Lisa M. Augustine

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.

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.

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.

Renal xenobiotic transporters are differentially expressed in mice following cisplatin treatment

The goal of this study was to identify alterations in mRNA and protein expression of various xenobiotic transport proteins in mouse kidney during cisplatin-induced acute renal failure. For this purpose, male C57BL/6J mice received a single dose of cisplatin (18 mg/kg, i.p.) or vehicle. Four days later, tissues were collected for assessment of plasma BUN, histopathological analysis of renal lesions, and mRNA and Western blot analysis of renal transporters including organic anion and cation transporters (Oat, Oct), organic anion transporting polypeptides (Oatp), multidrug resistance-associated proteins (Mrp), multidrug resistance proteins (Mdr), breast cancer resistance protein (Bcrp) and multidrug and toxin extrusion proteins (Mate). Cisplatin treatment caused necrosis of renal proximal tubules along with elevated plasma BUN and renal kidney injury molecule-1 mRNA expression. Cisplatin-induced renal injury increased mRNA and protein levels of the efflux transporters Mrp2, Mrp4, Mrp5, Mdr1a and Mdr1b. Uptake transporters Oatp2a1 and Oatp2b1 mRNA were also up-regulated following cisplatin. By contrast, expression of Oat1, Oat2, Oct2 and Oatp1a1 mRNA was reduced in cisplatin-treated mice. Expression of several uptake and efflux transporters was unchanged in cisplatin-treated mice. Apical staining of Mrp2 and Mrp4 proteins was enhanced in proximal tubules from cisplatin-treated mice. Collectively, these expression patterns suggest coordinated regulation of uptake and efflux pathways during cisplatin-induced renal injury. Reduced expression of basolateral and apical uptake transporters along with enhanced transcription of export transporters likely represents an adaptation to lower intracellular accumulation of chemicals, prevent their reabsorption and enhance urinary clearance.

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.

Induction of hepatobiliary efflux transporters in acetaminophen-induced acute liver failure cases.

Alterations in transporter expression may represent a compensatory mechanism of damaged hepatocytes to reduce accumulation of potentially toxic compounds. The present study was conducted to investigate the expression of hepatobiliary efflux transporters in livers from patients after toxic acetaminophen (APAP) ingestion, with livers from patients with primary biliary cirrhosis (PBC) serving as positive controls. mRNA and protein expression of multidrug resistance-associated protein (MRP) 1-6, multidrug resistance protein (MDR) 1-3/P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) in normal (n = 6), APAP overdose (n = 5), and PBC (n = 6) human liver samples were determined by branched DNA and Western blot analysis, respectively. Double immunohistochemical staining of P-gp and proliferating cell nuclear antigen (PCNA), a marker of proliferation, was performed on paraffin-embedded tissue sections. Compared with normal liver specimens, MRP1 and MRP4 mRNA levels were elevated after APAP overdose and in PBC. Up-regulation of MRP5, MDR1, and BCRP mRNA occurred in PBC livers. Protein levels of MRP4, MRP5, BCRP, and P-gp were increased in both disease states, with MRP1 and MRP3 protein also being induced in PBC. Increased P-gp protein was confirmed immunohistochemically and was found to localize to areas of PCNA-positive hepatocytes, which were detected in APAP overdose and PBC livers. The findings from this study demonstrate that hepatic efflux transporter expression is up-regulated in cases of APAP-induced liver failure and PBC. This adaptation may aid in reducing retention of byproducts of cellular injury and bile constituents within hepatocytes. The close proximity of P-gp and PCNA-positive hepatocytes during liver injury suggests that along with cell regeneration, increased efflux transporter expression is a critical response to hepatic damage to protect the liver from additional insult.

The Nrf2 activator oltipraz also activates the constitutive androstane receptor.

Oltipraz (OPZ) is a well known inducer of NAD(P)H:quinone oxidoreductase (NQO1) along with other enzymes that comprise the nuclear factor E2–related factor 2 (Nrf2) battery of detoxification genes. However, OPZ treatment also induces expression of CYP2B, a gene regulated by the constitutive androstane receptor (CAR). Therefore, this study was designed to determine whether OPZ induces gene expression in the mouse liver through activation of CAR in addition to Nrf2. OPZ increased the mRNA expression of both Cyp2b10 and Nqo1 in C57BL/6 mouse livers. As expected, in livers from Nrf2-/- mice, OPZ induction of Nqo1 was reduced, indicating Nqo1 induction is dependent on Nrf2 activation, whereas Cyp2b10 induction was unchanged. The robust induction of Cyp2b10 by OPZ in wild-type mice was completely absent in CAR-/- mice, revealing a CAR-dependent induction by OPZ. OPZ also induced transcription of the human CYP2B6 promoter-reporter containing the phenobarbital (PB) responsive element in mouse liver using an in vivo transcription assay. Additionally, OPZ induced in vivo nuclear accumulation of CAR at 3 h but, as with PB, was unable to reverse androstanol repression of mouse CAR constitutive activity in transiently transfected HepG2 cells. In summary, OPZ induces expression of Cyp2b10 and Nqo1 via the activation of CAR and Nrf2, respectively.

Drug transporter expression and localization in rat nasal respiratory and olfactory mucosa and olfactory bulb.

Uptake of drugs and other xenobiotics from the nasal cavity and into either the brain or systemic circulation can occur through several different mechanisms, including paracellular transport and movement along primary olfactory nerve axons, which extend from the nasal cavity to the olfactory bulb of the brain. The present study was conducted to expand knowledge on a third means of uptake, namely the expression of drug transporters in the rat nasal epithelium. We used branched DNA technology to compare the level of expression of nine transporters [(equilibrative nucleoside transporters (ENT)1 and ENT2; organic cation transporter (OCT)1, 2, and 3; OCTN1; organic anion-transporting polypeptide (OATP)3; and multidrug resistance (MRP)1 and MRP4] in nasal respiratory mucosa, olfactory mucosa, and olfactory bulb to the level of expression of these transporters in the liver and kidney. Transporters with high expression in the nasal respiratory mucosa or olfactory tissues were immunolocalized by immunohistochemistry. ENT1 and ENT2 expression was relatively high in nasal epithelia and olfactory bulb, which may explain the uptake of intranasally administered nucleoside derivatives observed by other investigators. OATP3 immunoreactivity was high in olfactory epithelium and olfactory nerve bundles, which suggests that substrates transported by OATP3 may be candidates for intranasal administration.

Induction of drug metabolism enzymes and transporters by oltipraz in rats

Coordinate regulation of Phase‐I and ‐II enzymes with xenobiotic transporters has been shown after treatment with microsomal enzyme inducers. The chemopreventive agent oltipraz (OPZ) induces Phase‐I and ‐II drug‐metabolizing enzymes such as CYP2B and NQO1. The purpose of this study was to examine the regulation of drug‐metabolizing enzymes and transporters in response to OPZ treatment and to investigate a potential role for constitutive androstane receptor (CAR) in OPZ‐mediated induction. Sprague‐Dawley rats treated with OPZ exhibited increased mRNA and protein levels of both Nqo1 and Cyp2b1/2 by 24 h. To examine whether OPZ activates transporter gene expression via CAR, sexually dimorphic male and female Wistar‐Kyoto (WKY) rats were treated with OPZ and mRNA levels quantified by bDNA signal amplification. OPZ induced Ugt1a6 and Ugt2b1 in males significantly higher than in females, indicating a CAR‐dependent mechanism of induction. However, OPZ induced microsomal epoxide hydrolase, NAD(P)H quinone oxidoreductase, and Cyp3a1/23 equally in both genders, indicating a CAR‐independent mechanism of induction of these genes. Similarly, the transporters Mdr1a, Mdr1b, Mrp3, and Mrp4 were induced by OPZ without any apparent difference between genders. In summary, OPZ coordinately increases multiple hepatic xenobiotic transporter mRNA levels, along with Phase‐I and ‐II enzymes some of which may occur through CAR‐dependent mechanisms.