Cellular distribution of cytochromes P-450 in the rat kidney.
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The distribution of several cytochrome P-450 (P-450) isoenzymes between proximal tubular (PT) and distal tubular (DT) cells of the rat kidney was determined. Western blot analysis of microsomes prepared from liver and kidney cortical homogenates revealed that CYP2E1 protein was expressed in rat kidney microsomes at approximately 10% of hepatic levels. Microsomes from renal cortical, PT, and DT cells all expressed CYP2E1, with DT microsomes expressing slightly higher levels than PT microsomes. In contrast, chlorzoxazone hydroxylation activity was markedly higher in microsomes from PT cells than in those from DT cells. Northern blot analysis of total RNA from PT and DT cells exhibited a pattern of CYP2E1 mRNA distribution similar to that of CYP2E1 protein. CYP2C11 protein expression in renal cortical microsomes was approximately 10% of that in liver microsomes but was significantly higher in microsomes from PT cells than in those from DT cells. CYP3A1/2 was not detected in microsomes from either cortical, PT, or DT cells, but was detected in microsomes isolated from total liver or kidney cortical homogenates. CYP2B1/2 expression was detected in all tissues tested. The peroxisomal proliferator clofibrate enhanced the level of CYP2B1/2 in microsomes from both total liver and kidney cortical homogenates but not in microsomes from cortical, PT, or DT cells. CYP4A2/3 protein and CYP4A mRNA expression were detected in microsomes from total liver and kidney cortical homogenates and from renal cortical, PT, and DT cells using Western and Northern blot analyses, respectively. Lauric acid hydroxylation activity, an indicator of CYP4A, was comparable in PT and DT cells. Clofibrate elevation of CYP4A in cortical, PT, and DT microsomes was not as great as that detected in total kidney cortical microsomes. These results establish the distribution of several P-450 isoenzymes between different cell populations of the rat kidney. Furthermore, these results present evidence that the level of induction of certain P-450 isoenzymes in the kidney is cell type-specific. (+info)
Effect of detergents on the N-and ring-hydroxylation of 2-acetamidofluorene by hamster liver microsomal preparations.
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Effects of detergents such as cholate, deoxycholate and Triton X-100 were studied on N-and ring-hydroxylation of 2-acetamidofluorene by reconstituted and unresolved microsomal systems from livers of hamsters pretreated with 3-methylcholanthrene. Triton X-100 (2.5 mg/nmol of cytochrome P-448) inhibited N-and ring-hydroxylation by wholemicrosomal preparations by 40 and 90% respectively Deoxycholate at the same concentration inhibited both hydroxylations completely, whereas cholate inhibited N-and ring-hydroxylation by 40 and 50% respectively. In reconstitution studies, the presence of Triton X-100(0.5-1.0mg/nmol of cytochrome P-448) along with unsolubilized cytochrome P-448 fraction and solubilized reductase fraction increased N-hydroxylation to an appreciable extent compared with ring-hydroxylation. Both cholate and deoxycholate at 0.5-1.0 mg concentrations had a greater stimulatory effect on ring-than on N-hydroxylation activity in such a reconstituted system. (+info)
Role of metabolic activation in the pathogenesis of chemically induced pulmonary disease: mechanism of action of the lung-toxic furan, 4-ipomeanol.
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Many xenobiotics produce hepatic injury due to their metabolism in the liver to highly reactive electrophile intermediates which form covalent conjugates with nucleophilic cellular constituents. This presentation describes studies indicating that the production of chemically reactive metabolites by pulmonary metabolism of xenobiotics can also play a fundamental role in the pathogenesis of chemically induced lung disease. (+info)
Analysis of the inhibition of N-nitroso-dimethylamine activation in the liver by N-nitro-dimethylamine using a new non-linear statistical method.
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N-nitro-dimethylamine (NTDMA) is carcinogenic to rats: it induces nasal cavity tumours. It can be demethylated to N-nitromethylamine and formaldehyde and reduced to N-nitroso-dimethylamine (NDMA): a potent liver carcinogen and also of the nasal cavity if activation in the liver is blocked. To explain the mechanism of NTDMA carcinogenicity we compared its demethylation with that of NDMA in liver microsomes from female and male rats, untreated, fasted or treated with ethanol to induce cytochrome P450 2E1 (CYP2E1). Kinetic parameters were analysed by nonlinear statistical methods, which yielded unbiased parameter estimates for the calculated Km and Vmax values. Km for both compounds was very similar in females (24-47 microM) whereas Vmax for NTDMA was consistently higher than for NDMA as substrate: 1.07-4.70 nmol formaldehyde/mg microsomal protein x min and 0.52-2.76 nmol, respectively. In liver microsomes from induced male rats NTDMA was found to be a much more effective inhibitor of NDMA activation (KEI 39.6-73.6 microM) than NDMA of NTDMA demethylation (KEI 224-286 microM). Nasal microsomes can demethylate both NDMA and NTDMA but the kinetics are vastly different. NTDMA is demethylated at a linear rate and approximately 10-fold more effectively than NDMA. The mechanism of carcinogenicity of ingested NTDMA, we propose, is a partial reduction to NDMA in the liver and inhibition of NDMA activation in the liver by residual NTDMA, which enables NDMA to reach the nasal mucosa where it is activated to DNA-alkylating species and the observed tumours are formed. (+info)
Purification and properties of a cholesteryl ester hydrolase from rat liver microsomes.
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This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes. CEH was purified 12.4-fold from reisolated microsomes using sequential solubilization by sonication, polyethylene glycol precipitation, fractionation with hydroxyapatite, anion exchange chromatography, and chromatography on hydroxyapatite, with an overall yield of 3.2%. CEH activity was purified 141-fold over nonspecific esterase activity and 56-fold over triacylglycerol lipase activity. In sharp contrast with most esterases and lipases, CEH did not bind to concanavalin A-Sepharose and heparin-Sepharose. After polyacrylamide gel electrophoresis, the purified enzyme exhibited two silver-stained bands, but only the protein electroeluted from the low mobility band had CEH activity. Affinity-purified polyclonal antibodies raised to electroeluted CEH inhibited 90% of the activity of liver microsomal CEH and reacted with a 106 kDa protein band on Western blot analysis. This 106 kDa CEH contains a unique N-terminal amino acid sequence. The purified enzyme had optimal activity at pH 6 and no taurocholate requirements, and was inhibited by the serine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfhydryl specific reagents. It hydrolyzed cholesteryl oleate much more efficiently than trioleine, and hydrolytic activity with p-nitrophenyl acetate was higher than with p-nitrophenyl butyrate. These results indicate that rat liver microsomes contain a bile salt-independent catalytic protein that is relatively specific for cholesteryl ester hydrolysis. (+info)
O- and N-demethylation of venlafaxine in vitro by human liver microsomes and by microsomes from cDNA-transfected cells: effect of metabolic inhibitors and SSRI antidepressants.
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The biotransformation of venlafaxine (VF) into its two major metabolites, O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) was studied in vitro with human liver microsomes and with microsomes containing individual human cytochromes from cDNA-transfected human lymphoblastoid cells. VF was coincubated with selective cytochrome P450 (CYP) inhibitors and several selective serotonin reuptake inhibitors (SSRIs) to assess their inhibitory effect on VF metabolism. Formation rates for ODV incubated with human microsomes were consistent with Michaelis-Menten kinetics for a single-enzyme mediated reaction with substrate inhibition. Mean parameters determined by non-linear regression were: Vmax = 0.36 nmol/min/mg protein, K(m) = 41 microM, and Ks 22901 microM (Ks represents a constant which reflects the degree of substrate inhibition). Quinidine (QUI) was a potent inhibitor of ODV formation with a Ki of 0.04 microM, and paroxetine (PX) was the most potent SSRI at inhibiting ODV formation with a mean Ki value of 0.17 microM. Studies using expressed cytochromes showed that ODV was formed by CYP2C9, -2C19, and -2D6. CYP2D6 was dominant with the lowest K(m), 23.2 microM, and highest intrinsic clearance (Vmax/K(m) ratio). No unique model was applicable to the formation of NDV for all four livers tested. Parameters determined by applying a single-enzyme model were Vmax = 2.14 nmol/min/mg protein, and K(m) = 2504 microM. Ketoconazole was a potent inhibitor of NDV production, although its inhibitory activity was not as great as observed with pure 3A substrates. NDV formation was also reduced by 42% by a polyclonal rabbit antibody against rat liver CYP3A1. Studies using expressed cytochromes showed that NDV was formed by CYP2C9, -2C19, and -3A4. The highest intrinsic clearance was attributable to CYP2C19 and the lowest to CYP3A4. However the high in vivo abundance of 3A isoforms will magnify the importance of this cytochrome. Fluvoxamine (FX), at a concentration of 20 microM, decreased NDV production by 46% consistent with the capacity of FX to inhibit CYP3A, 2C9, and 2C19. These results are consistent with previous studies that show CYP2D6 and -3A4 play important roles in the formation of ODV and NDV, respectively. In addition we have shown that several other CYPs have important roles in the biotransformation of VF. (+info)
Uptake of 13-hydroperoxylinoleic acid by cultured cells.
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Oxidized free fatty acids have profound effects on cultured cells. However, little is known about whether these effects depend on their uptake and metabolism by cells or primarily involve their interaction with cell-surface components. We determined the uptake and metabolism of unoxidized (linoleic or oleic acid) and oxidized linoleic acid (13-hydroperoxyoctadecadienoic acid, 13-HPODE) by endothelial cells, smooth muscle cells, and macrophages. We show that 13-HPODE is poorly taken up by cells. The levels of uptake were dependent on the cell type but were independent of the expression of CD36. 13-HPODE was also poorly used by microsomal lysophosphatidylcholine acyltransferase that is involved in the formation of phosphatidylcholine. Based on these results, we suggest that most of the biological effects of 13-HPODE and other oxidized free fatty acids on cells might involve a direct interaction with cell-surface components. Alternatively, very small amounts of oxidized free fatty acids that enter the cell may have effects, analogous to those of hormones or prostanoids. (+info)
Comparative metabolism of 1-, 2-, and 4-nitropyrene by human hepatic and pulmonary microsomes.
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Determining the capability of humans to metabolize the mononitropyrene (mono-NP) isomers 1-, 2-, and 4-NP and understanding which human cytochrome P450 (P450) enzymes are involved in their activation and/or detoxification is important in the assessment of individual susceptibility to these environmental carcinogens. We compared the ability of 15 human hepatic and 8 pulmonary microsomal samples to metabolize each of the three isomers. Human hepatic microsomes were competent in metabolizing all three isomers. Qualitatively similar metabolic patterns were observed, although at much lower levels, upon incubating mono-NP with pulmonary microsomes. Ring-oxidized metabolites (phenols and trans-dihydrodiols) were produced from all three isomers. However, the nitroreductive metabolism leading to the formation of aminopyrene was evident only with 4-NP. The role of specific P450 enzymes in the human hepatic microsomal metabolism of mono-NP was investigated by correlating the P450-dependent catalytic activities in each microsomal sample with the levels of individual metabolites formed by the same microsomes and by examining the effects of agents that can either inhibit or stimulate specific P450 enzymes in mono-NP metabolism. On the basis of these studies, we attribute most of the hepatic microsomal metabolism of 1- and 4-NP to P450 3A4, although a minor role for P450 1A2 cannot be ruled out. Specifically, P450 3A4 was responsible for the formation of 3-hydroxy-1nitropyrene from 1-NP and the formation of trans-9,10-dihydro-9,10dihydroxy-4-nitropyrene, 9(10)-hydroxy-4-nitropyrene, and 4-aminopyrene from 4-NP. None of the P450 enzymes examined (P450s 3A4, 1A2, 2E1, 2A6, 2D6, and 2C9) appeared to be involved in catalyzing the formation of trans-4,5-dihydro-4,5-dihydroxy-2-nitropyrene and 6-hydroxy-2-nitropyrene from 2-NP in human hepatic microsomes. These results, the first report on the comparative metabolism of mono-NP in humans, clearly demonstrate that the role of specific human P450 enzymes in catalyzing oxidative and reductive pathways of mono-NP is dependent upon the position of the nitro group. (+info)