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The choroid plexus (CP) and blood-brain barrier (BBB) control the movement of several drugs and endogenous compounds between the brain and systemic circulation. The multidrug resistance associated protein (Mrp) efflux transporters form part of these barriers. Several Mrp transporters are positively regulated by the transcription factor nuclear factor erythroid-2-related factor (Nrf2) in liver. The Mrps, Nrf2 and Nrf2-dependent genes are cytoprotective and our aim was to examine basal gender differences in expression of Mrp transporters, Nrf2 and Nrf2-dependent genes (Nqo1 and Ho-1) in the brain-barriers. Previous studies have shown higher expression of Mrp1, Mrp2 and Mrp4 in female mouse liver and kidney. We hypothesized that similar renal/hepatic gender-specific patterns are present in the brain-barrier epithelia interfaces. qPCR and immunoblot analyses showed that Mrp4, Ho-1 and Nqo1 expression was higher in female CP. Mrp1, Mrp2 and Nrf2 expression in the CP had no gender pattern. Female Mrp1, Mrp2 and Mrp4 mouse brain expressions in remaining brain areas, excluding CP, were higher than male. Functional analysis of Mrp4 in CP revealed active accumulation of the Mrp4 model substrate fluo-cAMP. WT female CP had 10-fold higher accumulation in the vascular spaces than males and 60% higher than Mrp4 -/- females. Probenecid blocked all transport. Methotrexate did as well except in Mrp4 -/- females where it had no effect, suggesting compensatory induction of transport occurred in Mrp4 -/- . Collectively, our findings indicate significant gender differences in expression of Mrp transporters and cytoprotective genes in the CP and BBB., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Flavin-containing monooxygenase-3 (FMO3) catalyzes metabolic reactions similar to cytochrome P450 monooxygenase, however, most metabolites of FMO3 are considered non-toxic. Recent findings in our laboratory demonstrated Fmo3 gene induction following toxic acetaminophen (APAP) treatment in mice. The goal of this study was to evaluate Fmo3 gene expression in other diverse mouse models of hepatic oxidative stress and injury. Fmo3 gene regulation by Nrf2 was also investigated using Nrf2 knockout (Nrf2 KO) mice. In our studies, male C57BL/6J mice were treated with toxic doses of hepatotoxicants or underwent bile duct ligation (BDL, 10 days). Hepatotoxicants included APAP (400 mg/kg, 24-72 h), alpha-naphthyl isothiocyanate (ANIT; 50 mg/kg, 2-48 h), carbon tetrachloride (CCl4; 10 or 30 μL/kg, 24 and 48 h) and allyl alcohol (AlOH; 30 or 60 mg/kg, 6 and 24 h). Because oxidative stress activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2), additional studies investigated Fmo3 gene regulation by Nrf2 using Nrf2 knockout (Nrf2 KO) mice. At appropriate time-points, blood and liver samples were collected for assessment of plasma alanine aminotransferase (ALT) activity, plasma and hepatic bile acid levels, as well as liver Fmo3 mRNA and protein expression. Fmo3 mRNA expression increased significantly by 43-fold at 12 h after ANIT treatment, and this increase translates to a 4-fold change in protein levels. BDL also increased Fmo3 mRNA expression by 1899-fold, but with no change in protein levels. Treatment of mice with CCl4 decreased liver Fmo3 gene expression, while no change in expression was detected with AlOH treatment. Nrf2 KO mice are more susceptible to APAP (400mg/kg, 72 h) treatment compared to their wild-type (WT) counterparts, which is evidenced by greater plasma ALT activity. The Fmo3 mRNA and protein expression increased in Nrf2 KO mice after APAP treatment. Collectively, not all hepatotoxicants that produce oxidative stress alter Fmo3 gene expression. Along with APAP, toxic ANIT treatment in mice markedly increased Fmo3 gene expression. While BDL increased the Fmo3 mRNA expression, the protein level did not change. The discrepancy with Fmo3 induction in cholestatic models, ANIT and BDL, is not entirely clear. Results from Nrf2 KO mice with APAP suggest that the transcriptional regulation of Fmo3 during liver injury may not involve Nrf2., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

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.

Mice pretreated with the peroxisome proliferator clofibrate (CFB) are resistant to acetaminophen (APAP) hepatotoxicity. Whereas the mechanism of protection is not entirely known, CFB decreases protein adducts formed by the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI). NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme with antioxidant properties that is responsible for the reduction of cellular quinones. We hypothesized that CFB increases NQO1 activity, which in turn enhances the conversion of NAPQI back to the parent APAP. This could explain the decreases in APAP covalent binding and glutathione depletion produced by CFB without affecting APAP bioactivation to NAPQI. Administration of CFB (500mg/kg, i.p.) to male CD-1 mice for 5 or 10 days increased NQO1 protein and activity levels. To evaluate the capacity of NQO1 to reduce NAPQI back to APAP, we utilized a microsomal activating system. Cytochrome P450 enzymes present in microsomes bioactivate APAP to NAPQI, which binds the electrophile trapping agent, N-acetyl cysteine (NAC). We analyzed the formation of APAP-NAC metabolite in the presence of human recombinant NQO1. Results indicate that NQO1 is capable of reducing NAPQI. The capacity of NQO1 to amelioriate APAP toxicity was then evaluated in primary hepatocytes. Primary hepatocytes isolated from mice dosed with CFB are resistant to APAP toxicity. These hepatocytes were also exposed to ES936, a high affinity, and irreversible inhibitor of NQO1 in the presence of APAP. Concentrations of ES936 that resulted in over 94% inhibition of NQO1 activity did not increase the susceptibility of hepatocytes from CFB treated mice to APAP. Whereas NQO1 is mechanistically capable of reducing NAPQI, CFB-mediated hepatoprotection does not appear to be dependent upon enhanced expression of NQO1.

Acetaminophen (APAP) toxicity involves both arylative and oxidative mechanisms. The shark bile salt, 5 beta-scymnol (5beta-S), has been demonstrated to act as an antioxidant and free radical scavenger in vitro. To determine if 5beta-S protects against either APAP-induced hepatic or renal toxicity, 3-4-month-old male Swiss Laca mice were given APAP (500 mg/kg), and 5beta-S (100 mg/kg) was given at 0 and 2 h after APAP. Plasma SDH at 12 h after APAP alone was 1630 U/l and BUN was 19 mg/dl versus 20 U/l and 10 mg/dl, respectively, in controls. Either simultaneous or 2 h delayed treatment with 5beta-S significantly decreased the APAP-induced SDH increase while only the simultaneous pretreatment prevented the BUN elevation. 5beta-S alone did not increase liver glutathione content. Western analysis of APAP covalent binding using anti-APAP antibodies indicated the 5beta-S did not alter protein arylation either qualitatively or quantitatively. These results suggest that 5beta-S treatment did not impair APAP activation and are consistent with 5beta-S protection that likely results from its antioxidant activity.

Pretreatment of mice with clofibrate (CFB) has been shown to protect against acetaminophen (APAP) hepatotoxicity. To determine if pretreatment with CFB prevents the toxicity of other model hepatotoxicants, male C57BL6J or CD-1 mice received 500 mg CFB/kg, i.p., daily for 10 days, and then were challenged with either 250 mg bromobenzene (BrB)/kg, 0.025 ml carbon tetrachloride (CCl4)/kg or 0.5 ml chloroform (CHCl3)/kg. Liver and kidney injury was assessed by plasma sorbitol dehydrogenase activity (SDH) and blood urea nitrogen (BUN), respectively and histopathology. Challenge with BrB significantly elevated plasma SDH activity in C57Bl6J mice. This was prevented in CFB pretreated mice receiving the same dose of BrB. Changes in BUN were not detected in either group of BrB treated mice. Similarly, pretreatment of male CD-1 mice with CFB significantly reduced CCl4-induced elevation in plasma SDH activity, with no BUN elevation detected in either group. CFB pretreatment also diminished elevation in plasma SDH activity produced by CHCl3 in CD-1 mice, while BUN was significantly elevated in both groups, indicating that CFB did not protect against CHCl3-induced nephrotoxicity. Histopathological examination of liver and kidney sections confirmed these results. This study shows that mice pretreated with CFB were protected from toxicity at 24 h after challenge with other model hepatotoxic agents besides APAP.

Fatty acid ethyl esters (FAEE) are the end products of a non-oxidative pathway for ethanol metabolism in a variety of human, rabbit, rat and murine tissues. Our objective was to determine the significance of this pathway in the metabolism of ethanol by the rat lung. In vitro, 14C-labeled ethyl oleate formation was assayed in the lung and compared with the pancreas, liver, heart and brain. Lipids were extracted with acetone, and 14C-labeled ethyl oleate was isolated and quantified by thin layer chromatography (TLC) and scintillation spectrometry. FAEE synthetic activity in the lungs (in vitro) was found to be intermediate among the organs examined. In vivo, male rats received 10% ethanol in their drinking water with or without daily i.p. injections of 4-methylpyrazole (1 mmol/kg body wt) for 15 days. Another group of male rats received 4 g/kg body wt ethanol as a 50% (v/v) solution by gavage every 12 h for 2 days. FAEE from the three organs with the highest in vitro activity for FAEE synthesis (pancreas, liver and lung) were extracted with acetone, isolated from normal lipids by TLC and separated by gas chromatography. The lung had lower FAEE-forming activity than the pancreas or the liver in the 15-day studies. However, in the 2-day study, the lung had higher activity than the liver but lower activity than the pancreas. Ethyl oleate, ethyl stearate and ethyl palmitate were the predominant FAEE formed in the intact organism. Ethanol-induced FAEE may play a role in the development of alcohol-related injuries to the lung.