Inhibition by SKF 525-A of 7-ethoxycoumarin O-deethylation in microsomal and the reconstituted monooxygenase systems from PCB-treated rat livers.
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Addition of diethylaminoethyl 2,2-diphenylvalerate-HCl (SKF 525-A) to the incubation mixture containing liver microsomes or purified cytochrome P-450 (PCB P-450) from PCB (KC-500)-treated rats resulted in non-competitive inhibition of 7-ethoxycoumarin O-deethylation activity whereas the addition to the incubation mixture containing purified cytochrome P-448 (PCB P-448) showed a competitive inhibition. Fortification of PCB-induced microsomes with purified NADPH-cytochrome P-450 reductase enhanced the O-deethylation activity. With the reductase-fortified microsomes, SKF 525-A inhibited the O-deethylation in a competitive manner. Based on these results, we confirmed that SKF 525-A inhibits non-competitively and competitively, depending on the species of cytochrome P-450. Our results also support the view that in microsomes from PCB-treated rats, PCB P-450 rather than PCB P-448 is mainly involved in the O-deethylation reaction, presumably due to the presence of a limited amount of NADPH-cytochrome P-450 reductase in microsomes. (+info)
Isolation of the membrane-binding peptide of NADPH-cytochrome P-450 reductase. Characterization of the peptide and its role in the interaction of reductase with cytochrome P-450.
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A peptide identified as the membrane-associated segment of NADPH-cytochrome P-450 reductase has been generated by steapsin protease treatment of vesicle-incorporated reductase and isolated by preparative gel electrophoresis. This peptide remains associated with vesicles when steapsin protease digests of vesicle-incorporated reductase were fractionated by Sepharose 4B chromatography, confirming its identity as the membrane-binding peptide. The molecular weight of the membrane-binding peptide was 6400 as determined by gel filtration in 8 M guanidine hydrochloride, and its amino acid content was not especially hydrophobic. The activity of reconstituted hydroxylation systems consisting of reductase, cytochrome P-446, and dilauroyl phosphatidylcholine was not inhibited by large molar excesses of purified membrane-binding peptide. Moreover, when purified reductase and cytochrome P-446 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptides were used. Similar results were obtained with reductase, cytochrome P-450, and detergent-solubilized liposomes (with or without the membrane-binding peptide). Thus, the membrane-binding peptide does not appear to interact with either of these two forms of the hemoprotein in a site-specific manner to prevent reconstitution of hydroxylation activity. (+info)
The roles of cytochrome b5 in reconstituted monooxygenase systems containing various forms of hepatic microsomal cytochrome P-450.
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The roles of cytochrome b5 in NADPH-dependent monooxygenase reactions catalyzed by reconstituted systems containing four forms of hepatic microsomal cytochrome P-450, i.e. P-450(1), P-450(2), P-448(1), and P-448(2), were examined. Various substrates were metabolized actively in the absence of cytochrome b5 by the system containing P-450(1), but the monooxygenase reactions were accompanied by oxidation of NADPH uncoupled to the product formation. When cytochrome b5 was included in the system, the product formation increased to various extents, depending on the substrates used, while NADPH oxidation changed much less, resulting in an improvement of the coupling efficiency. The increase was large when a substrate metabolized at a low velocity was employed. Evidence is presented that the second of two electrons required for the monooxygenase reactions could be introduced into P-450(1) via cytochrome b5. On the other hand, the rate of P-450(1) reduction was not affected by the addition of cytochrome b5 to the system and that of cytochrome b5 reduction by NADPH-cytochrome P-450 reductase was sufficient to support electron flow to cytochrome P-450 via cytochrome b5 as the second electron. The stimulatory effect of cytochrome b5 on the P-450(1)-catalyzed monooxygenase reactions can be explained by assuming that the rate of the second electron supply to the oxygenated P-450(1)-substrate complex from cytochrome b5 is higher than that directly from NADPH-cytochrome P-450 reductase. An electron flow from NADH via cytochrome b5 can be utilized as the second electron for the O-deethylase reaction of 7-ethoxycoumarin catalyzed by reconstituted systems containing P-450(2) and P-448(2) when both NADH-cytochrome b5 reductase and cytochrome b5 are included in the system, although the cytochrome has no stimulatory effect at all on the deethylase activity of these two cytochrome P-450's. It has been shown that the second electron for P-448(1) can also be supplied from NADH via cytochrome b5 in a reconstituted acetanilide p-hydroxylase system containing P-448(1), cytochrome b5, and the two reductases. (+info)
Induction of O-deethylase activity as an index to exposure to coal-derived products and trace environmental pollutants.
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The metabolism of the synthetic substrate 7-ethoxyresorufin is a selective measure of the activity of cytochrome P-448 monooxygenase, the subset of cytochrome P-450-mediated enzymes preferentially induced by polycyclic aromatic hydrocarbons and related compounds. 7-Ethoxycoumarin metabolism, on the other hand, reflects total (nonselective) cytochrome P-450 monooxygenase activity. Either substrate yields a single, highly fluorescent product, amenable to direct, sensitive assay with the portable centrifugal analyzer. We used three assays with liver microsomes from C57/BL6 mice for the short-term bioassay of the dose-dependent effects of exposure to selected environmental toxins, including petroleums, polychlorinated biphenyls, and their oxidative degradation products. (+info)
Cytochrome P-450-dependent O-dealkylase activity in mammalian skin.
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1 A modified technique for the measurement of O-dealkylase (ODA) activity in crude homogenates is reported, and its application to skin is described. 2 Large differences in ODA levels were found between different species, no activity being observed in the skin of primates. 3 Induction of cutaneous ODA in mice was achieved by the subcutaneous injection of phenobarbitone, hexachlorobenzene or 20-methylcholanthrene. 4 Attempts to induce ODA in vitro were unsuccessful. (+info)
Biotransformation of coumarin derivatives (1). 7-alkoxycoumarin O-dealkylase in liver microsomes.
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The in vitro metabolic fate of 7-alkoxycoumarin was studied using liver microsomes. Microsomal enzyme catalyzed dealkylation of 7-alkoxycoumarin to 7-hydroxycoumarin in the presence of NADPH and molecular oxygen as cofactors was found to be one of the metabolic pathways. The metabolite 7-hydroxycoumarin was further metabolized to unidentified metabolite(s) in the presence of NADPH and O2 at a very slow rate, while the formation of the conjugate of 7-hydroxycoumarin with glucuronic acid was observed in the presence of UDPGA. Microsomal 7-alkoxycoumarin O-dealkylase activity was altered by the substitution of the alkyl group of the substrate, and the substitutions to either an O-propyl or an O-butyl group resulted in a decrease of the enzyme activity. Species differences were observed in the substrate specificity of microsomal O-dealkylation. The O-dealkylase activities in rat liver microsomes were stimulated by pretreatment of the animals with phenobarbital, regardless of the O-alkyl substituent at the 7 position of the coumarin ring. On the other hand, pretreatment with 3-methylcholanthrene or beta-naphthoflavone resulted in marked increase of O-deethylation. O-depropylation and O-debutylation activities, but not of O-demethylation activity. Pretreatment of animals with beta-naphthoflavone also resulted in remarkable stimulation of 7-hydroxycoumarin-glucuronide formation by the microsomal enzyme, while the conversion of 7-hydroxycoumarin to unidentified metabolite(s) was activated by the pretreatment of rats with only phenobarbital. The O-dealkylation activities in liver microsomes from intact and phenobarbital pretreated rats were inhibited markedly by the addition of hexobarbital to the incubation mixture, but no inhibition was observed with alpha-naphthoflavone. On the other hand, the O-dealkylation activities in microsomes from beta-naphthoflavone-pretreated rats were inhibited remarkably by alpha-naphthoflavone. These results confirmed that several microsomal enzymes, including the cytochrome P-450's and UDP-glucuronyltransferase, participate in the biotransformation of 7-alkoxycoumarin, and these enzymes are regulated differently by inducers. (+info)
Phenotyping of cytochromes P-450 in human tissues with monoclonal antibodies.
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Cytochrome P-450 (P-450)-dependent aryl hydrocarbon hydroxylase (AHHase) and 7-ethoxycoumarin deethylase (ECDEtase) in human tissues were differentially inhibited by monoclonal antibodies (MAbs) that were prepared to inhibit and completely inhibited the activity of 3-methylcholanthrene-induced rat liver P-450. The AHHase and ECDEtase of placentas from individual women who smoked were inhibited by the MAbs by 83-90% and by 34-74%, respectively. Benz[a]anthracene (BaA)-induced AHHase and ECDEtase in lymphocytes were inhibited 18-65% and 30-78%, respectively. The enzymes in both control and BaA-induced human cells in culture were inhibited to different extents. Both the AHHase and ECDEtase in control and BaA-induced monocytes and in normal liver were largely unaffected by the MAb. Thus, we have with the MAbs: (i) identified P-450s with a common antigenic site in placenta, lymphocytes, and human cells in culture; (ii) identified two forms of hydrocarbon-induced P-450s in lymphocytes, at least one of which is common with the induced P-450s of placenta and with a P-450 form present in uninduced lymphocytes; (iii) identified two forms of P-450 responsible for smoking-induced ECDEtase activity in placenta, one of which is also responsible for AHHase activity; (iv) shown that the P-450s of liver, basal, and BaA-induced monocytes are different from the MAb-sensitive P-450s of placenta and lymphocytes; (v) quantitated in several human tissues the percentages of control and inducible AHHase and ECDEtase that are dependent on the MAb-sensitive P-450; and (vi) defined by HPLC the contribution of the MAb-sensitive P-450 to the formation of specific benzo[a]-pyrene metabolites. The results demonstrate the value of MAbs for defining antigenic site relatedness for different enzymatic functions of P-450s and for identifying and quantifying the amount of a specific enzyme activity in a tissue dependent on specific P-450s. This study may be a prototype for the use of MAbs for phenotyping and mapping of P-450s responsible for specific metabolic reactions and, thus, may be useful in determining the relationship of P-450 phenotype to individual differences in drug metabolism and carcinogen susceptibility. (+info)
Kinetic evidence for heterogeneous responsiveness of mixed function oxidase isozymes to inhibition and induction by allylisopropylacetamide in chick embryo liver.
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Changes in hepatic mixed function oxidase kinetics after administration of allylisopropylacetamide (AIA) to chick embryos indicate that the activities of different cytochrome P-450 isozymes, including those participating in the metabolism of the same substrates, can be simultaneously increased and inhibited by a single xenobiotic. Up to 4 h after administration in ovo, or in vitro, AIA exclusively inhibited mixed function oxidases. At 24 h after administration in ovo, AIA simultaneously decreased the Vmax of the isozymes active in 7-ethoxycoumarin deethylation and in biphenyl and antipyrine hydroxylations in control liver and caused new isozymes with higher Km and Vmax values to appear. At the same time, AIA increased the Vmax values for isozymes active in aminopyrine demethylation and decreased the Vmax for benzo(a)pyrene hydroxylation (EC 1.14.14.1). As an inhibitor, AIA did not exhibit substrate selectivity but tended to inhibit isozymes with higher substrate affinity noncompetitively and lower affinity isozymes competitively. Competitive mechanisms and generalized P-450 breakdown could only partially account for the inhibition of mixed function oxidases by AIA. The inhibition at low doses of AIA (0.1 to 0.3 mg/egg) occurred without any decrease in P-450 and at higher doses it exceeded and was more persistent than the decrease in P-450. The data indicate that in addition to the known mechanisms for mixed function oxidase inhibition by AIA there is another noncompetitive mechanism independent of P-450 breakdown. As an inducer, AIA, like phenobarbital rather than beta-naphthoflavone increased the metabolism of aminopyrine and the concentration of Mr = 50,000 and 51,000 proteins preferentially. However, unlike either, AIA selectively induced new high Km and Vmax isozymes active toward 7-ethoxycoumarin, biphenyl, and antipyrine and increased the concentration of a Mr = 53,000 protein. These actions distinguish AIA from either the phenobarbital or polycyclic hydrocarbon class of inducers. The simultaneous inhibition by AIA of higher affinity isozymes with selective induction of low affinity isozymes produced a "crossover effect" in which after AIA administration the rates of 7-ethoxycoumarin deethylase and biphenyl and antipyrine hydroxylases were decreased at low and increased at high substrate concentrations. The findings demonstrate the complexity and selectivity of AIA's actions as a mixed function oxidase inhibitor and inducer and illustrate the potential heterogeneity of responses that can occur in the mixed function oxidase system after exposure of an organism to a xenobiotic. (+info)