Rhiannon N. Hardwick

Diversity in Antioxidant Response Enzymes in Progressive Stages of Human Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD), which occurs in approximately 17 to 40% of Americans, encompasses progressive stages of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH). Inflammation and oxidative stress are known characteristics of NAFLD; however, the precise mechanisms occurring during disease progression remain unclear. The purpose of the current study was to determine whether the expression or function of enzymes involved in the antioxidant response, NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione transferase (GST), and glutamate cysteine ligase, are altered in the progression of human NAFLD. Human livers staged as normal, steatotic, NASH (fatty), and NASH (not fatty) were obtained from the Liver Tissue Cell Distribution System. NQO1 mRNA, protein, and activity tended to increase with disease progression. mRNA levels of the GST isoforms A1, A2, A4, M3, and P1 increased with NAFLD progression. Likewise, GST A and P protein increased with progression; however, GST M protein levels tended to decrease. Of interest, total GST activity toward the substrate 1-chloro-2,4-dinitrobenzene decreased with NAFLD progression. GSH synthesis does not seem to be significantly dysregulated in NAFLD progression; however, the GSH/oxidized glutathione redox ratio seemed to be reduced with disease severity, indicating the presence of oxidative stress and depletion of GSH throughout progression of NAFLD. Malondialdehyde concentrations were significantly increased with disease progression, further indicating the presence of oxidative stress. Nuclear immunohistochemical staining of nuclear factor E2-related factor 2 (Nrf2), an indicator of activation of the transcription factor, was evident in all stages of NAFLD. The current data suggest that Nrf2 activation occurs in response to disease progression followed by induction of specific Nrf2 targets, whereas functionality of specific antioxidant defense enzymes seems to be impaired as NAFLD progresses.

Analysis of Global and Absorption, Distribution, Metabolism, and Elimination Gene Expression in the Progressive Stages of Human Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is characterized by a series of pathological changes that range from simple fatty liver to nonalcoholic steatohepatitis (NASH). The objective of this study is to describe changes in global gene expression associated with the progression of human NAFLD. This study is focused on the expression levels of genes responsible for the absorption, distribution, metabolism, and elimination (ADME) of drugs. Differential gene expression between three clinically defined pathological groups—normal, steatosis, and NASH—was analyzed. Genome-wide mRNA levels in samples of human liver tissue were assayed with Affymetrix GeneChip Human 1.0ST arrays. A total of 11,633 genes exhibited altered expression out of 33,252 genes at a 5% false discovery rate. Most gene expression changes occurred in the progression from steatosis to NASH. Principal component analysis revealed that hepatic disease status was the major determinant of differential ADME gene expression rather than age or sex of sample donors. Among the 515 drug transporters and 258 drug-metabolizing enzymes (DMEs) examined, uptake transporters but not efflux transporters or DMEs were significantly over-represented in the number of genes down-regulated. These results suggest that uptake transporter genes are coordinately targeted for down-regulation at the global level during the pathological development of NASH and that these patients may have decreased drug uptake capacity. This coordinated regulation of uptake transporter genes is indicative of a hepatoprotective mechanism acting to prevent accumulation of toxic intermediates in disease-compromised hepatocytes.

Variations in ATP-Binding Cassette Transporter Regulation during the Progression of Human Nonalcoholic Fatty Liver Disease

Transporters located on the sinusoidal and canalicular membranes of hepatocytes regulate the efflux of drugs and metabolites into blood and bile, respectively. Changes in the expression or function of these transporters during liver disease may lead to a greater risk of adverse drug reactions. Nonalcoholic fatty liver disease (NAFLD) is a progressive condition encompassing the relatively benign steatosis and the more severe, inflammatory state of nonalcoholic steatohepatitis (NASH). Here, we present an analysis of the effect of NAFLD progression on the major ATP-binding cassette (ABC) efflux transport proteins ABCC1–6, ABCB1, and ABCG2. Human liver samples diagnosed as normal, steatotic, NASH (fatty), and NASH (not fatty) were analyzed. Increasing trends in mRNA expression of ABCC1, ABCC4–5, ABCB1, and ABCG2 were found with NAFLD progression, whereas protein levels of all transporters exhibited increasing trends with disease progression. Immunohistochemical staining of ABCC3, ABCB1, and ABCG2 revealed no alterations in cellular localization during NAFLD progression. ABCC2 staining revealed an alternative mechanism of regulation in NASH in which the transporter appears to be internalized away from the canalicular membrane. This correlated with a preferential shift in the molecular mass of ABCC2 from 200 to 180 kDa in NASH, which has been shown to be associated with a loss of glycosylation and internalization of the protein. These data demonstrate increased expression of multiple efflux transporters as well as altered cellular localization of ABCC2 in NASH, which may have profound effects on the ability of patients with NASH to eliminate drugs in an appropriate manner.

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.

Role of Hepatic Efflux Transporters in Regulating Systemic and Hepatocyte Exposure to Xenobiotics

Hepatic efflux transporters include numerous well-known and emerging proteins localized to the canalicular or basolateral membrane of the hepatocyte that are responsible for the excretion of drugs into the bile or blood, respectively. Altered function of hepatic efflux transporters due to drug-drug interactions, genetic variation, and/or disease states may lead to changes in xenobiotic exposure in the hepatocyte and/or systemic circulation. This review focuses on transport proteins involved in the hepatocellular efflux of drugs and metabolites, discusses mechanisms of altered transporter function as well as the interplay between multiple transport pathways, and highlights the importance of considering intracellular unbound concentrations of transporter substrates and/or inhibitors. Methods to evaluate hepatic efflux transport and predict the effects of impaired transporter function on systemic and hepatocyte exposure are discussed, and the sandwich-cultured hepatocyte model to evaluate comprehensively the role of hepatic efflux in the hepatobiliary disposition of xenobiotics is characterized.

Molecular Mechanism of Altered Ezetimibe Disposition in Nonalcoholic Steatohepatitis

Ezetimibe (EZE) lowers serum lipid levels by blocking cholesterol uptake in the intestine. Disposition of EZE and its pharmacologically active glucuronide metabolite (EZE-GLUC) to the intestine is dependent on hepatobiliary efflux. Previous studies suggested that hepatic transporter expression and function may be altered during nonalcoholic steatohepatitis (NASH). The purpose of the current study was to determine whether NASH-induced changes in the expression and function of hepatic transporters result in altered disposition of EZE and EZE-GLUC. Rats fed a methionine- and choline-deficient (MCD) diet for 8 weeks were administered 10 mg/kg EZE either by intravenous bolus or oral gavage. Plasma and bile samples were collected over 2 h followed by terminal urine and tissue collection. EZE and EZE-GLUC concentrations were determined by liquid chromatography-tandem mass spectrometry. The sinusoidal transporter Abcc3 was induced in MCD rats, which correlated with increased plasma concentrations of EZE-GLUC, regardless of dosing method. Hepatic expression of the biliary transporters Abcc2 and Abcb1 was also increased in MCD animals, but the biliary efflux of EZE-GLUC was slightly diminished, whereas biliary bile acid concentrations were unaltered. The cellular localization of Abcc2 and Abcb1 appeared to be internalized away from the canalicular membrane in MCD livers, providing a mechanism for the shift to plasma drug efflux. The combination of induced expression and altered localization of efflux transporters in NASH shifts the disposition profile of EZE-GLUC toward plasma retention away from the site of action. This increased plasma retention of drugs in NASH may have implications for the pharmacological effect and safety of numerous drugs.

Increased Susceptibility to Methotrexate-Induced Toxicity in Nonalcoholic Steatohepatitis

Hepatic drug metabolizing enzymes and transporters play a crucial role in determining the fate of drugs, and alterations in liver function can place individuals at greater risk for adverse drug reactions (ADRs). We have shown that nonalcoholic steatohepatitis (NASH) leads to changes in the expression and localization of enzymes and transporters responsible for the disposition of numerous drugs. The purpose of this study was to determine the effect of NASH on methotrexate (MTX) disposition and the resulting toxicity profile. Sprague Dawley rats were fed either a control or methionine-choline-deficient diet for 8 weeks to induce NASH, then administered a single ip vehicle, 10, 40, or 100 mg/kg MTX injection followed by blood, urine, and feces collection over 96 h with terminal tissue collection. At the onset of dosing, Abcc1-4, Abcb1, and Abcg2 were elevated in NASH livers, whereas Abcc2 and Abcb1 were not properly localized to the membrane, similar to that previously observed in human NASH. NASH rodents receiving 40-100 mg/kg MTX exhibited hepatocellular damage followed by initiation of repair, whereas damage was absent in controls. NASH rodents receiving 100 mg/kg MTX exhibited slightly greater renal toxicity, indicating multiple organ toxicity, despite the majority of the dose being excreted by 6 h. Intestinal toxicity in NASH however, was strikingly less severe than controls, and coincided with reduced fecal MTX excretion. Because MTX-induced gastrointestinal toxicity limits the dose escalation necessary for cancer remission, these data suggest a greater risk for life-threatening MTX-induced hepatic and renal toxicity in NASH in the absence of overt gastrointestinal toxicity.

Synergistic interaction between genetics and disease on pravastatin disposition.

BACKGROUND & AIMS A genome wide association study and multiple pharmacogenetic studies have implicated the hepatic uptake transporter organic anion transporting polypeptide-1B1 (OATP1B1) in the pharmacokinetics and musculoskeletal toxicity of statin drugs. Other OATP uptake transporters can participate in the transport of pravastatin, partially compensating for the loss of OATP1B1 in patients carrying the polymorphism. Non-alcoholic steatohepatitis (NASH) in humans and in a diet-induced rodent model alter the expression of multiple OATP transporters. METHODS To determine how genetic alteration in one Oatp transporter can interact with NASH-associated changes in Oatp expression we measured the disposition of intravenously administered pravastatin in Slco1b2 knockout (Slco1b2(-/-)) and wild-type (WT) mice fed either a control or a methionine and choline deficient (MCD) diet to induce NASH. RESULTS Genetic loss of Oatp1b2, the rodent ortholog of human OATP1B transporters, caused a modest increase in pravastatin plasma concentrations in mice with healthy livers. Although a diet-induced model of NASH decreased the expression of multiple hepatic Oatp transporters, it did not alter the disposition of pravastatin compared to WT control mice. In contrast, the combination of NASH-associated decrease in compensatory Oatp transporters and Oatp1b2 genetic loss caused a synergistic increase in plasma area under the curve (AUC) and tissue concentrations in kidney and muscle. CONCLUSIONS Our data show that NASH alters the expression of multiple hepatic uptake transporters which, due to overlapping substrate specificity among the OATP transporters, may combine with the pharmacogenetic loss of OATP1B1 to increase the risk of statin-induced adverse drug reactions.

Modeling Human Nonalcoholic Steatohepatitis-Associated Changes in Drug Transporter Expression Using Experimental Rodent Models

Nonalcoholic fatty liver disease is a prevalent form of chronic liver disease that can progress to the more advanced stage of nonalcoholic steatohepatitis (NASH). NASH has been shown to alter drug transporter regulation and may have implications in the development of adverse drug reactions. Several experimental rodent models have been proposed for the study of NASH, but no single model fully recapitulates all aspects of the human disease. The purpose of the current study was to determine which experimental NASH model best reflects the known alterations in human drug transporter expression to enable more accurate drug disposition predictions in NASH. Both rat and mouse NASH models were used in this investigation and include the methionine and choline deficient (MCD) diet model, atherogenic diet model, ob/ob and db/db mice, and fa/fa rats. Pathologic scoring evaluations demonstrated that MCD and atherogenic rats, as well as ob/ob and db/db mice, developed NASH. Liver mRNA and protein expression analyses of drug transporters showed that in general, efflux transporters were induced and uptake transporters were repressed in the rat MCD and the mouse ob/ob and db/db models. Lastly, concordance analyses suggest that both the mouse and rat MCD models as well as mouse ob/ob and db/db NASH models show the most similarity to human transporter mRNA and protein expression. These results suggest that the MCD rat and mouse model, as well as the ob/ob and db/db mouse models, may be useful for predicting altered disposition of drugs with similar kinetics across humans and rodents.

Experimental Nonalcoholic Steatohepatitis Increases Exposure to Simvastatin Hydroxy Acid by Decreasing Hepatic Organic Anion Transporting Polypeptide Expression

Simvastatin (SIM)-induced myopathy is a dose-dependent adverse drug reaction (ADR) that has been reported to occur in 18.2% of patients receiving a 40- to 80-mg dose. The pharmacokinetics of SIM hydroxy acid (SIMA), the bioactive form of SIM, and the occurrence of SIM-induced myopathy are linked to the function of the organic anion transporting polypeptide (Oatp) hepatic uptake transporters. Genetic polymorphisms in SLCO1B1, the gene for human hepatic OATP1B1, cause decreased elimination of SIMA and increased risk of developing myopathy. Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease, and is known to alter drug transporter expression and drug disposition. The purpose of this study was to assess the metabolism and disposition of SIM in a diet-induced rodent model of NASH. Rats were fed a methionine- and choline-deficient diet for 8 weeks to induce NASH and SIM was administered intravenously. Diet-induced NASH caused increased plasma retention and decreased biliary excretion of SIMA due to decreased protein expression of multiple hepatic Oatps. SIM exhibited increased volume of distribution in NASH as evidenced by increased muscle, decreased plasma, and no change in biliary concentrations. Although Cyp3a and Cyp2c11 proteins were decreased in NASH, no alterations in SIM metabolism were observed. These data, in conjunction with our previous data showing that human NASH causes a coordinated downregulation of hepatic uptake transporters, suggest that NASH-mediated transporter regulation may play a role in altered SIMA disposition and the occurrence of myopathy.