Induction of cytochrome P4502B: Role of regulatory elements and nuclear receptors

Cytochrome P450 of the 2B subfamily is easily induced by many xenobiotics. In spite of intensive investigations, the molecular mechanisms of regulation of the CYP2B genes are not clear. The nuclear receptor CAR is shown to play a crucial role in the activation of CYP2B genes by xenobiotics, but many problems of CAR activation in different animal species and humans remain unsolved. This review focuses on signaling pathways involved in the control of CYP2B gene expression in mammals.     

An Sp1 response element in the Kaposi's sarcoma-associated herpesvirus open reading frame 50 promoter mediates lytic cycle induction by butyrate

Kaposi's sarcoma-associated herpesvirus (KSHV) can be driven into the lytic cycle in vitro by phorbol esters and sodium butyrate. This report begins to analyze the process by which butyrate activates the promoter of KSHV open reading frame 50 (ORF50), the key viral regulator of the KSHV latency to lytic cycle switch. A short fragment of the promoter, 134 nucleotides upstream of the translational start of ORF50, retained basal uninduced activity and conferred maximal responsiveness to sodium butyrate. The butyrate response element was mapped to a consensus Sp1-binding site. By means of electrophoretic mobility shift assays, both Sp1 and Sp3 were shown to form complexes in vitro with the ORF50 promoter at the Sp1 site. Butyrate induced the formation of a group of novel complexes, including several Sp3-containing complexes, one Sp1-containing complex, and several other complexes that were not identified with antibodies to Sp1 or Sp3. Formation of all butyrate-induced DNA-protein complexes was mediated by the consensus Sp1 site. In insect and mammalian cell lines, Sp1 significantly activated the ORF50 promoter linked to luciferase. Chromatin immunoprecipitation experiments in a PEL cell line showed that butyrate induced Sp1, CBP, and p300 binding to the ORF50 promoter in vivo in an on-off manner. The results suggest that induction of the KSHV lytic cycle by butyrate is mediated through interactions at the Sp1/Sp3 site located 103 to 112 nucleotides upstream of the translational initiation of ORF50 presumably by enhancing the binding of Sp1 to this site.     

Involvement of the xenobiotic response element (XRE) in Ah receptor-mediated induction of human UDP-glucuronosyltransferase 1A1

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an important physiological role by contributing to the metabolism of endogenous substances such as bilirubin in addition to xenobiotics and drugs. The UGT1A1 gene has been shown to be inducible by nuclear receptors steroid xenobiotic receptor (SXR) and the constitutive active receptor, CAR. In this report, we show that in human hepatoma HepG2 cells the UGT1A1 gene is also inducible with aryl hydrocarbon receptor (Ah receptor) ligands such as 2,3,7,8-tetrachlodibenzo-p-dioxin (TCDD), beta-naphthoflavone, and benzo[a]pyrene metabolites. Induction was monitored by increases in protein and catalytic activity as well as UGT1A1 mRNA. To examine the molecular interactions that control UGT1A1 expression, the gene was characterized and induction by Ah receptor ligands was regionalized to bases -3338 to -3287. Nucleotide sequence analysis of this UGT1A1 enhancer region revealed a xenobiotic response element (XRE) at -3381/-3299. The dependence of the XRE on UGT1A1-luciferase activity was demonstrated by a loss of Ah receptor ligand inducibility when the XRE core region (CACGCA) was deleted or mutated. Gel mobility shift analysis confirmed that TCDD induction of nuclear proteins specifically bound to the UGT1A1-XRE, and competition experiments with Ah receptor and Arnt antibodies demonstrated that the nuclear protein was the Ah receptor. These observations reveal that the Ah receptor is involved in human UGT1A1 induction.     

Acetone-regulated synthesis and degradation of cytochrome P450E1 and cytochrome P4502B1 in rat liver [corrected

The regulation of CYP2E1 and 2B1 was studied by following mRNA levels, catalytic activities and the subcellular distribution of the apoproteins in rat liver 0, 6, 12, 24, 48 and 96 h after a single intragastric dose of acetone. No changes were observed in hepatic CYP2E1 mRNA levels at any time after acetone treatment, whereas rapid rises were observed in the microsomal amount of CYP2E1 protein and CYP2E1-catalyzed 4-nitrophenol hydroxylase and carbon-tetrachloride-initiated lipid-peroxidation activities. However, CYP2E1-dependent catalytic activities declined much faster than the immunodetectable CYP2E1 protein, suggesting that this cytochrome P-450 is inactivated prior to degradation. Similar results were seen in primary hepatocyte cultures. By contrast, concomitant changes in levels of CYP2B1 and CYP2B1-dependent O-depentylation of pentoxyresorufin were observed in the same microsomal preparations. Investigation of the degradative mechanism of both CYP2E1 and CYP2B1 by immunoquantitation of the proteins in lysosomes and by immunohistochemistry indicated their degradation via an autophagic-lysosomal pathway. The data suggest that CYP2E1 is acutely inactivated in the endoplasmic reticulum and that degradation of this isozyme occurs, at least in part, by the lysosomal route. By contrast, CYP2B1 is principally controlled at the level of synthesis.     

Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction.

The mouse cytochrome P1450 (CYP1A1) gene is responsible for the metabolism of numerous carcinogens and toxic chemicals. Induction by the environmental contaminant tetrachlorodibenzo-p-dioxin (TCDD) requires a functional aromatic hydrocarbon (Ah) receptor. We examined the 5'-flanking region of the CYP1A1 gene in mouse hepatoma Hepa-1 wild-type cells and a mutant line having a defect in chromatin binding of the TCDD-receptor complex. We identified two cis-acting elements (distal, -1071 to -901 region; proximal, -245 to -50 region) required for constitutive and TCDD-inducible CYP1A1 gene expression. Three classes of DNA-protein complexes binding to the distal element were identified: class I, found only in the presence of TCDD and a functional Ah receptor, that was heat labile and not competed against by simian virus 40 (SV40) early promoter DNA; class II, consisting of at least three constitutive complexes that were heat stable and bound to SV40 DNA; and class III, composed of at least three constitutive complexes that were thermolabile and were not competed against by SV40 DNA. Essential contacts for these proteins were centered at -993 to -990 for the class I complex, -987, -986, or both for the class II complexes, and -938 to -927 for the class III complexes. The proximal element was absolutely essential for both constitutive and TCDD-inducible CYP1A1 gene expression, and at least two constitutive complexes bound to this region. These data are consistent with the proximal element that binds proteins being necessary but not sufficient for inducible gene expression; interaction of these proteins with those at the distal element was found to be required for full CYP1A1 induction by TCDD.     

Alcohol steatosis and cytotoxicity: The role of cytochrome P4502E1 and autophagy

The goal of the current study was to evaluate whether CYP2E1 plays a role in binge-ethanol induced steatosis and if autophagy impacts CYP2E1-mediated hepatotoxicity, oxidative stress and fatty liver formation produced by ethanol. Wild type (WT), CYP2E1 knockin (KI) and CYP2E1 knockout (KO) mice were gavaged with 3g/kg body wt ethanol twice a day for four days. This treatment caused fatty liver, elevation of CYP2E1 and oxidative stress in WT and KI mice but not KO mice. Autophagy was impaired in ethanol-treated KI mice compared to KO mice as reflected by a decline in the LC3-II/LC3-I ratio and lower total LC-3 and Beclin-1 levels coupled to increases in P62, pAKT/AKT and mTOR. Inhibition of macroautophagy by administration of 3-methyladenine enhanced the binge ethanol hepatotoxicity, steatosis and oxidant stress in CYP2E1 KI, but not CYP2E1 KO mice. Stimulation of autophagy by rapamycin blunted the elevated steatosis produced by binge ethanol. Treatment of HepG2 E47 cells which express CYP2E1 with 100mM ethanol for 8 days increased fat accumulation and oxidant stress but decreased autophagy. Ethanol had no effect on these reactions in HepG2 C34 cells which do not express CYP2E1. Inhibition of autophagy elevated ethanol toxicity, lipid accumulation and oxidant stress in the E47, but not C34 cells. The antioxidant N-acetylcysteine, and CYP2E1 inhibitor chlormethiazole blunted these effects of ethanol. These results indicate that CYP2E1 plays an important role in binge ethanol-induced fatty liver. We propose that CYP2E1-derived reactive oxygen species inhibit autophagy, which subsequently causes accumulation of lipid droplets. Inhibition of autophagy promotes binge ethanol induced hepatotoxicity, steatosis and oxidant stress via CYP2E1.     

Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley-Bixler syndrome variants of cytochrome P450 oxidoreductase

Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR^null, POR^wt, POR^YH, and POR^VE, for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR^null, not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in POR^YH and POR^VE models than in POR^wt, indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.     

Polymorphisms of alcohol metabolizing enzyme and cytochrome P4502E1 genes in mongolian population

Although the genetic polymorphism of the alcohol-metabolizing enzymes was extensively studied at the molecular level by many investigators, the genetic polymorphism studies for ethanolmetabolizing enzymes in Mongolians are very rare. The present study was therefore performed to determine the genetic distribution of various forms of alcohol-metabolizing enzymes such as alcohol dehydrogenase 2 (ADH2, currently accepted nomenclature ADH1B), ADH3 (ADH1C), aldehyde dehydrogenase 2 (ALDH2) and cytochrome P4502E1 (CYP2E1) in 300 healthy Mongolian males. Genetic polymorphisms were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. The allele frequencies ofADH2 *1 andADH2 *2 were 0.24 and 0.76;ADH3 *1 andADH3 *2 were 0.92 and 0.08;ALDH2 *1 andALDH2 *2 were 0.96 and 0.04; andCYP2E1 *C andCYP2E1 *D were 0.15 and 0.85, respectively. Compared to the results reported by other investigators, the allele frequencies ofALDH2 *2 andCYP2E1 *C among Mongolian subjects were much lower than among East Asians (Korean, Japanese, and/or Han-Chinese), while those ofADH2 andADH3 were more similar. Interestingly, this study shows that the ineffectiveALDH2 gene (ALDH2*2 allele) among Mongolians is not as common as among East Asians.     

Stereoselective metabolism of methadone N-demethylation by cytochrome P4502B6 and 2C19

Methadone is a clinically used opioid agonist that is oxidatively metabolized by cytochrome P450 (CYP) isoforms to a stable metabolite, EDDP. Methadone is a chiral drug administered as the racemic mixture of (R)-(-)- and (S)-(+)-methadone, but (R)-methadone is the active isomer. The cytochrome P450 (CYP) isoform involved in methadone's metabolism is thought to be CYP3A4, but human drug-drug interaction studies are not consistent with this. The ability of the common human drug-metabolizing CYPs (obtained from baculovirus-infected insect cell supersomes) to generate 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrilidine (EDDP) from racemic methadone was examined and then determined if the CYP isoforms metabolized methadone stereoselectively. Only CYP2B6, 2C19, and 3A4 generated measurable EDDP from 1 microg/ml of racemic methadone. The hierarchy of EDDP generation was CYP2B6 > CYP2C19 >/= CYP3A4. At 10 microg/ml of methadone, CYP2C9 and CYP2D6 also generated EDDP, but in at least 10-fold lower quantities than CYP2B6. Michaelis-Menten kinetic data demonstrated that CYP2B6 had the highest V(max) (44 ng/min/10pmol) and the lowest K(m) (12.6 microg/ml) for EDDP formation of all the CYP isoforms. In human liver microsomes with high and low CYP2B6 expression but equivalent CYP3A4 expression, high CYP2B6 expression microsomes generated twice the amount of EDDP from 10 microg/ml of methadone than low CYP2B6 expression microsomes. When stereoselective metabolism of racemic methadone by CYP2B6, 2C19, and 3A4 was examined using an enantiospecific methadone assay, CYP2B6 preferentially metabolized (S)-methadone, CYP2C19 preferentially metabolized (R)-methadone, and CYP3A4 showed no preference. These data suggest that multiple CYPs metabolized methadone but CYP2B6 had the highest V(max)/K(m). In addition, only CYP2B6 and 2C19 showed stereoselective metabolism. Our data could explain why the plasma concentration ratio of R/S methadone is variable and why drugs that induce CYP2B6 such as nevirapine and efavirenz also induce methadone metabolism, while the CYP3A4 inducer rifabutin has no effect on methadone pharmacokinetics.     

Catalyzation of cocaine N-demethylation by cytochromes P4502B,P4503A,and P4502D in fish liver

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.