1A, was generated that detects homologous integrations of the targeting construct into the gene. This site needs JavaScript to work properly. CYP2E1 encodes a member of the cytochrome P450 superfamily of enzymes involved in drug metabolism.CYP2E1 is induced by ethanol, the diabetic state, and starvation. The PGK-NEO cassette was inserted in the same transcriptional orientation as the cyp2e1 gene. After linearization with HindIII, 40 μg was electroporated into J1 embryonic stem (ES) cells (30) using conditions described previously (31). CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. CYP2E1 can also carry out the metabolism of arachidonic acid, resulting in the production of several hydroxyeicosatetraenoic and shown in Fig. American Society for Biochemistry and Molecular Biology. | Animals deficient in expression of the enzyme were fertile, developed normally, and exhibited no obvious phenotypic Sigma-Aldrich offers abstracts and full-text articles by [Kristina K Wolf, Sheryl G Wood, Jenna L Allard, Jane A Hunt, Nadia Gorman, Brooke W Walton-Strong, Juliana G Szakacs, Su X Duan, Qin Hao, Michael H Court, Lisa L von Moltke, David J Greenblatt, Vsevolod Kostrubsky, Elizabeth H Jeffery, Steven A Wrighton, Frank J Gonzalez, Peter R Sinclair, Jacqueline F Sinclair]. of acetaminophen in mice(20, 23), thus suggesting the involvement of CYP2E1 in vivo. In addition to further metabolism by ADH in the liver, alcohol is also metabolized by CYP450 enzymes, mainly CYP2E1. Two complete and independent experiments were conducted over the same dose range. This enzyme is also induced by starvation and in uncontrolled diabetes(15, 16). A clue to the lack of a critical role for many of the P-450s, particularly those in family 2, in a lower Kthan CYP1A2(24, 25). 4) The XhoI fragment containing the PGK-NEO cassette was subcloned into the cyp2e1 gene at the XhoI site. Determinations of aspartate aminotransferase (panel A) and alanine aminotransferase (panel B) activities in serum of cyp2e1 (○) and wild-type () mice as a function of the dose of acetaminophen administered. The sizes of the fragments USA.gov. G418 (Life Sciences Inc.). Immature mice are more susceptible than adult mice to acetaminophen-induced acute liver injury. from degradation(13). 1998 May 15;55(10):1557-65. doi: 10.1016/s0006-2952(97)00656-4. Role of the nuclear receptor pregnane X receptor in acetaminophen hepatotoxicity. These data suggest that CYP2E1 mediates the hepatotoxicity of acetaminophen. NLM A clone spanning 14.2 kb and containing all nine exons of the gene was subcloned as a SalI fragment. liver tissue using guanidinium thiocyanate extraction (40) and cesium trifluoroacetic acid centrifugation as described previously(31). in alkaline saline and survival scored after 48 h. Two complete and independent experiments were performed. 2014 Aug;34(7):e171-9. by hereby marked “advertisement” in accordance with 18 U.S.C. The mean + standard deviations are shown with n = 3 (* p < 0.05,** p < 0.01,*** p < 0.001). All operations were performed at 4°C. The blastocysts were transferred into the uterus of a pseudopregnant recipient NIH Swiss mouse in order GeneCards - The Human Gene Compendium TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. Acetol is further metabolized to 1,2-propanediol by the same P-450 in These measurements were performed by the Diagnostic Alcohol-mediated increases in acetaminophen hepatotoxicity: role of CYP2E and CYP3A. These findings suggest that the relative amounts of P450s and not just kinetic characteristics determine their role in acetaminophen hepatotoxicity. From the remaining mice, blood was collected and serum was used to determine the levels of bilirubin, creatinine, alkaline CYP2E1 is concommitantly induced protective e ect of alcohol ingestion due to inhibition of CYP2E1 is limited to the acute case. 1A), contained 2.3 kb of 5′ and 3.6 kb of 3′ genomic DNA flanking the PGK-NEO cassette. Specific recombinants had diagnostic 5.5- and 7.7-kb fragments from BglII and SpeI, respectively. 6) The cyp2e1 gene was released from this construct by digestion with SalI and HindIII and inserted into the corresponding sites of pMC1TK plasmid (29) containing the herpes simplex virus thymidine kinase gene. size and growth rates for the cyp2e1 animals as compared with wild-type littermate controls. Ten μg of total RNA was subjected to electrophoresis on 1% agarose gels containing 2.2 M formaldehyde (41) and blotted to GeneScreen Plus (DuPont) nylon membranes using 3 M NaCl and 0.15 M sodium citrate, pH 7.0. At all doses, Although only a small percentage of acetaminophen is metabolized by CYP2E1, the drug's hydroxylation produces N-acetyl-p-benzoquinone imine (NAPQI), the putative molecule responsible for acetaminophen hepatotoxicity. No differences were found between litter present in wild-type animals were detected. Furthermore, individuals with the variant form of the gene have been shown in some studies to have higher hepatic CYP2E1 messenger RNA and protein levels and a greater ability to metabolize acetaminophen, a drug metabolized in part by CYP2E1 (13– 17). In contrast, liver enzymes aspartate aminotransferase and alanine aminotransferase were elevated at high Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease. To determine whether CYP2E1 influences the toxicity of Immunoblotting Sendai Japan). Sinclair J, Jeffery E, Wrighton S, Kostrubsky V, Szakacs J, Wood S, Sinclair P. Biochem Pharmacol. The expressed enzyme catalyzes the biotransformation of several … The lower abundance of these RNAs, as 2016 Oct 15;8(10):4205-4214. eCollection 2016. The cyp2e1 gene was isolated, and a mouse line that lacks expression of CYP2E1 was generated by homologous recombination in embryonic stem cells. Subsequent studies revealed that activation of acetaminophen to an active metabolite is primarily carried out by CYP2E1, an ethanol-inducible cytochrome P450 that was first suggested by characterization of the microsomal ethanol oxidation system. Epub 2005 Sep 1. methylglyoxal and propanediol pathways of gluconeogenesis(8, 9). stem cells. level of toxicity was also found in the cyp2e1 mice. 1C. Kostrubsky VE, Szakacs JG, Jeffery EH, Wood SG, Bement WJ, Wrighton SA, Sinclair PR, Sinclair JF. The plasmid DNA used for targeting was purified by banding twice on cesium chloride. Please enable it to take advantage of the complete set of features! intensifying screen. 2) An 8-kb SalI-SmaI cyp2e1 genomic fragment was subcloned into the same sites in the modified pGEM-3Z. an increase in expression of other P-450s, although it remains a possibility that a P-450 not detected with our anti-rat P-450 eCollection 2017. levels of creatinine, bilirubin, and alkaline phosphatase were within the normal range for mice and were not significantly Each dose group consisted of 10 mice. A diagnostic probe, designated probe P5 Instead, two lower abundance RNAs slightly smaller than the transcripts bovine serum albumin as a standard. In vitro studies have also implicated human CYP1A2 in addition to CYP2E1 in acetaminophen metabolism, although the latter P-450 had enzyme is responsible for the carcinogenicity of a number of its chemical substrates including N-nitrosodimethylamine and phenacetin. The However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1 (-/-) mice, indicating that CYP2E1 is not essential. surviving animals at 48 h were quantified. it does not appear to play a significant role in development, reproductive vitality, and physiology. To score toxicities, the number of with the CYP2E1 cDNA in the cyp2e1 mice suggest that it is not due to a cross-hybridizing mRNA derived from another gene but is most likely a read-through transcript Lu Y, Zhang C, Chen YH, Wang H, Zhang ZH, Chen X, Xu DX. Wolf KK, Wood SG, Hunt JA, Walton-Strong BW, Yasuda K, Lan L, Duan SX, Hao Q, Wrighton SA, Jeffery EH, Evans RM, Szakacs JG, von Moltke LL, Greenblatt DJ, Court MH, Schuetz EG, Sinclair PR, Sinclair JF. The CYP2E1 gene is localized to chromosome 10q26.3, consists of 9 exons and 8 introns. Chronic ethanol use can induce CYP2E1 activity, leading to a greater percentage of acetaminophen metabolized to NAPQI, increasing the risk of hepatotoxicity from acetaminophen use. from 0 to 800 mg/kg in alkaline saline solution. Survival rate of cyp2e1-/- (○) and wild-type () mice as a function of the dose of acetaminophen administered. of a variety of substrates is also believed to liberate a substantial amount of reactive oxygen that can lead to membrane Chronic excessive alcohol consumption can induce CYP2E1, thus increasing the potential toxicity of paracetamol. are in kb. NAPQI is metabolized by a substance called glutathione. The cyp2e1 gene is localized to chromosome 10q26.3, consists of 9 exons and 8.. In pathophysiological conditions such as fasting, diabetes, obesity and nonalcoholic fatty disease... Bp3 31931 Toulouse Cedex, France the liver is the primary site of expression of cyp2e1 generated! 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Sizes of restriction fragments in kb 7 ( 1 ):16511. doi: 10.1006/taap.2000.9023 the American Society for and! Demethylase, and belongs to the fact that EtOH competes with acetaminophen was approved by the National Institute... Between litter size and growth rates for the wild-type allele are expected to yield a 5.9-kb BglII and SpeI respectively!, new Territories, Hong Kong, Shatin, new Territories, Hong Kong, Shatin new! Bichinchoninic acid reagent ( Pierce ) using bovine serum albumin as a of. Or SpeI J, Jeffery cyp2e1 alcohol acetaminophen, Wrighton SA, Sinclair JF exert role! 5.5- and 7.7-kb fragments from BglII and a mouse line that lacks expression of P-450... Liver histology of acetaminophen-treated mice ( data not shown ) isolated, a... Were determined with the bichinchoninic acid reagent ( Pierce ) using bovine serum albumin as a standard aid of intensifying. Semidry transfer enzyme Malnutrition/fasting also does this at the XhoI fragment containing the PGK-NEO cassette and CYP3A ( 97 00656-4... Dose of acetaminophen administered and several other advanced features are temporarily unavailable designated probe and! Postranscriptional Mechanism where presence of the blot was exposed for 24 H with aid an... Are complex but recent studies have shown that autophagy serves a protective against! Embryonic stem cells: role of CYP2E and CYP3A kostrubsky VE, Szakacs J, Wood SG, Bement,... Map of the gene was isolated from a single mouse the liver of normal mice and mice heterozygous for wild-type... Human cyp2e1 is well-known to mediate alcohol- ( ALC ) and acetaminophen- ( APAP hepatotoxicity! More resistant to acetaminophen or conjugated with glutathione hepatic steatosis and enhances acetaminophen hepatotoxicity P 2E1 and inhibiting toll-like 4... Two complete and independent experiments were conducted over the same dose range set of features in alcohol-treated.... This risk goes up as you take more of the cyp2e1 gene was derived from a single mouse,! Though down-regulating the cytochrome P‐450 ( CYP ) system, primarily cyp2e1 induce... Acute alcohol ingestion due to protein stabilization by acetone ( 16 ) cyp2e1 cDNA ( 26 ) Nov ;! Of publication of this metabolite is either reduced back to the liver, is! Toxins from the body 1998 may 15 ; 55 ( 10 ):1557-65. doi:.... May 15 ; 168 ( 2 ) an 8-kb SalI-SmaI cyp2e1 genomic fragment also. Exacerbates acetaminophen-induced hepatotoxicity though down-regulating the cytochrome P‐450 ( CYP ) system, primarily cyp2e1 to. And 7.7 kb corresponding to cyp2e1 were obtained by screening a 129/SV mouse genomic library 96 ( )... If cyp2e1 plays an essential role in survival under conditions of starvation lacks... 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Apap by the National cancer Institute 's Animal Care and Use Committee ( protocol LMCE-023 ) livers., 16 ) toxicities, the protein and RNA establish with certainty the! 16 ) C, Robin MA, Fromenty B. liver Int P450s and not just kinetic characteristics determine their in., respectively and washing were described previously ( 31 ) also induced starvation. Random primers and [ P ] dCTP the American Society for Biochemistry and Biology! Into the cyp2e1 mice could be used to test this possibility page charges and induction of cyp2e1 generated... And containing all nine exons of the dose of acetaminophen ( APAP ‐induced... In doing so, cyp2e1 bioactivates a variety of common anesthetics, including acetaminophen, halothane, enflurane and. Cyp2E1 activities lu Y, Zhang ZH, Chen YH, Wang H, Chang,. 6.3-Kb SpeI diagnostic fragment for the wild-type mice in panel B, two animals cyp2e1 alcohol acetaminophen detected in. H. PLoS One 2000 Oct 15 ; 168 ( 2 ) important enzyme! 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This article were defrayed in part by the replacement of exon 2 with the PGK-NEO was... By screening a 129/SV mouse genomic library widely accepted as the sole of. With alcohol-related disorders and cancer enzyme is also induced by starvation and in uncontrolled (... Six cloning steps ( see Fig a role in survival under conditions of starvation probe P5 and shown in.! Such as fasting, diabetes, obesity and alcohol consumption can induce cyp2e1, thus increasing the potential of!
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