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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We prepared primary monolayer cultures of adult rat hepatocytes and measured the losses of cytochromes P-450 with the use of specific antibodies directed against purified forms of hepatic cytochrome P-450 which predominate in untreated rats (P-450UT-A, P-450UT-F) or in rats treated with phenobarbital (P-450PB-B/D, P-450PB-C, P-450PB/PCN-E) or with 3-methylcholanthrene (P-450 beta NF-B, P-450 beta NF/ISF-G). In hepatocytes prepared from an untreated rat and incubated in control medium, total cytochrome P-450, measured spectrally as CO-binding hemoprotein, declined 68% during the first 72 hr in culture. However, the sum of the immunoreactive cytochromes P-450 declined only 24%, indicating that loss of heme rather than of protein accounts for much of the well-known loss of cytochromes P-450 in hepatocyte cultures. In cultures prepared from untreated rats or from rats treated with phenobarbital or with 3-methylcholanthrene, individual forms of cytochrome P-450 declined at markedly differing rates. Incubation of cultures in three different media previously reported to maintain levels of total cytochrome P-450 failed to prevent the decline in total cytochrome P-450 during the first 24 to 72 hr in culture. However, in cultures incubated in medium containing metyrapone, the level of holocytochrome P-450 was maintained at the initial value during the first 72 hr, apparently by preventing the net loss of cytochrome P-450 heme and by increasing the concentrations of immunoreactive P-450PB/PCN-E and P-450 beta NF-B. Medium containing nicotinamide increased the proportion of P-450 beta NF-B relative to the other forms of cytochrome P-450, whereas cysteine-free medium increased P-450UT-F. We conclude that loss of cytochrome P-450 in cultured hepatocytes involves loss of its heme moiety coupled with changes in the concentrations of the individual forms. Recognition of these changes as influenced by specific components of the culture medium is important when using primary hepatocyte cultures for study of xenobiotic metabolism and toxicity in the liver.
Mol Pharmacol 1985 Jan
PMID:Changes in the concentration of seven forms of cytochrome P-450 in primary cultures of adult rat hepatocytes. 396 24

The metabolic clearance of circulating benzo[a]pyrene (B[a]P) by liver and lung of control and 3-methylcholanthrene (3MC)-pretreated rats was predicted according to the perfusion-limited model from apparent enzyme kinetic constants determined in microsomal incubations. These predictions were tested in isolated organs perfused at normal organ flow. From microsomal incubations the apparent enzyme kinetic constants of B[a]P metabolism were determined. The apparent Km of liver microsomes was decreased 100 times by pretreatment with 3MC, while the Km of lung microsomes remained at about 0.2 microM. Maximal velocity of B[a]P metabolism was much greater in microsomes from liver than in those from lung of both control and 3MC-pretreated rats. Liver was found to have a far greater capacity for B[a]P metabolism (intrinsic free clearance) than lung. However, this large disparity was not evident in the predicted clearances. Perfused organs had B[a]P clearances very close to those predicted from the model. At normal (in vivo) organ flows, control rat lung had a clearance of 1.0 +/- 0.1 ml/min, whereas liver had a clearance of 5.9 +/- 0.2 ml/min. Corresponding clearances in organs from 3 MC-pretreated rats were 8.9 +/- 0.5 and 6.7 +/- 0.6 ml/min for lung and liver, respectively. Small discrepancies between predicted and observed values could not be explained by non-uniform distribution of B[a]P or shunting of flow. These results suggest that enzyme kinetic data can be used to assess accurately the ability of lung and liver to clear xenobiotic compounds such as B[a]P and that, despite the great disparity in their metabolic capacity, under certain conditions these two organs may function equally well in the removal of circulating compounds from the blood.
Mol Pharmacol 1983 Sep
PMID:The prediction of benzo[a]pyrene clearance by rat liver and lung from enzyme kinetic data. 631 Mar 65

The microsomal fraction of ram seminal vesicles (RSV), when fortified with arachidonic acid, catalyzed the dealkylation of various N-methyl compounds. These included an analogous series of monomethyl- and dimethyl-substituted anilines as well as the drugs aminopyrine and benzphetamine. In contrast, S-alkyl and O-alkyl compounds were poor substrates for dealkylation by RSV microsomes fortified with fatty acid. RSV microsomal N-dealkylation was completely dependent on enzyme and arachidonic acid and could be inhibited by the prostaglandin synthetase inhibitors indomethacin, phenylbutazone, and flufenamic acid as well as by anaerobic conditions. Butylated hydroxyanisole also inhibited the reaction, whereas SKF-525A and metyrapone, which are inhibitors of cytochrome P-450-dependent N-dealkylation, did not. In addition to arachidonic acid, N-dealkylation was elicited by 15-hydroperoxyarachidonic acid, tert-butyl-hydroperoxide, and hydrogen peroxide; these latter reactions were not inhibited by either prostaglandin synthetase inhibitors or anaerobic conditions but did require the presence of microsomal protein. The time course of RSV N-dealkylation, which paralleled O2 consumption by this tissue (an indicator of prostaglandin biosynthesis) implied arachidonic acid-dependent irreversible self-inactivation of catalytic activity. Apparently, oxidizing agents are formed during the interaction of hydroperoxide intermediates of prostaglandin biosynthesis with prostaglandin synthetase, with the oxidizing agents then causing both substrate N-dealkylation and destruction of the enzyme. The metabolism of N-alkyl compounds during the biosynthesis of prostaglandins may provide an additional xenobiotic oxidation pathway to cytochrome P-450-dependent monooxygenases.
Mol Pharmacol 1982 Jan
PMID:Metabolism of N-alkyl compounds during the biosynthesis of prostaglandins. N-Dealkylation during prostaglandin biosynthesis. 681 75

The basic helix-loop-helix containing dioxin receptor mediates dioxin signal transduction. The ligand-activated receptor complex binds to specific sequences termed xenobiotic response elements and regulates transcription of target genes such as the gene for cytochrome P450IA1. This study demonstrates that induction of cytochrome P450IA1 and P450IB1 gene expression by a dioxin receptor ligand is repressed by camptothecin, an inhibitor of the topoisomerase I enzyme. However, a transiently transfected reporter construct under control of an xenobiotic response element-containing promoter was not affected by the topoisomerase inhibitor. In agreement with this observation, ligand-dependent activation of the dioxin receptor to its DNA-binding form is not altered by camptothecin as analyzed by electrophoretic mobility shift assay. Moreover, the inhibitory effect of camptothecin cannot be exerted once the P450IA1 gene has been activated. These results imply that topoisomerase I activity is necessary for the primary P450IA1 induction response, possibly involving dioxin-dependent alterations in chromatin structure of the P450IA1 promoter.
Mol Pharmacol 1995 Oct
PMID:Differential effects of a topoisomerase I inhibitor on dioxin inducibility and high-level expression of the cytochrome P450IA1 gene. 747 85

Functional domains of the mouse aryl hydrocarbon receptor (Ahr) were investigated by deletion analysis. Ligand binding was localized to a region encompassing the PAS B repeat. The ligand-mediated dissociation of Ahr from the 90-kDa heat shock protein (HSP90) does not require the aryl hydrocarbon receptor nuclear translocator (Arnt), but it is slightly enhanced by this protein. One HSP90 molecule appears to bind within the PAS region. The other molecule of HSP90 appears to require interaction at two sites: one over the basic helix-loop-helix region, and the other located within the PAS region. Each mutant was analyzed for dimerization with full-length mouse Arnt and subsequent binding of the dimer to the xenobiotic responsive element (XRE). In order to minimize any artificial steric hindrances to dimerization and XRE binding, each Ahr mutant was also tested with an equivalently deleted Arnt mutant. The basic region of Ahr is required for XRE binding but not for dimerization. Both the first and second helices of the basic helix-loop-helix motif and the PAS region are required for dimerization. These last results are analogous to those previously obtained for Arnt (Reisz-Porszasz, S., Probst, M.R., Fukunaga, B. N., and Hankinson, O. (1994) Mol. Cell. Biol. 14, 6075-6086) compatible with the notion that equivalent regions of Ahr and Arnt associate with each other. Deletion of the carboxyl-terminal half of Ahr does not affect dimerization or XRE binding but, in contrast to an equivalent deletion of Arnt, eliminates biological activity as assessed by an in vivo transcriptional activation assay, suggesting that this region of Ahr plays a more prominent role in transcriptional activation of the cyp1a1 gene than the corresponding region of Arnt.
 ...
PMID:Identification of functional domains of the aryl hydrocarbon receptor. 749 58

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration of guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17 alpha-hydroxylase and 21-hydroxylase activities were decreased to 20-25% of control values by the higher dose of ABT. Mitochondrial 11 beta-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3 beta-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.
J Steroid Biochem Mol Biol 1995 Sep
PMID:Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs. 757 11

We investigated the use of multiplex polymerase chain reaction (PCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWW0), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWW0, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10(0) to 10(6) cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 microL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradative cells, at a lower detection limit of approximately 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.
Mol Ecol 1995 Oct
PMID:Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis. 758 66

Dehydroepiandrosterone sulphate (DHEAS) is a major adrenal secretory product, particularly in the fetus where it serves as a substrate for oestrogen biosynthesis by the placenta. The enzyme in the adrenal responsible for synthesising DHEAS, hydroxysteroid sulphotransferase (HST), is therefore essential for human development. We have isolated a full-length cDNA clone, encoding human fetal adrenal HST, and constructed a stable cell line expressing it by transfection into V79 Chinese hamster lung fibroblast cells. This cDNA was essentially identical to that isolated from adult human liver, where the role of HST is less well understood. This recombinant cell line allowed determination of the substrate specificity and kinetic properties of this enzyme towards various steroid hormones, and by comparison of these activities with human liver cytosol we have shown that HST is the major sulphotransferase responsible for the sulphation of DHEA, androsterone and pregnenolone in man and that, functionally, the hepatic and adrenal enzymes are very similar. The expressed HST was also active with testosterone, cortisol (although at low levels) and the xenobiotic 17 alpha-ethinyloestradiol, but not with oestrone or 1-naphthol. We have therefore created a valuable resource for the study of this important enzyme.
Mol Cell Endocrinol 1995 Jul
PMID:Human fetal adrenal hydroxysteroid sulphotransferase: cDNA cloning, stable expression in V79 cells and functional characterisation of the expressed enzyme. 758 85

The 2,3,7,8-tetrachlorodibenzo-p-dioxin-transformed aryl hydrocarbon receptor (AHR) complex binds to xenobiotic-responsive element (XRE) sequences in the 5' flanking region of the CYP1A1 gene, resulting in initiation of transcription. Both components of the transformed AHR complex [the ligand-binding AHR monomer and the AHR nuclear translocator (ARNT)] directly contact the XRE. These proteins belong to a novel subclass of basic helix-loop-helix transcription factors. The binding sites of AHR and ARNT on the asymmetric XRE were determined using nuclear extracts of 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated Hepa-1c1c7 cells and a panel of double-stranded oligonucleotides containing XRE1 of the CYP1A1 gene (5'-TTGCGTGAGAA-3'), in which all combinations of three, two, or one of the thymines indicated were substituted by the photoreactive thymine analog 5-bromodeoxyuracil. Covalent cross-linking analysis and immunoprecipitation with antibodies specific for AHR or ARNT demonstrated that ARNT directly contacts the 3'-most thymine position, that AHR directly contacts the second thymine position, and that neither protein contacts the 5'-most thymine position. The thymine position contacted by ARNT lies within a three-nucleotide sequence (5'-GTG-3') identical to a half-site of an E-box element (5'-CACGTG-3') that is recognized by a number of other basic helix-loop-helix transcription factors. AHR binds to a portion of the XRE that does not resemble an E-box. Additional experiments demonstrated that neither protein loops over to contact residues located beyond the other's binding site.
Mol Pharmacol 1995 Mar
PMID:Orientation of the heterodimeric aryl hydrocarbon (dioxin) receptor complex on its asymmetric DNA recognition sequence. 770 Feb 40

In this report we characterized the transcriptional regulation of the rat mdr1b gene by xenobiotics. The expression of this gene was increased in primary rat hepatocytes and in the H4-II-E hepatoma cell line by exposure to carcinogens such as aflatoxin B1, N-acetoxy-2-acetylaminofluorene, and methyl methanesulfonate. Nuclear run-on experiments indicated that the higher steady-state levels of mdr1b mRNA were due to an increase in transcription. The 5'-flanking region of the mdr1b gene was isolated, sequenced, and functionally characterized in transient and stable transfection assays. A single transcription start site was identified for this gene; no alternate start sites were used after induction with aflatoxin B1. Deletion analysis of this promoter demonstrated that the sequence between nt -214 and -178 was critical for basal promoter activity. This region did not contain any consensus-binding sites for previously identified transcription factors. A negative regulatory region was also identified between nt -940 and -250. No specific carcinogen-responsive element was identified; the xenobiotic response required a large part of the promoter. These data suggest that the carcinogen induction of mdr1b expression is mediated through sequences that overlap or that are identical to the basal promoter element.
Mol Carcinog 1995 May
PMID:Characterization of the basal and carcinogen regulatory elements of the rat mdr1b promoter. 776 10


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