Andrew J. Lickteig

Hepatic Cytochrome P450 Enzyme Alterations in Humans with Progressive Stages of Nonalcoholic Fatty Liver Disease

Members of the cytochrome P450 (P450) enzyme families CYP1, CYP2, and CYP3 are responsible for the metabolism of approximately 75% of all clinically relevant drugs. With the increased prevalence of nonalcoholic fatty liver disease (NAFLD), it is likely that patients with this disease represent an emerging population at significant risk for alterations in these important drug-metabolizing enzymes. The purpose of this study was to determine whether three progressive stages of human NALFD alter hepatic P450 expression and activity. Microsomes isolated from human liver samples diagnosed as normal, n = 20; steatosis, n = 11; nonalcoholic steatohepatitis (NASH) (fatty liver), n = 10; and NASH (no longer fatty), n = 11 were analyzed for P450 mRNA, protein, and enzyme activity. Microsomal CYP1A2, CYP2D6, and CYP2E1 mRNA levels were decreased with NAFLD progression, whereas CYP2A6, CYP2B6, and CYP2C9 mRNA expression increased. Microsomal protein expression of CYP1A2, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 tended to decrease with NAFLD progression. Likewise, functional activity assays revealed decreasing trends in CYP1A2 (p = 0.001) and CYP2C19 (p = 0.05) enzymatic activity with increasing NAFLD severity. In contrast, activity of CYP2A6 (p = 0.001) and CYP2C9 (diclofenac, p = 0.0001; tolbutamide, p = 0.004) was significantly increased with NAFLD progression. Increased expression of proinflammatory cytokines tumor necrosis factor α and interleukin 1β was observed and may be responsible for observed decreases in respective P450 activity. Furthermore, elevated CYP2C9 activity during NAFLD progression correlated with elevated hypoxia-induced factor 1α expression in the later stages of NAFLD. These results suggest that significant and novel changes occur in hepatic P450 activity during progressive stages of NAFLD.

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.

Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of diagnoses ranging from simple fatty liver (SFL), to non-alcoholic steatohepatitis (NASH). This study aimed to determine the effect of moderate and severe NAFLD on hepatic transporter expression and function in vivo. Rats were fed a high-fat diet (SFL model) or a methionine-choline-deficient diet (NASH model) for eight weeks. Hepatic uptake transporter function was determined by bromosulfophthalein (BSP) disposition. Transporter expression was determined by branched DNA signal amplification assay and western blotting; inflammation was identified by immunostaining of liver slices for interleukin 1 beta (IL-1beta). MC- rats showed significant retention of BSP in the plasma when compared to control rats. Hepatic NTCP, OATP1a1, 1a4, 1b2 and 2b1; and OAT 2 and 3 mRNA levels were significantly decreased in high-fat and MC- diet rats when compared to control. Protein expression of OATP1a1 was significantly decreased in high-fat animals, while OATP1a1 and OATP1b2 expressions were significantly lower in MC- rats when compared to control. Liver tissue from high-fat and MC- rats stained positive for IL-1beta, a pro-inflammatory cytokine known to decrease expression of NTCP, OATP and OAT transporters, suggesting a plausible mechanism for the observed transporter alterations. These data suggest that different stages of NAFLD result in altered hepatic uptake transporter expression that can lead to a functional impairment of xenobiotic uptake from the blood. Furthermore, NAFLD may alter the plasma retention time of clinically relevant drugs that are reliant on these transporters and may increase the potential drug toxicity.

Tissue distribution, ontogeny and induction of the transporters Multidrug and toxin extrusion (MATE) 1 and MATE2 mRNA expression levels in mice.

Transporters are expressed in a wide variety of tissues where they perform the critical function of enabling anionic and cationic chemicals of exogenous and endogenous origin to cross otherwise impermeable cell membranes. The Multidrug and toxin extrusion (MATE) transporters mediate cellular efflux of a variety of organic cations, including many drugs. The purpose of the current study was to determine (1) constitutive expression levels of MATE mRNA in various tissues, (2) whether there are gender differences in the expression of MATEs, (3) the ontogenic expression pattern of MATE1 in kidney and (4) whether MATEs are pharmacologically inducible in liver via activation of known transcription factors. In both male and female mice, MATE1 mRNA levels were highest in the kidney, where male expression was higher than female. MATE2 mRNA expression levels were the highest in the testis, where high expression was localized to Sertoli cells, a critical cell type of the blood testis barrier. In female mice, MATE2 mRNA levels were expressed most highly in the colon. The ontogenic pattern of expression of MATE1 mRNA in the kidneys of both males and females was gradual, with levels increasing steadily from prenatal day -2 to 45 days of age, and a gender difference appearing at day 30. Of the transcription factor activators examined (AhR, CAR, Nrf2, PPARalpha and PXR), none were capable of altering MATE1 or MATE2. The current findings support a potential role for MATE1 and MATE2 in a wide range of tissues and, notably, a unique role for MATE2 in the blood-testis barrier.

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.

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.

Tissue distribution, ontogeny, and chemical induction of aldo-keto reductases in mice.

Aldo-keto reductases (Akrs) are a conserved group of NADPH-dependent oxido-reductase enzymes. This study provides a comprehensive examination of the tissue distribution of the 16 substrate-metabolizing Akrs in mice, their expression during development, and whether they are altered by chemicals that activate distinct transcriptional factor pathways. Akr1c6, 1c14, 1c20, and 1c22 are primarily present in liver; Akr1a4, 1c18, 1c21, and 7a5 in kidney; Akr1d1 in liver and kidney; Akr1b7 in small intestine; Akr1b3 and Akr1e1 in brain; Akr1b8 in testes; Akr1c14 in ovaries; and Akrs1c12, 1c13, and 1c19 are expressed in numerous tissues. Liver expression of Akr1d1 and Akr1c is lowest during prenatal and postnatal development. However, by 20 days of age, liver Akr1d1 increases 120-fold, and Akr1c mRNAs increase as much as 5-fold (Akr1c19) to 1000-fold (Akr1c6). Treatment of mice with chemical activators of transcription factors constitutive androgen receptor (CAR), pregnane X receptor (PXR), and the nuclear factor-erythroid-2 (Nrf2) transcription factor alters liver mRNAs of Akrs. Specifically, CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases mRNAs of Akr1b7, Akr1c6, Akr1c19, and Akr1d1, whereas PXR activation by 5-pregnenolone-16α-carbonitrile (PCN) increases the mRNA of Akr1b7 and suppresses mRNAs of Akr1c13 and Akr1c20. The Nrf2 activator 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) induces mRNAs of Akr1c6 and Akr1c19. Moreover, Nrf2-null and Nrf2 overexpressing mice demonstrate that this induction is Nrf2-dependent.

Identification of a Functional Antioxidant Response Element within the Eighth Intron of the Human ABCC3 Gene

The ATP-binding cassette (ABC) family of transporters, including ABCC3, is a large family of efflux pumps that plays a pivotal role in the elimination of xenobiotics from the body. ABCC3 has been reported to be induced during hepatic stress conditions and through the progression of some forms of cancer. Several lines of evidence have implicated the transcription factor nuclear factor (erythroid-derived 2)–like 2 (Nrf2) in this induction. However, although rodent models have been investigated, a functional antioxidant response element (ARE) in the human ABCC3 gene has not been identified. The purpose of this study was to identify and characterize the ARE(s) responsible for mediating the Nrf2-dependent induction of the human ABCC3 gene. A high-throughput chromatin immunoprecipitation-sequencing analysis performed in A549 cells revealed a specific interaction between Nrf2 and the eighth intron of the human ABCC3 gene rather than the more prototypical flanking region of the gene. Subsequent in silico analysis of the intron identified two putative ARE elements that contained the core consensus ARE sequence commonly found in several Nrf2-responsive genes. Functional characterization of these two AREs using luciferase-reporter constructs with ARE mutant constructs revealed that one of these putative AREs is functionally active. Finally, DNA pull-down assays confirmed specific binding of these intronic AREs by Nrf2 in vitro. Our findings identify a functional Nrf2 response element within the eighth intron of the ABCC3 gene, which may provide mechanistic insight into the induction of ABCC3 during antioxidant response stimuli.

Gender divergent expression of Nqo1 in Sprague Dawley and August Copenhagen x Irish rats.

In the mammalian liver, there is an abundance of enzymes that function to enable the safe and efficient elimination of potentially harmful xenobiotics that are encountered through environmental exposure. A variety of factors, including gender and genetic polymorphisms, contribute to the variation between an individual systems detoxification capacity and thus its ability to protect itself against oxidative stress, cellular damage, cell death, etc. NAD(P)H:quinone oxidoreducatase 1 (Nqo1) is an antioxidant enzyme that plays a major role in reducing reactive electrophiles, thereby protecting cells from free‐radical damage and oxidative stress. The goal of this study was to determine the gender‐specific expression and inducibility of Nqo1 in the Sprague Dawley (SD) and August Copenhagen x Irish (ACI) rat strains, two strains that are commonly used in drug metabolism and drug‐induced enzyme induction, toxicity, and carcinogenesis studies. Nqo1 mRNA, protein, and activity levels were determined through 96 h in SD and ACI males and females following treatment with known Nqo1 inducers oltipraz and butylated hydroxyanisole. In the SD strain, gender dimorphic expression of Nqo1 was observed with female mRNA, protein, and activity levels being significantly higher than in males. In contrast, there were minimal differences in Nqo1 mRNA, protein, and activity levels between ACI males and females. The gender dimorphic expression of Nqo1 in the SD rats was maintained through the course of induction, with female‐induced levels greater than male‐induced levels indicating that SD females may have a greater capacity to protect against oxidative stress and thus a decreased susceptibility to carcinogens.

Activation of Constitutive Androstane Receptor (CAR) in Mice Results in Maintained Biliary Excretion of Bile Acids Despite a Marked Decrease of Bile Acids in Liver

Activation of Constitutive Androstane Receptor (CAR) protects against bile acid (BA)-induced liver injury. This study was performed to determine the effect of CAR activation on bile flow, BA profile, as well as expression of BA synthesis and transport genes. Synthetic CAR ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) was administered to mice for 4 days. BAs were quantified by UPLC-MS/MS (ultraperformance liquid chromatography-tandem mass spectrometry). CAR activation decreases total BAs in livers of male (49%) and female mice (26%), largely attributable to decreases of the 12α-hydroxylated BA taurocholic acid (T-CA) (males (M) 65%, females (F) 45%). Bile flow in both sexes was increased by CAR activation, and the increases were BA-independent. CAR activation did not alter biliary excretion of total BAs, but overall BA composition changed. Excretion of muricholic (6-hydroxylated) BAs was increased in males (101%), and the 12α-OH proportion of biliary BAs was decreased in both males (37%) and females (28%). The decrease of T-CA in livers of males and females correlates with the decreased mRNA of the sterol 12α-hydroxylase Cyp8b1 in males (71%) and females (54%). As a response to restore BAs to physiologic concentrations in liver, mRNA of Cyp7a1 is upregulated following TCPOBOP (males 185%, females 132%). In ilea, mRNA of the negative feedback regulator Fgf15 was unaltered by CAR activation, indicating biliary BA excretion was sufficient to maintain concentrations of total BAs in the small intestine. In summary, the effects of CAR activation on BAs in male and female mice are quite similar, with a marked decrease in the major BA T-CA in the liver.