Angela L. Slitt

Oxidative and electrophilic stress induces multidrug resistance–associated protein transporters via the nuclear factor‐E2–related factor‐2 transcriptional pathway

Multidrug resistance–associated proteins (Mrps) are adenosine triphosphate–dependent transporters that efflux chemicals out of cells. In the liver, Mrp2 transports bilirubin‐glucuronide, glutathione (GSH), and drug conjugates into bile, whereas Mrp3 and Mrp4 efflux these entities into blood. The purpose of this study was to determine whether oxidative conditions (that is, the disruption of hepatic GSH synthesis) or the administration of nuclear factor‐E2–related factor‐2 (Nrf2) activators (oltipraz and butylated hydroxyanisole) can induce hepatic Mrp transporters and whether that induction is through the Nrf2 transcriptional pathway. Livers from hepatocyte‐specific glutamate‐cysteine ligase catalytic subunit–null mice had increased nuclear Nrf2 levels, marked gene and protein induction of the Nrf2 target gene NAD(P)H:quinone oxidoreductase 1, as well as Mrp2, Mrp3, and Mrp4 expression. The treatment of wild‐type and Nrf2‐null mice with oltipraz and butylated hydroxyanisole demonstrated that the induction of Mrp2, Mrp3, and Mrp4 is Nrf2‐dependent. In Hepa1c1c7 cells treated with the Nrf2 activator tert‐butyl hydroquinone, chromatin immunoprecipitation with Nrf2 antibodies revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of mouse Mrp2 [−185 base pairs (bp)], Mrp3 (−9919 bp), and Mrp4 (−3767 bp). Conclusion: The activation of the Nrf2 regulatory pathway stimulates the coordinated induction of hepatic Mrps. (HEPATOLOGY 2007.)

Induction of Drug-Metabolizing Enzymes by Garlic and Allyl Sulfide Compounds via Activation of Constitutive Androstane Receptor and Nuclear Factor E2-Related Factor 2

Garlic oil (GO) contains several linear sulfur compounds, including diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), that induce drug-metabolizing enzymes such as CYP2B and NAD(P)H quinone oxidoreductase 1 (NQO1). CYP2B and NQO1 are primarily regulated by constitutive androstane receptor (CAR) and nuclear factor E2-related factor 2 (Nrf2) transcription factors, respectively. The purpose of this study was to determine whether GO and its specific constituents induce these two enzymes via CAR and Nrf2 activation. Female Wistar-Kyoto (WKY) rats express little CAR protein and exhibit less induction of CYP2B1/2 than males. GO, DAS, and DADS, but not DATS, induced CYP2B1/2 mRNA levels to a greater extent in WKY males than in females, suggesting CAR activation. Conversely, DAS induced NQO1 levels equally in WKY males and females, indicating CAR-independent induction in rats. DAS, but not GO, DADS, or DATS, induced CYP2B10 mRNA levels 530-fold in wild-type (WT) mice, whereas this induction was attenuated in CAR-/- mice. DAS induced NQO1 in WT and CAR-/- mice equally, suggesting CAR-independent induction in mice. DAS induced NQO1 5-fold in WT mice, whereas induction was completely absent in Nrf2-/- mice, indicating DAS also activates Nrf2. DAS induction of CYP2B10 mRNA was independent of Nrf2 presence or absence. In in vivo transcription assays, DAS activated the human CYP2B6 promoter, and the antioxidant response element of the human NQO1 promoter, respectively. These studies indicate that GO constituents, particularly DAS, activate CAR and Nrf2 to induce drug-metabolizing enzymes.

Enhanced Nrf2 Activity Worsens Insulin Resistance, Impairs Lipid Accumulation in Adipose Tissue, and Increases Hepatic Steatosis in Leptin-Deficient Mice

The study herein determined the role of nuclear factor erythoid 2–related factor 2 (Nrf2) in the pathogenesis of hepatic steatosis, insulin resistance, obesity, and type 2 diabetes. Lepob/ob-Keap1-knockdown (KD) mice, which have increased Nrf2 activity, were generated. Markers of obesity and type 2 diabetes were measured in C57Bl/6J, Keap1-KD, Lepob/ob, and Lepob/ob-Keap1-KD mice. Lepob/ob-Keap1-KD mice exhibited less lipid accumulation, smaller adipocytes, decreased food intake, and reduced lipogenic gene expression. Enhanced Nrf2 activity impaired insulin signaling, prolonged hyperglycemia in response to glucose challenge, and induced insulin resistance in Lepob/ob background. Nrf2 augmented hepatic steatosis and increased lipid deposition in liver. Next, C57Bl/6J and Keap1-KD mice were fed a high-fat diet (HFD) to determine whether Keap1 and Nrf2 impact HFD-induced obesity. HFD-induced obesity and lipid accumulation in white adipose tissue was decreased in Keap1-KD mice. Nrf2 activation via Keap1-KD or sulforaphane suppressed hormone-induced differentiation and decreased peroxisome proliferator–activated receptor-γ, CCAAT/enhancer–binding protein α, and fatty acid–binding protein 4 expression in mouse embryonic fibroblasts. Constitutive Nrf2 activation inhibited lipid accumulation in white adipose tissue, suppressed adipogenesis, induced insulin resistance and glucose intolerance, and increased hepatic steatosis in Lepob/ob mice.

Drug-metabolizing enzyme and transporter expression in a mouse model of diabetes and obesity

Obesity and type II diabetes pose a serious human health risk. Obese or diabetic patients usually take prescription drugs that require hepatic and renal metabolism and transport, and these patients sometimes display different pharmacokinetics of these drugs. Therefore, mRNA and protein expression of drug-metabolizing enzymes (DMEs) and transporters was measured in livers and kidneys of adult wild-type and ob/ob mice, which model obesity and diabetes. mRNA expression of numerous DMEs increased by at least 2-fold in livers of male ob/ob mice, including Cyp4a14, Cyp2b10, NAD(P)H:quinone oxidoreductase 1 (Nqo1), and sulfotransferase 2a1/2. In general, expression of uptake transporters was decreased in livers of ob/ob mice, namely organic anion-transporting polypeptides (Oatps) and sodium/taurocholate cotransporting polypeptide (Ntcp). In particular, Oatp1a1 mRNA and protein expression in livers of ob/ob mice was diminished to <5% and <15% of that in wild-types, respectively. Generally, the mRNA and protein expression of efflux transporters multidrug resistance-associated proteins (Mrps) was increased in livers of ob/ob mice, particularly with Mrp4 expression being elevated by at least 6-fold and Mrp2 expression at least 3-fold in livers of ob/ob mice. In kidney, Nqo1, Mrp3, 4, Oatp1a1, and organic anion transporter 2 (Oat2) showed significant alterations with mRNA expression levels in ob/ob mice, being increased for Nqo1 and Mrp4 and decreased for Mrp3, Oatp1a1, and Oat2. In summary, the expression of a number of DMEs and transporters was significantly altered in livers and kidneys of ob/ob mice. Since expression of some DMEs and transporters is regulated similarly between mouse and human, the data from this study suggest that transporter expression in liver and kidney may be changed in patients presenting with obesity and/or type II diabetes.

Efflux transporter expression and acetaminophen metabolite excretion are altered in rodent models of nonalcoholic fatty liver disease

Efflux transporters are responsible for the excretion of numerous xenobiotics and endobiotics and thus play an essential role in proper liver and kidney function. Nonalcoholic fatty liver diseases (NAFLDs) comprise a spectrum of disorders that range from simple fatty liver (SFL) to nonalcoholic steatohepatitis (NASH). Although the precise events leading to NAFLD are unclear, even less is known about the effects on efflux transporter expression and drug disposition. The purpose of this study was to determine the effect of NAFLD on efflux transporter expression in rat liver as well as on acetaminophen (APAP) metabolite excretion. To simulate SFL and NASH, rats were fed either a high-fat (HF) or a methionine- and choline-deficient (MCD) diet for 8 weeks. In the livers of MCD rats, there were striking increases in both mRNA and protein levels of multidrug resistance-associated protein (Mrp) 3, Mrp4, and breast cancer resistance protein, as well as increased Mrp2 protein. After administration of a nontoxic dose of APAP, biliary concentrations of APAP-sulfate, APAP-glucuronide (APAP-GLUC), and APAP-glutathione were reduced in MCD rats. The effects of the HF diet on both transporter expression and APAP disposition were by comparison far less dramatic than the MCD diet-induced alterations. Whereas APAP-sulfate levels were also decreased in MCD rat plasma, the levels of the Mrp3 substrate APAP-GLUC were elevated. Urinary elimination of APAP metabolites was identical between groups, except for APAP-GLUC, the concentration of which was 80% higher in MCD rats. These studies correlate increased hepatic Mrp3 protein in the MCD model of NASH with increased urinary elimination of APAP-GLUC. Furthermore, the proportional shift in elimination of APAP metabolites from bile to urine indicates that MCD-induced alterations in efflux transporter expression can affect the route of drug elimination.

Altered UDP-Glucuronosyltransferase and Sulfotransferase Expression and Function during Progressive Stages of Human Nonalcoholic Fatty Liver Disease

The UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) represent major phase II drug-metabolizing enzymes that are also responsible for maintaining cellular homeostasis by metabolism of several endogenous molecules. Perturbations in the expression or function of these enzymes can lead to metabolic disorders and improper management of xenobiotics and endobiotics. Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Because the liver plays a central role in the metabolism of xenobiotics, the purpose of the current study was to determine the effect of human NAFLD progression on the expression and function of UGTs and SULTs in normal, steatosis, NASH (fatty), and NASH (not fatty/cirrhosis) samples. We identified upregulation of UGT1A9, 2B10, and 3A1 and SULT1C4 mRNA in both stages of NASH, whereas UGT2A3, 2B15, and 2B28 and SULT1A1, 2B1, and 4A1 as well as 3′-phosphoadenosine-5′-phosphosulfate synthase 1 were increased in NASH (not fatty/cirrhosis) only. UGT1A9 and 1A6 and SULT1A1 and 2A1 protein levels were decreased in NASH; however, SULT1C4 was increased. Measurement of the glucuronidation and sulfonation of acetaminophen (APAP) revealed no alterations in glucuronidation; however, SULT activity was increased in steatosis compared with normal samples, but then decreased in NASH compared with steatosis. In conclusion, the expression of specific UGT and SULT isoforms appears to be differentially regulated, whereas sulfonation of APAP is disrupted during progression of NAFLD.

UDP-Glucuronosyltransferase Expression in Mouse Liver Is Increased in Obesity- and Fasting-Induced Steatosis

UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lepob/ob (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance.

Nuclear factor-E2-related factor 2 expression in liver is critical for induction of NAD(P)H:quinone oxidoreductase 1 during cholestasis

Abstract Bile duct ligation (BDL) causes hepatocellular oxidative stress and injury. The transcription factor nuclear factor-E2-related factor (Nrf2) induces expression of numerous genes including NAD(P)H:quinone oxidoreductase 1 (Nqo1) during periods of oxidative stress. Therefore, we hypothesized that BDL increases liver expression of mouse antioxidant genes in an Nrf2-dependent manner. BDL or sham surgeries were performed on male C57BL/6, Nrf2-null, and wild-type mice. Livers were collected at 1, 3, and 7 days after surgery for analysis of messenger ribonucleic acid (mRNA) levels of Nrf2-responsive genes as well as Nqo1 protein and activity. BDL increased mRNA expression of multiple Nrf2 genes in mouse liver, compared to sham-operated controls. Follow-up studies investigating protein expression, enzyme activity, and Nrf2 dependency were limited to Nqo1. Nqo1 protein expression and activity in mouse livers was increased 2- to 3-, and 4- to 5-fold at 3 and 7 days after BDL, respectively. Studies also showed that BDL increases Nqo1 mRNA, protein expression, and enzyme activity in livers from wild-type mice, but not in Nrf2-null mice. In conclusion, expression of Nrf2-dependent genes is increased during cholestasis. These studies also demonstrate that Nqo1 expression and activity in mouse liver are induced via an Nrf2-dependent mechanism.

Differential Regulation of Hepatic Transporters in the Absence of Tumor Necrosis Factor-α, Interleukin-1β, Interleukin-6, and Nuclear Factor-κB in Two Models of Cholestasis

Hepatic transporters are responsible for uptake and efflux of bile acids and xenobiotics as an essential aspect of liver function. When normal vectorial transport of bile acids by the apical uptake and canalicular excretion transporters is disrupted, cholestasis ensues, leading to accumulation of toxic bile constituents and considerable hepatocellular damage. The purpose of this study was to assess the role of cytokines and nuclear factor-κB (NF-κB) in the transcriptional regulation of transporters in two models of cholestasis, lipopolysaccharide (LPS) administration and bile duct ligation (BDL). In wild-type (WT) and knockout mouse strains lacking tumor necrosis factor (TNF) receptor-1, interleukin (IL)-1 receptor I, IL-6, or inhibitor of κB(IκB) kinase β, transporter mRNA levels in liver were determined using branched DNA signal amplification 16 h after LPS administration or 3 days after BDL. In WT mice, LPS administration tended to decrease mRNA levels of organic anion-transporting polypeptide (Oatp) 2, Na+-taurocholate cotransporting polypeptide (Ntcp), Oatp1, Oatp4, bile salt excretory protein (Bsep), multidrug resistance-associated protein (Mrp) 2, and Mrp6 compared with saline treatment, whereas it increased Mrp1, 3, and 5 levels. Similar changes were observed in each knockout strain after LPS administration. Conversely, BDL decreased only Oatp1 expression in WT mice, meanwhile increasing expression of Mrp1, 3, and 5 and Oatp2 expression in both WT and knockout strains. Because the transcriptional effects of BDL- and LPS-induced cholestasis reflect dissimilarity in hepatic transporter regulation, we conclude that these disparities are not due to the individual activity of TNF-α, IL-1, IL-6, or NF-κB but to the differences in the mechanism of cholestasis.

Bile salt export pump is dysregulated with altered farnesoid X receptor isoform expression in patients with hepatocellular carcinoma

As a canalicular bile acid effluxer, the bile salt export pump (BSEP) plays a vital role in maintaining bile acid homeostasis. BSEP deficiency leads to severe cholestasis and hepatocellular carcinoma (HCC) in young children. Regardless of the etiology, chronic inflammation is the common pathological process for HCC development. Clinical studies have shown that bile acid homeostasis is disrupted in HCC patients with elevated serum bile acid level as a proposed marker for HCC. However, the underlying mechanisms remain largely unknown. In this study, we found that BSEP expression was severely diminished in HCC tissues and markedly reduced in adjacent nontumor tissues. In contrast to mice, human BSEP was regulated by farnesoid X receptor (FXR) in an isoform‐dependent manner. FXR‐α2 exhibited a much more potent activity than FXR‐α1 in transactivating human BSEP in vitro and in vivo. The decreased BSEP expression in HCC was associated with altered relative expression of FXR‐α1 and FXR‐α2. FXR‐α1/FXR‐α2 ratios were significantly increased, with undetectable FXR‐α2 expression in one third of the HCC tumor samples. A similar correlation between BSEP and FXR isoform expression was confirmed in hepatoma Huh7 and HepG2 cells. Further studies showed that intrahepatic proinflammatory cytokines, such as interleukin‐6 (IL‐6) and tumor necrosis factor alpha (TNF‐α), were significantly elevated in HCC tissues. Treatment of Huh7 cells with IL‐6 and TNF‐α resulted in a marked increase in FXR‐α1/FXR‐α2 ratio, concurrent with a significant decrease in BSEP expression. Conclusion: BSEP expression is severely diminished in HCC patients associated with alteration of FXR isoform expression induced by inflammation. Restoration of BSEP expression through suppressing inflammation in the liver may reestablish bile acid homeostasis. (HEPATOLOGY 2013)