columbia according to the IACUC regulations at the University of Missouri Columbia as previously described (Rosenfeld et al., 2013). Livers from this study (Rosenfeld et al., 2013) were used for the study herein.. Animal exposure and breeding was performed as previously described by Rosenfeld et al., 2013(Rosenfeld et al., 2013. Briefly, virgin females (C57Bl/6, a/a) were fed one of the following five diets 1) Corn oil (control), 2) BPA 50 µg/ Kg diet (low dose), 3) BPA 50mg/Kg diet (high dose), 4) Ethniyl estradiol (EE), 0.1 µg/ Kg diet, 5) BPA 50mg/Kg and Genistein 250mg/Kg diet (BPA-G). Dams were fed the diet through gestation and lactation period. Pups were weaned and fed control diet, such that they were exposed to BPA only during the period of gestation and lactation. Livers were collected from male pups after postnatal day 135 days (n=5-6 per group).

84
Transcription factor binding assay: Nuclear extracts obtained from livers were quantified for transcription factor binding to a prototypical ARE consensus sequence using a Procarta TF 9-plex custom array (Affymetrix, CA) according to the manufacturer's instructions and quantified using a Biorad Bioplex, which utilizes a luminex platform (Xu et al., 2012a).

Hepatic bile acid levels:
Hepatic bile acids were extracted from using tertiary-butanol extraction method and quantified as described previously (Donepudi et al., 2012) using a bile acid assay kit (Bioquant, San Diego, CA, USA).

Hepatic Glutathione levels:
Hepatic reduced glutathione (GSH) levels were measured using GSH-Glo TM Glutathione assay kit (Promega). Briefly, 20mg of liver tissue was homogenized in 2ml of 2mM EDTA in phosphate buffered saline (PBS) and homogenate was centrifuged. The resulting supernatant was analyzed for hepatic glutathione levels. Hepatic GSH levels were analyzed according to manufacturer's protocol. Luminescence was measured using a GloRunnerTM microplate luminometer interfaced with GloRunner DXL Software (Turner Biosystems, Sunnywale, CA, USA).

Dibromosulfophthalein (DBSP) disposition:
was injected into mice. Mice were euthanized 45 min after DBSP injection and DBSP concentration was determined in gallbladder bile. Concentration of DBSP was quantified spectrophotometrically at 575 nm after alkalinization of the samples with 0.1M NaOH.

Statistical Analysis:
The statistical significance between groups was Similar to body weights, exposure to the various diets did not change the liver weights. Figure   2A illustrates the effect of developmental BPA exposure on hepatic BPA glucuronidation. In rodents, Ugt2b1 and Sul1a1 are Phase-II enzymes involved in BPA metabolism .

Effect of developmental exposure of BPA on Phase-II conjugation.
Both BPA and EE developmental exposure decreased Ugt2b1 expression in pups to 35 and 31%, and Sult2a1 mRNA levels by 48 and 27 % of control dietexposed pups respectively. BPA-Gluc is the predominant BPA metabolite formed by humans and rodents, formed through glucuronidation . Figure 2B illustrates the effect of BPA developmental exposure on hepatic BPA glucronidation capacity at a relatively low BPA concentration.
Similar to mRNA levels of Ugt2b1, BPA glucuronidation levels decreased with BPA developmental exposure. BPA high dose and EE developmental exposure decreased BPA glucuronidation in pups to about 74% and 81% of control, respectively. Genistein co-administration reversed the observed decrease caused by BPA developmental exposure. The BPA concentration (1 µm) in the reaction vessels was chosen because it is a more relevant 87 concentration and lower or as low as previously described .
The observed percent decrease in BPA glucuronidation is less than the observed decrease in Ugt2b1 expression, and this is likely due to the low concentration of BPA used, which is less than Km (Mazur et al., 2010). Abcg2, and Bsep mRNA levels were decreased to 54, 40 and 41 % of control diet-exposed group respectively. EE developmental exposure decreased Abcc2, Abcg2, and Bsep mRNA levels about to 36, 20 and 22% of control diet group respectively. Similar to mRNA levels, BPA developmental exposure decreased Abcc2 protein expression in liver. Abcg2 protein expression was similar to controls and unaltered with BPA and EE exposure, EE exposure increased Bsep protein expression, whereas Bsep expression was similar between BPA exposed and control groups.

Developmental BPA exposure perturbs bile acid and GSH levels in liver.
Hepatic Abc transporters are determinants for the excretion and enterohepatic circulation of bile acids. Altered hepatic Abcc2 expression was observed during cholestasis (Geier et al., 2003), a pathological condition caused by impedence of bile acids leading to bile acid accumulation in liver. Exposure to the low BPA dose significantly increased hepatic bile acid concentration by 30% compared to controls, but not at the higher BPA dose. Abcc2 transports utilizes GSH as cofactor to drive biliary excretion. Moreover Abcc2 -/mice have increased hepatic total glutathione levels (Chu et al., 2006). BPA exposure increased GSH levels in liver by 60 and 80%, respectively, compared to concentrations on livers of controls ( Figure 5B).

Effect of developmental exposure of BPA on DBSP disposition. DBSP
disposition is used as a estimation of hepatic transport function (Dhumeaux et al., 1974) and targeted Abcc2 deletion significantly decreased DBSP levels in mice (Chu et al., 2006), illustrating DBSP as a substrate for mouse Abcc2.
Both BPA exposures significantly reduced DBSP levels in bile to 24 and 33%, of control diet group respectively ( Figure 5C). The observed decrease in DBSP concentration in gallbladder bile likely reflects the corresponding decrease in liver Abcc2 expression.
Developmental BPA exposure decreases transcription factor expression and binding. Figure 6 illustrates the effect of developmental BPA exposure on gene expression and binding activity of transcription factors involved in regulation of drug transporters and Phase-II enzymes.
In rodents, transcription factors such as Ahr, Car, Fxr and Nrf2 are known to regulate drug transporters and Phase-II enzyme expression (Rushmore and Kong, 2002;Nakata et al., 2006). BPA, at both concentrations, decreased Ahr, Car, Fxr  Figure 7 illustrates Class I Hdac protein expression in nuclear extracts obtained from livers of adult sons from dams that were fed control, BPA, EE, or BPA-G diet. Class I Hdac proteins such, as Hdac 1, 2 and 3, nuclear levels was increased with both doses of BPA and EE developmental exposure compared to control group. Genistein coadministration slightly increased Hdac1, 2 and 3 nuclear levels but not to the level of BPA and EE developmental exposure groups. 91

Discussion:
BPA a xenoestrogen present ubiquitously in the environment , and is known to cause several adverse effects . BPA exposure in humans is variable as studies report different values of estimated human exposure . Toxicological studies identify 1000 mg BPA/kg body weight (BW)/day as maximum tolerable dose in humans ). According to EPA, the daily tolerable intake or no observed adverse effect levels (NOAEL) of BPA is 50 µg/kg BW/ day . In this study, two concentrations BPA were supplemented in maternal feed, one below NOAEL, one higher than the NOAEL. Several studies documented, in rodent models, that BPA exposure is associated with physiological, behavioral and gene expression changes at or below NOAEL (Alonso-Magdalena et al., 2006;Wei et al., 2011;Patisaul et al., 2012), indicating BPA can causes several adverse effect below NOAEL.
Despite liver being important for elimination of endocrine disrupting compounds and determining systemic hormone levels, few studies address the effect of BPA on hepatic processes like metabolism. BPA undergoes extensive Phase-II conjugation, which is essential for decreasing BPA body burden  and conjugated BPA does not have endocrine disruption effects caused by BPA (Matthews et al., 2001). BPA primarily undergoes glucuronidation, whereas sulfonation can occur, but is considered to have a minor, but appreciable role . Species-specific differences were observed in BPA elimination pathways. In rodents, BPA-Gluc primarily undergoes biliary excretion whereas in humans, BPA undergoes urinary excretion . In vitro studies using ATPase assays have identified ABCC3 in humans as a possible drug transporter involved in transport of BPA-Gluc , whereas in rodents Abcc2 is major transporter of BPA-Gluc .
In the present study, the effects of developmental exposure of BPA on liver function and hepatic metabolic processes in adult were documented.
Developmental BPA exposure downregulated key enzymes and transporters involved in BPA metabolism and excretion. Developmental BPA exposure also decreased activity or function of genes involved in BPA metabolism and excretion. Effects observed in this study by developmental BPA exposure are similar to EE developmental exposure although there were no changes in estrogen levels in these mice ( Figure 10). BPA and EE developmental exposure significantly down-regulated hepatic Phase II enzymes such as Ugt2b1 and Sult1a1, and hepatic efflux drug transporters such as Abcc2, 3, and 4 expressions. Developmental BPA and EE exposure also decreased BPA glucuronidation capacity of pups, which supports decrease in Phase II enzyme expression in liver. Decrease in hepatic efflux transporters, primarily Abcc2 expression was supported by increase total hepatic glutathione levels and decrease of DBSP levels in bile. As mentioned previously, glutathione conjugates and DBSP are substrates for Abcc2 (Chu et al., 2006).
Interestingly, changes in hepatic gene expression such Phase II enzymes and 93 Abc transporter expression due to developmental BPA exposure was reversed by genistein co-administration. Serum BPA levels in dams were unaltered with genistein supplementation (Figure 8), indicating genistein coadministration did not changed BPA exposure to dams and effects shown by genistein co-administration did not involve differences in BPA exposure.
Decrease in hepatic metabolism genes and transcription factors by BPA developmental exposure in this study were supported by similar observation in CD-1 male pups, which are exposed to BPA at perinatal, and peripubertal stages ( Figure 11). Hepatic Ugt2b1is a key enzyme involved in conjugation of endogenous androgens and xenobiotics . Although Phase II enzymes particularly Ugt2b1 expression is minimally induced with transcription factors activators, Nrf2-nullmice showed significant decrease in Ugt2b1 expression indicating basal levels of Ugt2b1 expression is regulated by Nrf2 (Buckley and 94 Klaassen, 2009;Reisman et al., 2009 Nrf2 (Maher et al., 2005). In mice, prototypical activators of these transcription factors increase hepatic efflux transporter expression (Maher et al., 2005). In mice, activation of Nrf2 by oxidative stress and chemical activators increased hepatic Abc transporters expression such as Abcc2, 3 and 4. Moreover, hepatic efflux transporters such as Abcc2, 3, 4 and Abcg2 expression decrease significantly in Nrf2-null mice whereas there expression significantly increased in Keap1 knock down and Keap1 heaptocyte knockout mice (Reisman et al., 2009;Wu et al., 2012). Similar to these studies, developmental BPA at both doses and EE exposure decreased hepatic efflux transporter expression and only Nrf2 expression and activity in same pattern, indicating Nrf2 role in BPA induced down regulation of hepatic Abcc2, 3 and 4 transporters.
In mice, developmental BPA exposure is known to induce epigenetic changes such as hypomethylation and histone modification in different tissues at different promoter regions (Kundakovic and Champagne, 2011). Epigenetic 95 changes specifically hypomethylation of intracisternal A particle (IAP) retrotransposon of agouti promoter region caused by developmental BPA exposure is reversed with methyl donor and genistein supplementation (Dolinoy et al., 2007a). Moreover, as mentioned earlier effects observed in this study by BPA are similar to EE indicating these changes are caused due to endocrine component involved in it which is known to cause epigenetic changes in pups . In our study we observe a decrease in methyl cytosine levels in genomic DNA isolated from liver with developmental exposure of BPA although these changes are not reversed with genistein co-administration ( Figure 9). Apart from DNA methylation, histone modification such as methylation and acetylation also can regulate nuclear receptor inducible gene transcription (Biddie, 2011). Hdac proteins are known to remodel chromatin structure and act as co-repressors for nuclear receptor inducible gene transcription (Biddie, 2011). Results presented in this study showthat developmental BPA and EE exposure increased nuclear Hdac levels whereas genistein co-administration did not as much as BPA. Increase in class I Hdac nuclear levels indicate decrease in nuclear receptor inducible gene transcription. In vitro studies had shown role of class I Hdac proteins in regulation of efflux transporter expression Xu et al., 2012b).
Moreover studies have shown that Hdac proteins can regulate Nrf2 induced transcription and expression (Yu et al., 2010;Lee et al., 2012   mice per group). An asterisk (*) represents a statistical difference between pups from control diet fed dams to pups from BPA, EE and BPA-G exposed dams (p≤0.05).    group. An asterisk (*) represents a statistical difference between pups from control diet fed dams to pups from BPA, EE and BPA-G exposed dams (p≤0.05).  . Serum BPA levels in dams exposed to different diets. Data was presented as average total BPA concentration (ng/ml) ± S.E.M (n=4-6 mice per group). An asterisk (*) represents a statistical difference between pups from control diet fed dams to pups from BPA, EE and BPA-G exposed dams (p≤0.05). Data was presented as average percentage of (5-methyl cytosine) ± S.E.M (n=5-6 mice per group). An asterisk (*) represents a statistical difference between pups from control diet fed dams to pups from BPA and BPA-G exposed dams (p≤0.05).  Post weaning pups were exposed to similar BPA doses through drinking water. Tissues from female offspring were collected on postnatal day 32. Total RNA was isolated from livers of male pups that are developmentally exposed to DMSO and BPA and mRNA levels were quantified by quantitative PCR. Data was normalized to 18S rRNA and presented as a mean arbitary units + S.E.M. (n=9-10 mice per group). An asterisk (*) represents a statistical difference between pups from control to pups from BPA exposed dams (p≤0.05).

INTRODUCTION
BPA is a bi-product of plastic manufacturing, available ubiquitously in environment and exposed to humans through several forms . According National Health and Nutrition Examination Survey (NHANES) 92.6% of people analyzed have BPA in their urine . Rodent studies documented, BPA can cause several adverse effects such as obesity, insulin resistance, oxidative stress, endocrine disruptors and behavioral changes . BPA is highly metabolized in liver and eliminated by urinary and fecal excretion . BPA-glucuronide (BPA-Gluc) and BPA-sulfate (BPA-S) are the major metabolites formed in humans and mice The UGT2B family and SULT enzymes are major biotransformation enzymes involved in BPA conjugation Hanioka et al., 2008). In rodents, Abcc2 is the predominant transporter, which mediates BPA-Gluc excretion from liver into bile .
Obesity is a hallmark disease of metabolic syndrome and widely considered as a complex condition. Obesity is characterized by body mass index (BMI> 30 kg/m 2 ). There is growing concerns of obesity affecting people worldwide and approximately above 30% of US population was effected by obesity of which 5% are considered as morbidly obese (Ogden et al., 2006;Flegal et al., 2010). The CDC estimates that more than 86% of US adults will be overweight and more than 50% obese by the year 2030 (Wang et al., 2008). One of the major complications during obesity is NAFLD, 15-39% of US population are 127 effected by NAFLD (Younossi et al., 2002;. Obese people have altered drug absorption, distribution, metabolism and elimination process. Several studies documented altered metabolism and drug disposition in both obesity and NAFLD disease conditions (Naik et al., 2013). During obesity altered pharmacokinetic parameter are related to several factors such as change volume of distribution, fat mass and clearance .
Apart from these factors hepatic metabolism, which includes phase I, II and III (drug transporter) proteins also plays important role in pharmacokinetics aspects in obese population . Both in humans and rodents, altered phase I and, II enzymes and drug transporter expression were observed during NAFLD (Naik et al., 2013). Moreover, during obesity and non-alcoholic fatty liver disease (NAFLD) conditions phase II enzymes and drug transporters involved in BPA metabolism are altered (More and Hardwick et al., 2013).
There is very less known about whether changes in hepatic metabolic pathways in obese population alters endocrine disruptors disposition. In this study we want to determine how obese population handle BPA body burden.
In this study we also want to determine whether changes in phase II enzymes and drug transporters during metabolic syndrome conditions alter BPA metabolism and disposition. We used lean and obese zucker rat and injected BPA to identify how obese population handle high amount of BPA exposure.  (Coughlin et al., 2011). In this study data represented from bile and urine sample have n= 6 samples per group whereas plasma samples are n= 5-6 per group.

Statistical Analysis:
The statistical significance between groups was determined using one-tailed student t-test. Data are presented as mean ± SE, with P ≤0.05 considered statistically significant. 132

Results:
Enzymatic BPA glucuronidation and Sulfation: Figure 1 illustrates hepatic glucuronidation and sulfation in both lean and obese rats. Hepatic glucuronidtation was significantly reduced in obese rats compared to leans by 42% ( Figure 1A). In Vitro sulfation assay showed 1.4-fold increase in hepatic sulfation in obese zucker rats compared to lean zucker rats ( Figure 1B). 133 Figure 3 shows cumulative levels of BPA and its metabolites in plasma.

Effect of obesity on BPA and its metabolite disposition:
Cumulative plasma BPA levels did not altered in obese rats compared to lean rats. Cumulative BPA-Gluc levels in plasma showed an increasing trend in obese compared to lean rats ( figure 3B). Cumulative BPA-S levels in plasma were unaltered in obese rats ( figure 3C). Figure 4 shows cumulative levels of BPA and its metabolites in urine. Interestingly, although no significant changes were observed with plasma BPA-Gluc and BPA-S levels urine BPA-Gluc and BPA-S levels increased significantly.
substrate mycophenolic acid conjugation was decreased during diabetes (Dostalek et al., 2011). These changes indicate altered metabolism especially hepatic BPA glucuronidation during obesity, diabetes and NAFLD, which are components of metabolic syndrome.
Both in humans and rodents BPA sulfation is a minor pathway, which is catalyzed by SULT1A1 predominantly . In rodents, diet induced obesity increased Sult1a1 expression, which is similar to SULT1A1 expression in human steatotic livers .
SULT1A1 is involved in sulfation of phenolic compounds like acetaminophen.
Studies have documented during NAFLD both in human and rodents acetaminophen sulfation increased Hardwick et al., 2013). This indicates both humans and rodents have increased BPA sulfate formation during obesity.
In humans BPA is rapidly metabolized and excreted through urine whereas, in rodents BPA metabolites are excreted through feces .
Transporters involved in BPA metabolites excretion are different in human and in rodents . BPA-Gluc, a major metabolite formed in BPA metabolism is transported by Abcc2 in rodents .
Transporter assays using membrane ATPase assays identified in humans ABCC3 might be involved in BPA-Gluc transport . ABCC3 is efflux drug transporter present on basolateral membrane of hepatocyte and inovolved in excretion of endogenous and xenobiotics from liver into blood . In obese zucker rats hepatic Abcc2 137 expression was significantly downregulated (Geier et al., 2005). Decrease in hepatic phase II and III (drug transporter) expression in obese rats play major role in altered disposition of BPA. Decrease cumulative BPA-Gluc levels in bile is may be due to decreased Abcc2 expression. Studies have documented that rats lacking Abcc2 show decreased BPA-Gluc biliary excretion . Decreased biliary BPA-Gluc excretion resulted in increase of plasma and urine cumulative BPA-Gluc levels.
Changes in humans, during NAFLD hepatic altered phase II and III protein expression was observed (Hardwick et al., 2011;Hardwick et al., 2013). As mentioned previously, in humans during obesity increased glucuronide conjugate formation of lorazepam, and oxazepam was observed, which are substrates for UGT2B15 .
Increase in UGT2B15 activity in obese people indicates that obese patient can detoxify BPA more efficiently than normal population. Moreover in humans, hepatic ABCC3 expression increased with increase in progression of NAFLD (Hardwick et al., 2011;. This indicates obese people will have high clearance of BPA-Gluc from liver, which ultimately results in increased BPA-Gluc urinary excretion. As mentioned previously, in humans urinary BPA levels are correlated with obesity. In most of these studies only total urinary BPA levels were observed and these studies did not specified ratio of BPA and its metabolites in human urine samples (Carwile and Michels, 2011;Trasande et al., 2012;Wang et al., 2012). May be high amount of total BPA in obese human urine may be result 138 of altered metabolism and disposition of BPA. Correlation between obesity and urinary BPA levels observed from previous studies may need more evaluation. Detailed evaluation of BPA and its metabolites in human urine gives more information whether obesity and BPA exposure are interlinked in humans or not.
In conclusion, hepatic clearance plays a major role in detoxifying and elimination of BPA from body. We think changes in phase II and III protein during obesity will alter hepatic clearance of BPA and its metabolites. Our model of obesity in which altered hepatic phase II and III protein expression resulted in altered BPA metabolism and disposition. Although our model did not show same changes that are observed in obese human population, it strongly indicates that BPA metabolism and disposition will be altered during obesity. In humans, correlation between total BPA levels in urine cannot be correlated to obesity, as obese population is known to have altered ADME. In depth evaluation of BPA levels such as ratio of metabolite to parent compound are needed to correlate BPA exposure to occurrence of obesity.    These results supports that altered Phase II and Abc drug transporter expression in liver during obesity will results in altered disposition of endocrine disruptors and increases body burden of BPA.