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Find video protocols related to scientific articles indexed in Pubmed.
Identification of a Functional Antioxidant Response Element within the Eighth Intron of the Human ABCC3 Gene.
Drug Metab. Dispos.
PUBLISHED: 10-27-2014
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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, while 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 ChIP-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.
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H1-antihistamines exacerbate high-fat diet-induced hepatic steatosis in wild-type but not in apolipoprotein E knockout mice.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 05-22-2014
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We examined the effects of two over-the-counter H1-antihistamines on the progression of fatty liver disease in male C57Bl/6 wild-type and apolipoprotein E (ApoE)-/- mice. Mice were fed a high-fat diet (HFD) for 3 mo, together with administration of either cetirizine (4 mg/kg body wt) or fexofenadine (40 mg/kg body wt) in drinking water. Antihistamine treatments increased body weight gain, gonadal fat deposition, liver weight, and hepatic steatosis in wild-type mice but not in ApoE-/- mice. Lobular inflammation, acute inflammation, and necrosis were not affected by H1-antihistamines in either genotype. Serum biomarkers of liver injury tended to increase in antihistamine-treated wild-type mice. Serum level of glucose was increased by fexofenadine, whereas lipase was increased by cetirizine. H1-antihistamines reduced the mRNA expression of ApoE and carbohydrate response element-binding protein in wild-type mice, without altering the mRNA expression of sterol regulatory element-binding protein 1c, fatty acid synthase, or ApoB100, in either genotype. Fexofenadine increased both triglycerides and cholesterol ester, whereas cetirizine increased only cholesterol ester in liver, with a concomitant decrease in serum triglycerides by both antihistamines in wild-type mice. Antihistamines increased hepatic levels of conjugated bile acids in wild-type mice, with the effect being significant in fexofenadine-treated animals. The increase was associated with changes in the expression of organic anion transport polypeptide 1b2 and bile salt export pump. These results suggest that H1-antihistamines increase the progression of fatty liver disease in wild-type mice, and there seems to be an association between the severity of disease, presence of ApoE, and increase in hepatic bile acid levels.
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Synergistic interaction between genetics and disease on pravastatin disposition.
J. Hepatol.
PUBLISHED: 01-31-2014
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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.
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Intestine-specific deletion of SIRT1 in mice impairs DCoH2-HNF-1?-FXR signaling and alters systemic bile acid homeostasis.
Gastroenterology
PUBLISHED: 01-07-2014
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Sirtuin 1 (SIRT1), the most conserved mammalian oxidized nicotinamide adenine dinucleotide-dependent protein deacetylase, is an important metabolic sensor in many tissues. However, little is known about its role in the small intestine, which absorbs and senses nutrients. We investigated the functions of intestinal SIRT1 in systemic bile acid and cholesterol metabolism in mice.
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Tissue distribution, ontogeny, and chemical induction of aldo-keto reductases in mice.
Drug Metab. Dispos.
PUBLISHED: 05-09-2013
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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-16?-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.
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Hepatic cytochrome P450 enzyme alterations in humans with progressive stages of nonalcoholic fatty liver disease.
Drug Metab. Dispos.
PUBLISHED: 08-03-2009
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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 alpha and interleukin 1beta 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 1alpha 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.
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Expression and characterization of functional dog flavin-containing monooxygenase 3.
Drug Metab. Dispos.
PUBLISHED: 07-27-2009
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Mammalian flavin-containing monooxygenase (FMO) enzymes catalyze oxidation at nucleophilic, heteroatom centers and are important for drug, xenobiotic, and endogenous substrate metabolism. In human liver, human FMO3 (hFMO3) is the most abundant FMO isoform and is known to contribute to the hepatic clearance of a variety of clinical drugs. The purpose of the current study was to express and compare the dog (beagle) FMO3 (dFMO3) to hFMO3. A full-length dFMO3 cDNA was obtained from liver by reverse transcription-polymerase chain reaction. Using a baculovirus expression system in Spodoptera frugiperda insect cells, dFMO3 was expressed to protein levels of 0.50 nmol/mg, as determined by liquid chromatography-fluorescence detection. Expressed dFMO3 displayed Michaelis-Menten kinetics, catalyzing NADPH-dependent N-oxidation of benzydamine, with K(m) and V(max) values of 18.6 microM and 0.63 nmol N-oxide formed/min/nmol of enzyme, respectively. Benzydamine N-oxidation catalyzed by hFMO3 showed values of 42.6 microM (K(m)) and 3.56 nmol/min/nmol of enzyme (V(max)). Human FMO3 was observed to catalyze the S-oxidation of sulindac sulfide, with respective K(m) and V(max) values of 69.3 microM and 35.4 nmol/min/nmol of enzyme. dFMO3 also catalyzed sulindac sulfide S-oxidation with 6.8 nmol/min/nmol of enzyme being the highest velocity observed. Finally, Western blot analysis indicated protein expression levels of dFMO3 in pooled dog liver and lung microsomes to be 27 and 9 pmol/mg, respectively. In summary, dFMO3 appears to be a functional enzyme expressed at appreciable levels in liver, but one with some kinetic properties that are substantially different from its human homolog hFMO3.
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Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats.
Eur. J. Pharmacol.
PUBLISHED: 03-22-2009
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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.
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