Role of Nuclear Factor E2 Related Factor 2 (NRF2) in Development of Steatosis and Drug Transporter Alterations

Steatosis is fat deposition in liver arising from conditions like obesity, diabetes, and/or alcohol consumption. It is a benign condition with normal liver function, and can often be reversed. Both alcoholic and non-alcoholic liver steatosis can further progress to irreversible steatohepatitis to cirrhosis and substantial loss of liver function. Nuclear factor E2 related factor 2 (Nrf2) is a transcription factor known to combat oxidative stress in the cell. The contribution of Nrf2 to other cellular functions, such as lipid homeostasis is emerging. The work herein assessed how enhanced Nrf2 activity impacts progression of hepatic steatosis with long-term high fat diet (HFD) feeding. C57BL/6 and Keap1Knockdown (Keap1-KD) mice, which exhibit enhanced Nrf2 activity, were fed a HFD for 24 weeks. Keap1-KD mice had higher body weight, liver weight and higher hepatic fat deposition. Lipogenic gene expression was also higher in livers of Keap1-KD mice fed HFD. Next, the work herein studied effect of steatosis and cirrhosis on Nrf2 and drug transporter expression in human livers. Transporters aid in hepatobiliary excretion of many drugs and toxic chemicals, and can be determinants of drug-induced liver injury. Alcohol cirrhosis increased efflux transporter mRNA and protein expression in human livers as compared to normal non-steatotic livers. It was observed that transporter expression alterations with steatosis were much less severe as compared to cirrhosis. In order to demonstrate the effects of these drug transporter and metabolizing enzyme alterations on pharmacokinetics, we conducted oral Bisphenol A (BPA) disposition study in diet-induced obese mice. The mice were administered deuterated BPA orally and blood levels were detected for BPA and BPA metabolites at times after BPA administration. Increased BPA clearance was observed in DIO mice, as compared to lean controls, attributed to increased phase II conjugation enzyme Ugt and biliary efflux transporter Abcc2 expression. In conclusion, constitutive activity of Nrf2 increases susceptibility to mice to develop liver steatosis; and human livers with steatosis and alcohol cirrhosis have altered expression of drug transporters, which may result in xenobiotic disposition alterations.


NRF2:
Nuclear factor E2 related factor 2, Nrf2, is a transcription factor very well known for combating oxidative stress by inducing a battery of antioxidant genes. Nrf2 protects against oxidative stress at baseline levels, as well as, upon challenge by reactive oxygen species (ROS). ROS are integral part of normal physiological mechanisms. However, loss of redox balance causes generation of excess ROS, which can lead to cytotoxicity (1). ROS play a vital role in pathogenesis of variety of diseases, such as cirrhosis, diabetes, hypertension, and cancer, along with neurological disorders including Parkinson's disease (2), and Schizophrenia (3). As reviewed by Naik and Dixit (2011), ROS can also lead to deregulated inflammation, arising from production of pro-inflammatory cytokines (4).
Kelch like associated protein 1 (Keap1) acts as an inhibitor of Nrf2 by preventing its entry into nucleus to interact with antioxidant response element (ARE) (Fig. 1) (5). Keap1 contains multiple cysteine residues in its structure, which are excellent sites for electrophillic attack. The hinge and latch model described that one molecule of Nrf2 is sequestered to two molecules of Keap1, with one having more affinity for Nrf2 than other. Upon induction, the loose interaction between Nrf2 and one of the Keap1 is broken, and stronger one remains intact.
Because of this structural modification of protein complex, degradation of Nrf2 by proteosome 26S is inhibited. This results in ! $! Nrf2 accumulation to the extent that passes sequestering capacity of Keap1, and excess Nrf2 moves to nucleus (6).

NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD):
Owing to increasing prevalence of obesity and diabetes, NAFLD is becoming the most common liver disease (7). Youssef and McCullough reviewed the connections between obesity and NAFLD (8). The prevalence of NAFLD is 10-fold more in obese patients than in general population. Non-alcoholic steatohepatitis (NASH) is severe form of NAFLD, which can progress to fibrosis and cirrhosis. Insulin resistance is considered as key pathogenic factor in progression of NASH (9). NASH, the most severe form of NAFLD, is known to develop by two "hits" (10). The first hit is steatosis, which is fat deposition in liver, followed by second hit involving oxidative stress. Even though progression of liver from steatosis to NASH is incompletely understood, it is clear that oxidative stress plays the major role in the process (11). So, it is likely that Nrf2 exerts its protective actions in the second hit of the NASH, but acting to bolster expression of genes that encode for cytoprotective enzymes, which can counter oxidative stress, such as glutathione cysteine ligase, superoxide dismutase, and glutathione peroxidase. However, very little is known about whether Nrf2 plays any role in steatosis, the first hit of NASH.

ALCOHOLIC FATTY LIVER DISEASE:
Alcoholic liver disease is a spectrum of conditions ranging from simple steatosis to alcoholic steatohepatitis to alcoholic cirrhosis. Similar to obesity driven steatosis, alcoholic steatosis is also a reversible condition, and can be suppressed by abstinence from alcohol (12). Continued consumption of larger quantities of alcohol causes increase in inflammatory cytokine levels, increased bile acid levels in liver, leading to alcoholic hepatitis. There is severe hepatocyte ballooning because of excessive amount of water (13).
There is increase in oxidative stress in the liver, and this may lead to fibrosis of liver. Excessive scar tissue formation leads to cirrhotic liver, wherein significant loss of liver function occurs. Alcoholic cirrhosis is completely irreversible damage of the liver tissue, and eventually leads to liver failure (13).
According to Center for Disease Control and prevention (CDC), more than 15,000 Americans die every year from alcoholic liver cirrhosis (National Vital Statistics Report, Volume 60, No 3).
About 30% of cirrhotic patients also suffer from diabetes (14). Acute, as well as chronic alcohol consumption leads to development of insulin resistance, which can progress to diabetes mellitus (15). Disruption of normal functions of the liver in cirrhosis may lead to hepatogenous diabetes (16). Additionally, obesity and diabetes mellitus increase the severity of alcoholic liver disease (17). Owing to interplay between diabetes and cirrhosis, the two conditions often co-present clinically (18).

Introduction
Metabolic syndrome is described as a cluster of risk factors that increase risk for developing cardiovascular disease [1]. Some of the risk factors include central obesity, atherogenic dyslipidemia (elevated triglycerides and low HDL cholesterol), insulin resistance (with or without glucose intolerance), and a proinflammatory state. In 2003-2009, in an analytic sample that consisted of 3,423 adults, 20 years of age and over, 34% of American adults met the criteria for metabolic syndrome [2].
Nuclear factor E2 related factor 2 (Nrf2) is a basic leucine zipper transcription factor, which regulates basal and inducible expression of multiple antioxidant and biotransformation genes [3]. Kelch-like ECH-associated protein 1 (Keap1) is a cysteine rich protein that binds Nrf2 in the cytosol, and is a critical determinant for Nrf2 nuclear accumulation. Dose-dependent accumulation of Nrf2 in nucleus and increasing Nrf2 target gene expression occurs in Nrf2knockout, Keap1-knockdown and liver-specific Keap1 knockout mice [4]. The effects of Nrf2 and Keap1 knockout/knockdown are well described in models of liver injury caused by acetaminophen, diquat, cadmium, alcohol, or oxidative stress [5][6][7][8][9][10]. But the effects are largely undescribed for hyperlipidemia and tissues important to metabolic syndrome, such as adipose tissue and skeletal muscle (SKM).
Central obesity is a major hallmark of metabolic syndrome. Multiple nuclear receptors influence stem cell differentiation to adipocytes and adipocyte maturation. For example, multiple CCAAT-enhancer-binding protein isoforms (Cebp! and Cebp") are required at various stages of adipocyte differentiation [11]. Peroxisome proliferator-activated receptor-gamma (Ppar-#) is known as a key regulator of fat synthesis, which regulates additional genes that contribute to lipid storage, such as Fatty acid binding protein 4 (Fabp4), Cluster of Differentiation 36 (Cd36, fatty acid translocase), Lipoprotein lipase (Lpl) and steroyl CoA desaturase (Scd1) [12]. Acetyl CoA carboxylase 1 (Acc1) catalyzes formation of malonyl CoA, which is a vital substrate for fatty acid biosynthesis [13]. Malonyl CoA also inhibits "-oxidation of fatty acids.
Phosphorylated Acc1 (pAcc1) is an inactive form of Acc1. Fatty acid synthase (Fas) uses precursors like acetyl CoA and malonyl CoA to synthesize long chain saturated fatty acids. Steroyl CoA desaturase 1 (Scd1) catalyzes synthesis of unsaturated fatty acids from saturated fatty acids [13].
Lipoprotein lipase (Lpl) breaks down triglycerides (TG) from lipoproteins to release free fatty acids [14]. In summary, all of the abovementioned enzymes/ enzyme complexes are responsible for fatty acid levels in the tissues as well as serum.
Adipocytes function to not only store fat, but also produce and secrete 'adipocytokines' that include bioactive products such as inflammatory mediators (e.g. Interleukin-6, IL-6; monocyte chemoattractant protein, Mcp1; tumor necrosis factor, Tnf), which are considered to be a cause of insulin resistance and non-alcoholic fatty liver disease [15,16]. Obesity increases the presence of M1 pro-inflammatory macrophages in adipose tissue, increases secretion of pro-inflammatory cytokines, and increases M1 hepatic macrophages and inflammation [17].
In adipose tissue, Nrf2 binds to an ARE present in the Ppar-# promoter to promote adipocyte differentiation [18]. Nrf2 knockout mice were protected against hepatic steatosis induced by high fat diet (HFD) feeding [19], indicating that Nrf2 presence is needed for hepatic lipid accumulation. Huang et al. The study herein describes the effect of chronic HFD-feeding on markers of metabolic syndrome including 1) WAT mass and hepatic steatosis, 2) glucose clearance, and 3) WAT and liver inflammation in C57BL/6 and Keap1knockdown mice. Overall, Keap1-KD mice exhibited increased markers of metabolic syndrome with long-term HFD feeding.

Hepatic triglyceride (TG) quantification.
Total lipids were extracted from liver tissue by methanol-chloroform extraction according to [25] and TGs were quantified using a kit from Pointe Scientific Inc (Canton, MI) according to manufacturer's protocol. Signaling Inc (Danvers, MA). The membrane was then incubated with ECL+ (GE Healthcare, Waukesha, WI) and chemiluminescence was exposed to Xray film. The resulting bands on autoradiography films were evaluated using Quantity One® software from BioRad.

Statistical Analysis.
Groups were analyzed by a one-way ANOVA followed by a Duncan's Multiple Range post hoc test and planned comparison between C57BL/6 and Keap1-KD groups were performed among HFD groups after performing the one-way ANOVA. Different letters indicate statistically significant difference between the groups (p<0.05).

Effect of Keap1-KD on body, WAT, and liver weight and food
consumption with long-term HFD feeding. Figure 1A depicts body weight change over 24 weeks. There was no significant difference in body weight between C57BL/6 and Keap1-KD mice fed the LFD. Keap1-KD mice fed HFD had significantly higher body weight between weeks 17-24, compared to C57BL/6 mice fed HFD. At weeks 8 and 9, the HFD did not increase body weight in Keap1-KD mice as much as C57BL/6 mice. However, around 11 th week feeding the HFD, the trends in body weight gain appeared to reverse, with Keap1-KD mice having body weight higher than C57BL/6 mice. Food consumption ( Fig. 1B) for the LFD groups stayed within the range of 15-20 g/week per mouse for entire duration of the study. For HFD fed mice, it was noted that food consumption appeared slightly higher in C57BL/6, as compared to Keap1-KD mice throughout the study (no statistical significance).
Blood glucose levels of the mice throughout the course of study were observed to remain in the range of 100 to 200 mg/dL, with no significant difference between any of the groups (data not shown).
HFD feeding increased WAT weight ( Fig 1C) significantly higher in Keap1-KD compared to C57BL/6 mice. At 24 weeks of feeding the LFD or HFD, Keap1-KD mice also had an increased liver-to-body weight ratio as compared to C57BL/6 mice on the respective diet.

Keap1 knockdown increases liver steatosis with chronic HFD
feeding. As depicted in Fig. 2A, the HFD increased lipid accumulation in the liver compared to the LFD. Keap1-KD mice fed the HFD had a higher degree of steatosis compared to C57BL/6 mice, as seen with hematoxylin and eosin staining. Oil red O staining of neutral lipids also revealed that the HFD significantly increased hepatic steatosis, with higher levels being observed in Keap1-KD mice (Fig. 2B). Correspondingly, the HFD increased hepatic triglycerides (Fig. 2C); with significantly higher TG levels being detected in livers of Keap1-KD mice compared to C57BL/6 mice.

Keap1 knockdown increases lipogenic gene and protein expression
in liver. Protein expression of similar adipogenic targets also tended to increase livers of Keap1-KD mice fed HFD (Fig. 4). HFD slightly increased Ppar$# protein expression in Keap1-KD mice, however the change did not reach statistical significance. Phosphorylated acetyl CoA carboxylase 1 (pAcc1), Acc1, and Scd1 protein levels were increased in Keap1-KD mice fed HFD compared to C57BL/6 mice. Fatty acid synthase (Fas) protein expression was equivalent among all groups; however, HFD groups displayed an increasing trend in expression (not statistical), as compared to LFD.

Keap1-KD increases liver and WAT tissue inflammation. Neutrophil
staining of paraffin-embedded liver sections revealed increased infiltration in the HFD fed mice, with even more neutrophils in Keap1-KD mice fed HFD After chronic HFD feeding, WAT from Keap1 mice had increased cellularity and inflammation compared to C57BL/6 mice, as determined by histopathological analysis. Messenger RNA levels of proinflammatory macrophage M1-marker Tnf was elevated in WAT of Keap1-KD mice fed HFD as compared to C57BL/6 mice fed HFD (Fig. 6B). Mcp1 and Cd11c mRNA levels were higher in HFD fed groups, but there was no significant difference between the C57BL/6 and Keap1-KD mice. In accordance with the GTT, the expression of insulin signaling target insulin receptor substrate 1 (Irs1) was also down regulated in SKM. In HFD fed mice, Keap1-KD mice had decreased mRNA expression of Irs1 compared to C57BL/6 mice (Fig. 7C). However, Glut4 mRNA and protein expression was similar between all the groups, as determined by QGP 2.0 assay and western blot respectively ( Fig. 7C and 7D).

Discussion
Metabolic syndrome is considered to be a manifestation of obesity, characterized by increased central abdominal mass, dyslipidemia (e.g. increased serum triglycerides), increased hepatic steatosis and markers of systemic inflammation, and dysregulation of glucose tolerance [31]. To date, no study has evaluated the effect of Keap1 knockdown on development of metabolic syndrome. The present study demonstrates that Keap1 knockdown increased some markers of metabolic syndrome after long term HFD feeding.
Along with increased body weight and WAT mass, Keap1-KD mice fed a HFD displayed increased hepatic and white adipose markers of inflammation, hepatic steatosis, increased adipose cellularity, and altered glucose homeostasis. Taken together, these data suggest that Keap1 knockdown, and perhaps persistent Nrf2 activation, are associated with increased metabolic syndrome risk with HFD challenge.
The present data indicate that Keap1-KD mice had significantly higher body weight and adipose tissue mass compared to C57BL/6 mice with chronic longterm HFD feeding, which are in line with other published findings. Pi et al.
described adipose tissue changes in Nrf2 -/mice [18]. The body weight of Nrf2 -/mice was significantly lower than wild type mice fed an ad libitum diet.
Abdominal fat pad mass, and adipocyte size was also significantly smaller in mice with Nrf2 -/mice. Nrf2 -/mice were also resistant to diet-induced obesity, when fed 41% kCal fat diet for 12 weeks after weaning. Also, adipocytes derived from Nrf2 -/mouse embryonic fibroblasts accumulated less lipids compared to those derived from Nrf2 +/+ mouse embryonic fibroblasts [18].
Another study by Huang et al. also demonstrated that deletion of Nrf2 (Nrf2 -/-) in mice resulted in reduced body weight in Nrf2 -/mice fed a HFD for approximately three months. These mice also had lower hepatic TG content when challenged with HFD, compared to Nrf2 +/+ mice [19]. might also be activated [24,37]. One must also consider the absorption, metabolism, and disposition of the chemical inducers being administered in comparison to a genetically manipulated mouse model that has whole body Keap1 knockdown.
The present study also demonstrated that constitutive Nrf2 activation altered The reason why increased Nrf2 activation might promote lipogenesis and inflammation with HFD feeding is intriguing. It remains to be determined whether lipid accumulation preceded inflammation, but it is likely. Nrf2 has been shown to be a positive regulator of the mouse Ppar-# promoter, increase Ppar-# expression, and promote adipogenesis [18], with a similar mechanism occurring in liver [33]. Thus, the persistent Nrf2 activation in liver appeared to promote lipid accumulation via upstream Ppar-# activation. Perhaps an increased biotransformation due increased Nrf2 activity resulted in increased lipids that caused tissue injury and inflammation. The data clearly demonstrate increased inflammation in liver and WAT, yet an underlying mechanism for the increased inflammation remains to be determined.       consumption of C57BL/6 (C57) and Keap1-KD mice fed a LFD or HFD from weaning age to 27 weeks (starting at age 6 weeks). C) Abdominal adipose tissue weight and liver to body weight ratio. Differences between the groups were analyzed by a one-way ANOVA followed by a Duncan's post hoc test.
Different letters indicate statistically significant difference between the groups (p<0.05). For example, letter "a" is significantly different from "b", but not different from "a". Also, "a" is significantly different from "b,c" but not different from "a,b".  letter "a" is significantly different from "b", but not different from "a". Also, "a" is significantly different from "b,c" but not different from "a,b".

Introduction
Hepatobiliary excretion is an integral function necessary to excrete bile acids, bilirubin, conjugated hormones, as well as, drugs and chemicals from liver Other major causes of cirrhosis include chronic viral hepatitis, non-alcoholic steatohepatitis (NASH), and damaged or blocked bile flow (Anand, 1999).
About 30% of cirrhotic patients also suffer from diabetes (Hickman and Macdonald, 2007). Acute, as well as chronic alcohol consumption leads to development of insulin resistance, which can progress to diabetes mellitus  Laboratories, Hercules, CA).  Table 2 provides the antibody source and western blot conditions. OATP1B1 and 1B3

Western blot analysis. Western blots
protein expression by Western blot was not determined due to lack of high quality commercially available antibodies.

Statistical analysis.
Raw data from mRNA quantification was normalized to housekeeping gene hypoxanthine phosphoribosyl transferase 1 (HPRT1). Log transformed normalized data was more approximately normally distributed as compared with non-transformed data. Within each gene, pairwise comparison of expression between disease groups was tested a oneway ANOVA followed by a Tukey Honestly Significant Difference (HSD) test.
Data from protein quantification was plotted as percent expression and analyzed by one-way ANOVA followed by Dunnett's post hoc test. Difference of p! 0.05 was considered statistically significant. Asterisks (*) represent a statistical difference (p!0.05) from normal non-steatotic livers, and dots (") represent outliers. Hierarchical clustering analysis with Pearson correlation as a similarity measurement was also done to discover potential groups of genes with high correlation.

Transporter mRNA expression in liver is altered by alcohol
cirrhosis and diabetic-cirrhosis. Alcohol cirrhosis altered mRNA expression of some transporters (Fig. 1A). SLCO1B1 mRNA expression was similar among all groups examined. SLCO1B3 mRNA expression was significantly decreased in livers from alcohol cirrhosis patients compared to normal nonsteatotic livers. In contrast, SLCO2B1 mRNA expression was increased with alcohol cirrhosis compared to normal non-steatotic livers.

ABCC1, 4 and 5 mRNA expression was increased in alcohol cirrhotic livers
compared to normal non-steatotic livers (Fig. 1B). ABCC2 mRNA expression remained unchanged between the groups compared, whereas ABCG2 expression was increased in livers from subjects with alcohol cirrhosis (Fig.   1B). Diabetic-cirrhosis decreased ABCC3 expression compared to normal non-steatotic livers. ABCC6 mRNA expression was similar among from normal, steatotic, alcohol cirrhotic, diabetic-cirrhotic, and diabetic livers.

Transporter protein expression is altered in livers from subjects
with steatosis, alcohol cirrhosis, and diabetic-cirrhosis. Fig. 2 illustrates the effect of steatosis, alcoholic cirrhosis, and diabetic-cirrhosis on transporter protein expression in fractions from intact human liver tissue (representative blots). Alcoholic cirrhosis and diabetic-cirrhosis increased ABCC1, 3, and 5 protein expression compared to normal non-steatotic livers. ABCC2 protein remained unchanged between all the groups. ABCC4 and ABCG2 protein expression was increased in livers with steatosis, alcohol cirrhosis and diabetic cirrhosis. In contrast to other ABC transporters, ABCC6 protein expression decreased in livers with alcohol cirrhosis and diabetic cirrhosis. Other CYP and UGT isoforms including CYP2D6, UGT1A1, 1A4 mRNA expressions were also studied, and remained unchanged between the groups (data not shown).

Alcohol cirrhosis increases NRF2, NQO1, and Glutathione
Peroxidase protein expression. NRF2 protein expression in liver fractions was correspondingly increased in alcohol cirrhotic and diabetic-cirrhotic livers compared to normal non-steatotic livers ( Fig. 3C and 3D). NQO1 and Glutathione Peroxidase 1 (GPX1), enzymes, which are regulated via NRF2, were also quantified at protein level. NQO1 protein expression was increased in steatotic, alcohol cirrhotic and diabetic-cirrhotic livers compared to normal livers, with the most prominent increase present in alcohol cirrhosis. GPX1 protein expression was increased in liver fractions from subjects with alcohol cirrhosis and diabetic-cirrhosis.
TNF" mRNA expression was increased in both steatosis and alcohol cirrhosis groups, as compared to normal non-steatotic livers. IL1# expression was increased only with steatosis as compared to normal livers.

Hierarchical cluster analysis of transporter and transcription factor
mRNA expression. Fig. 5 depicts the correlations between transcription factor and transporter mRNA expression. ABCG2 and SLCO2B1 expression were closely related to CAR expression. Similarly, expression of ABCC4, ABCC5 and NRF2 were closely related. Expression of ABCC2 and PXR were also closely related, and more distantly related to SLCO1B3 and 1B1 expression.

Discussion
This study demonstrated predominant increased mRNA and protein of efflux transporters, such as ABCG2, ABCC1, 3-5 in intact livers of human subjects with alcohol cirrhosis. Uptake transporter expression was less consistent, with decreased SLCO1B3 and increased SLCO2B1 mRNA expression occurring in livers with alcoholic cirrhosis. Transcription factors that regulate transporter expression were also correspondingly altered. NRF2 mRNA and protein expression was increased in alcoholic cirrhotic livers, whereas FXR mRNA expression was decreased.

Hierarchical cluster analysis of transcription factors and transporters obtained
in this study is in agreement with the findings in literature. In rodents as well as in humans, NRF2 is known to regulate expression of efflux transporters ABCC2-5 (Klaassen and Slitt, 2005). In the cluster analysis in the present study, ABCC4 and 5 were expressed together with NRF2. Similarly, SLCO2B1 and CAR were expressed together, as observed in rodents (Cheng et al., 2005). ABCC2 and PXR were also clustered together, as also described (Klaassen and Slitt, 2005). SLCO1B1 and 1B3 are reported to be The present study had results consistent with this observation -UGT1A1, 1A3, 1A4, and 2B7 expression was remained unchanged between normal and alcohol cirrhotic livers, although it should be noted that UGT1A3 was decreased in diabetic-cirrhosis livers and UGT2B7 was decreased in diabetic livers.
In summary, we demonstrate that alcohol cirrhosis significantly alters transporter expression in human liver, most notably altering ABCC3, ABCC4, and, ABCC5, which was associated with altered NRF2, CAR, and FXR mRNA expression. Significant correlations between transporter and nuclear receptor expression were observed in the cohort of livers analyzed. Overall, the data herein illustrate alterations in hepatic transporter expression in the alcohol cirrhotic liver that correlates to changes in nuclear receptor expression.
Alterations in nuclear receptor and drug transporter expression in alcoholic liver should be given consideration when evaluating altered drug toxicities.   Protein bands were quantified using Quantity One! software v4.6.3 (Biorad, Hercules, CA). Asterisks (*) represent a statistical difference (p!0.05) from normal non-steatotic livers and dots (") represent outliers. Steatosis increased ABCC4 and ABCG2 protein expression compared to normal livers. ABCC1, 3, 5 protein expression was increased, whereas ABCC6 was decreased in alcohol cirrhotic and diabetic-cirrhotic livers as compared to normal non-steatotic livers. ABCC4 and ABCG2 expression was increased in livers with steatosis, alcohol cirrhosis and diabetic-cirrhosis.   Genes were clustered as a group with bigger . ABCG2 and SLCO2B1 expression were closely related to CAR expression. Similarly, expression of ABCC4, ABCC5 and NRF2 were closely related to each other.
Expression of ABCC2 and PXR were also closely related, and further related to SLCO1B3 and 1B1 expression.

Introduction:
Bisphenol A (BPA) is an industrial chemical used in the polycarbonate plastic and epoxy resins manufacture. Plastic water bottles, food and beverage cans and dental sealant liners made with epoxy resins release the monomer BPA in the contents of the container in smaller amounts that leads to human exposure (1). Polyvinyl chloride plastics and thermal paper recycling also uses BPA.
Although consumption through diet and water is the primary source of human exposure, BPA can also get into body through skin contact with dust, water and air (2). Owing to use of baby bottles, and other plastic products, infants and children have higher exposure as compared to adult population (2). more than 90% of the US population is chronically exposed to BPA (4).
Therefore, in order to assess the susceptibility of all population groups, knowing the metabolism and disposition pathways of BPA becomes important (4).
Absorption of BPA to blood is very rapid and thorough, as peak concentrations of total BPA were obtained within 20 minutes of administering orally in rats. It is absorbed from small intestine, and there is conjugation of BPA to BPA glucuronide in the enterocytes (5). Plasma protein binding of BPA is extensive with the unbound fraction measuring as low as 0.046 in rats after oral administration (6). Monoglucuronide is the major metabolite of BPA. When studied in human liver microsomes, and in recombinant human UGT isoforms, it was demonstrated that UGT2B7 and 2B15 were the isoform primarily involved in BPA glucuronidation (7). In rats, BPA is predominantly conjugated by Ugt2b1 isoform (8). Membrane transporters involved in disposition of BPAglucuronide are multidrug resistance associated protein 3 (ABCC3) in humans and Abcc2 in rats (9 We previously demonstrated that DIO mice are characterized by alterations in the hepatic drug transporter expression (13). As reviewed by Klaassen and Aleksunes, drug transporters are the membrane proteins, which aid in exchange of drugs, endogenous chemicals and/or metabolites exchange across the cell membrane (14). Alterations in transporter expression are known to cause differences in disposition of certain xenobiotics including acetaminophen, ezetimibe, morphine, and raloxifene (15)(16)(17)(18) (19). Briefly, the weighed amount of BPA-d6 was dissolved in 95% ethanol before dilution in water. The solution was administered by oral gavage to mice to get 100µg/kg body weight. For each time point, the blood was collected from mice and spun to get serum.

BPA-d6 analysis by LC-ES/MS/MS:
The detailed method for the detection of BPAd-6 is described elsewhere (20

Total RNA extraction and mRNA quantification:
Total RNA was extracted from liver and intestinal tissues by phenol-chloroform extraction as described previously (21). One microgram of total RNA was converted to single-stranded cDNA by using oligo(dT) 18

Results and discussion:
As demonstrated in fig. 1 Lower levels of total BPA in DIO mice could result from the combination of multiple factors. This experiment was conducted with parallel dosing of 6 lean and 6 DIO mice per time point. So the blood levels are not from serial draws from same animal, rather these are different animals at each time point. The mice had access to food throughout the duration of the study. As mentioned earlier, DIO mice are fed a 60% kCal fat diet, whereas lean mice get diet containing only 10% kCal fat. BPA is a moderately hydrophobic with its noctanol/ water partition coefficient more than 3. Presence of high fat diet in the gut lumen may play a role in lowered absorption of orally administered BPA.
After absorption, the BPA may undergo glucoronidation within the enterocytes. !

96!
It was reported that rat intestinal microsomal preparations possessed higher conjugation activity for BPA as compared to human intestinal microsomes (22). However, little is known about extent to which mouse enterocytes glucuronidate BPA.
Because of hydrophobic nature of BPA, its transport through the membrane is thought to occur by passive diffusion (23). However, there are reports of BPA aglycone being a substrate for rat Abcc2, human ABCC2, ABCG2 and ABCC3 (24). BPA-glucuronide is a substrate for rat Abcc2, human ABCC3, but is a non-substrate/ inhibitor for human ABCC2, ABCB1, ABCG2, rat Abcb1a, Abcb1b, and Abcg2 (24). As reviewed by Willhite et al., BPA-glucuronide is preferentially excreted in bile in rats, whereas in urine in humans. This can be attributed to localization of rat Abcc2 on the apical membrane and human ABCC3 on the basolateral membrane in hepatocytes (14). aglycone. This aglycone is then absorbed back into the enterocytes. Now as this concentration of BPA will be much lower than original orally administered concentration, the extent of intestinal glucuronidation will me more (25). After intestinal glucuronidation, the conjugate will be preferably transported to serosal blood supply with the aid of increased basolateral efflux transporters Abcc3 and 4. This may explain why DIO mice have better enterohepatic recirculation of BPA as compared to lean mice.

Conclusion:
Overall body burden of BPA in DIO mice was significantly less as compared to lean mice.  B) Ugt1a1, 1a6, 2a3, 2b1, 2b5, 3a1, and 3a2 mRNA expression in liver.
*indicates statistical significance in AUC between lean and DIO mice (p<0.05).  2. Along with role in combating oxidative stress, it appears that Nrf2 also regulates some key events in steatosis of the liver, and more studies are needed to clarify the mechanism behind these observations.