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PubMed1983

[Reconstitution of membranous microsomal monooxygenase system during gel filtration on Sephadex LH-20].

Solubilization of liver microsomes of intact, phenobarbital-treated and 3-methylcholantrene-treated rats was investigated. It was shown that treatment of microsomes with 4% sodium cholate leads to separation of proteins and phospholipids from the microsomal membrane. The microsomal solubilizate was passed through lipophilic Sephadex LH-20 and the particles whose properties are close to those of the microsomal ones were obtained. The high efficiency of self-assembly of the microsome-like particles, e. g. lack of cytochrome P-450 conversion into cytochrome P-420, high microsomal protein yield and the high level of metabolizing activity of microsomal monooxygenase, was demonstrated.

Animals↗
PubMed1983

Effect of nonspecific phospholipid transfer protein on cholesterol esterification in microsomes from Morris hepatomas.

The content of cholesterol and cholesterol ester as well as the levels of acyl coenzyme A:cholesterol acyltransferase activity were determined in the microsomes from Morris hepatomas 7777, 5123D, and 7787. The free cholesterol content, expressed per mg microsomal protein, was significantly increased only in the microsomes from Morris hepatoma 7777 [47.8 +/- 0.4 micrograms (S.D.); p less than 0.001] and hepatoma 7787 (37.6 +/- 6.2 micrograms; p less than 0.01) as compared to normal liver (28.8 +/- 2.4 micrograms). The cholesterol ester content in the microsomes of the three different tumors did not significantly differ from that of normal liver (2.1 +/- 1.2 micrograms cholesterol per mg microsomal protein). The microsomal acyl coenzyme A:cholesterol acyltransferase activity was decreased in Morris hepatoma 7777 (8.6 +/- 2.3 pmol/min/mg protein; p less than 0.01) and in hepatoma 5123D (7.5 +/- 1.7 pmol/min/mg protein; p less than 0.02), and was normal in the hepatoma 7787 (16.5 +/- 7.8 pmol/min/mg protein) as compared to rat liver (16.0 +/- 2.9 pmol/min/mg protein). In a previous study (B. J. H. M. Poorthuis and K. W. A. Wirtz, Biochim. Biophys. Acta, 710: 99-105, 1982), this acyltransferase activity was shown to be stimulated by preincubation of rat liver microsomes with cholesterol-containing vesicles and the nonspecific phospholipid transfer protein. In this paper, a similar 4-fold stimulation of activity was observed for the microsomes of the various hepatomas investigated. The possible role of the nonspecific phospholipid transfer protein in intracellular cholesterol esterification is discussed.

Acyl Coenzyme A↗
PubMed1982

Retinol esterification by rat liver microsomes. Evidence for a fatty acyl coenzyme A: retinol acyltransferase.

To explore the nature of retinyl ester synthesis by liver microsomes, membranes prepared from rat or cat liver were incubated under various conditions with [3H] retinol dispersed in dimethyl sulfoxide. When [3H]retinol, buffer, and microsomes were incubated together (basal conditions), some [3H]retinol esterification was consistently observed. However, the rate of esterification could be increased 6- to 11-fold by addition of either palmitoyl-CoA (100 microM) or a fatty acyl CoA-generating system. To determine whether the fatty acid used to esterify [3H]retinol under basal conditions might be derived from an endogenous pool of fatty acyl-CoA associated with the microsomal preparation, microsomes were pretreated at pH 7.4 with 0.5 M hydroxylamine, a reagent that reacts with coenzyme A thioesters to form hydroxamates. This pretreatment reduced the basal reaction by 69%. However, hydroxylamine-treated microsomes still retained acyltransferase activity, as shown by a 24- to 40-fold increase in retinyl ester synthesis after addition of palmitoyl-CoA. When microsomes were incubated with both [3H]retinol and [14C]palmitoyl-CoA of known specific radioactivities, the ratio of 14C to 3H in newly synthesized retinyl palmitate was essentially equal to that of its putative substrates, indicating that [14C]palmitate did not undergo significant isotope dilution prior to acylation of [3H]retinol. These experiments provide direct evidence for retinol esterification catalyzed by a microsomal acyl-CoA:retinol acyltransferase and indirect evidence for a pool of fatty acyl-CoA in isolated liver microsomes that is available to react with [3H]retinol to form esterified retinol.

Acyltransferases↗
PubMed1982

[Transmembrane migration of phosphatidylcholine and peroxidation of lipids in rat liver microsomes].

The exchangeability of phosphatidylcholine in microsomes differing in the degree of lipid peroxidation was determined by incubation of microsomes labelled in vivo with [methyl-14C]choline chloride with an excess of phosphatidylcholine liposomes and phosphatidyl-exchange protein from beef liver. The phosphatidylcholine peroxidation products were shown to act as substrates for the phosphatidylcholine-exchange protein. The level of exchangeable microsomal phosphatidylcholine did not depend on the degree of lipid peroxidation in the microsomes. Stimulation of lipid peroxidation by Fe2+-ascorbate or inhibition of lipid peroxidation by alpha-tocopherol did not influence the exchange of microsomal phosphatidylcholine. However, with highly peroxidized phosphatidylcholine liposomes as acceptor particles the exchange of microsomal phosphatidylcholine in the presence of phosphatidylcholine-exchange protein was strongly inhibited. In all cases the percentage of microsomal phosphatidylcholine exchangeable within 1-2 hrs was 85-90%. It is concluded that lipid peroxidation is not responsible for the rapid translocation (flip-flop) of phosphatidylcholine in rat liver microsomes. A possible involvement of membrane proteins in this process is proposed.

Androgen-Binding Protein↗
PubMed1981

Species-specific enhancement by 7,8-benzoflavone of hepatic microsomal metabolism of benzo[e]pyrene 9,10-dihydrodiol to bay-region diol epoxides.

Metabolism of benzo[e]pyrene 9,10-dihydrodiol to the bay-region 9,10-diol-11,12-epoxides by hepatic microsomes from human, rat, mouse, guinea pig, hamster, and rabbit has been examined in the presence and absence of 7,8-benzoflavone. In the absence of 7,8-benzoflavone, the formation of bay-region diol epoxides from benzo[e]pyrene 9,10-dihydrodiol was low in all species except the hamster. With hamster liver microsomes, greater than 60% of total metabolites formed were bay-region diol epoxides, whereas human and mouse liver formed less than 5% of total metabolites as bay-region diol epoxides. Addition of 7,8-benzoflavone to the microsomal incubations stimulated the formation of diol epoxides, but this stimulation was species dependent. The most dramatic stimulation was observed with human and rabbit liver microsomes. In a parallel study, metabolic activation of benzo[e]pyrene 9,10-dihydrodiol to mutagens toward Salmonella typhimurium strain TA 100 by hepatic microsomes from the above species was examined in the presence and absence of 7,8-benzoflavone. In the absence of 7,8-benzoflavone, hepatic microsomes from all the species only weakly activated benzo[e]pyrene 9,10-dihydrodiol to mutagens. 7,8-Benzoflavone enhanced the metabolic activation catalyzed by microsomes from all species except rats and hamsters. Particularly high stimulation was observed with human and rabbit liver microsomes. 9,10-Dihydroxy-9,10,11,12-tetrahydrobenzo[e]pyrene, a compound which cannot be metabolized to a bay-region diol epoxide, was not metabolically activated to mutagenic metabolites in the presence or absence of 7,8-benzoflavone by any of the species examined. These results indicated that the effect of 7,8-benzoflavone on the enhanced mutagenic activity of benzo[e]pyrene 9,10-dihydrodiol is mediated by bay-region diol epoxides, which is consistent with the metabolism studies.

Animals↗
PubMed1980

Microsomal mixed-function oxidase and activities of some related enzymes in hyperplastic nodules induced by long-term griseofulvin administration in mouse liver.

Hepatic hyperplastic nodules induced in mice by long-term griseofulvin administration were examined for selected microsomal activities and responses to enzyme inducers. Despite a decrease in microsomal cytochrome P-450 in hyperplastic nodules, aminopyrine N-demethylase was at control levels. Benzopyrene hydroxylase activity was slightly lower in microsomes derived from hyperplastic nodules than in those of control liver. Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase was at control level, but reduced nicotinamide adenine dinucleotide- and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductases and the NADPH-ferricyanide reductase were increased. NADPH-supported lipid peroxidation was lower in microsomes from hyperplastic nodules than in those from control liver, whereas microsomal stearoyl coenzyme A desaturase activity was almost doubled in the nodules. NADPH-cytochrome c reductases isolated and semipurified from hyperplastic nodule and from control liver microsomes showed almost identical affinity for NADPH. Microsomal enzymes of hyperplastic nodules responded readily to phenobarbital induction, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis disclosed differences in the polypeptide patterns in the molecular weight range from 47,000 to 54,000 between microsomes derived from hyperplastic nodules and control livers.

Animals↗
PubMed1980

The nature of NADPH-dependent lipid peroxidation in rat kidney microsomes.

The NADPH-dependent lipid peroxidation activity of rat kidney microsomes was one-third of that of liver microsomes. This activity of kidney microsomes was inhibited by EDTA, cytochrome c, Co2+, Mn2+, Cr6+, and aminopyrine, in agreement with the results reported by several investigators for liver microsomes. However, the Km for NADPH, the effect of PCMB or NEM, and the inhibitory effect of carbon tetrachloride on lipid peroxidation of kidney microsomes were different from those in liver microsomes. These results reveal that the NADPH-dependent peroxidizing system of kidney microsomes is appreciably different from that of liver microsomes.

Animals↗
PubMed1995

Cytochrome P-450 2E1 is not the sole catalyst of chlorzoxazone hydroxylation in rat liver microsomes. off.

The contribution of individual cytochrome P-450 isozymes in the hydroxylation of the centrally acting skeletal muscle relaxant chlorzoxazone was determined in rat liver microsomes. The hydroxylation rate of chlorzoxazone was found to be 50% greater in male than female microsomes. Kinetic studies using control male microsomes showed that chlorzoxazone hydroxylation was biphasic with a calculated low Km (33 microM) and high Km (116 microM). Liver microsomes from isoniazid-, beta-naphthoflavone- or dexamethasone-treated male rats produced a Km of 93, 69 and 26 microM, respectively. When chlorzoxazone hydroxylation activity was measured at a high substrate concentration (200 microM), treatment of male rats with isoniazid, acetone, beta-naphthoflavone and dexamethasone produced increases in the activity rate of 124%, 117%, 81% and 32%, respectively. However, when the activity was measured at a low substrate concentration (2 microM), liver microsomes from dexamethasone-treated male and female rats produced 5- and 10-fold induction, respectively. In immunoinhibition studies at 200 microM of chlorzoxazone, antibodies specific for cytochrome P-450 2E1 inhibited the rate of chlorzoxazone hydroxylation in microsomes from control and isoniazid-treated male rats by 68% and 79%, respectively. A monoclonal antibody (C8) against P-450 1A1 inhibited 67% of the activity in microsomes from beta-naphthoflavone-treated male rats but was ineffective inhibiting chlorzoxazone hydroxylation in microsomes from control or dexamethasone-treated male rats. In liver from control female rats, antibodies against cytochrome P-450 2E1 inhibited 80% of chlorzoxazone hydroxylation, whereas it inhibited only 47% of the activity in dexamethasone-treated females.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Malonyl-CoA-sensitive and -insensitive carnitine palmitoyltransferase activities of microsomes are due to different proteins.

A carnitine palmitoyltransferase (CPT), extracted from microsomes with octyl glucoside, was purified and characterized as a 54-kDa protein and was found to show no malonyl-CoA inhibition (Murthy, M. S. R., and Bieber, L. L. (1992) Protein Exp. Purif. 3, 75-79). We show here that the malonyl-CoA-sensitive CPT of microsomes associates with their membrane, whereas the above 54-kDa CPT is a soluble luminal protein. Western blot probing with antibody to the 54-kDa CPT was found to show a positive response with the soluble microsomal fraction but not with their membranes. 2-Tetradecylglycidyl-CoA inhibited the membrane-associated CPT activity irreversibly, whereas the inhibition of the soluble CPT was largely reversible. Exposure of microsomes to [3H]etomoxir, ATP, and CoA led to the labeling of a approximately 47-kDa peptide that associated with membranes, whereas no such peptide labeling was seen with the soluble microsomal fraction. These and other results show (a) that microsomes have malonyl-CoA-sensitive, as well as malonyl-CoA-insensitive, CPT activities, (b) that these two activities are due to distinct proteins, (c) that the malonyl-CoA-sensitive CPT of microsomes is a previously uncharacterized CPT isoform, and (d) that the [3H]etomoxir-labeled approximately 47-kDa peptide is a likely candidate for the microsomal malonyl-CoA-sensitive CPT or its regulatory subunit.

Animals↗
PubMed1994

Microsomal retinoic acid metabolism. Effects of cellular retinoic acid-binding protein (type I) and C18-hydroxylation as an initial step.

This report extends our observation that cellular retinoic acid-binding protein type I (CRABP) serves as substrate for retinoic acid metabolism by testis microsomes. Retinoic acid bound to excess CRABP was metabolized at 70% of the unbound retinoic acid rate with testis microsomes and at the same rates as unbound retinoic acid with kidney and lung microsomes. Chromatography of testis, lung, kidney, and liver microsomal incubations provided two sets of metabolites each, P1 and P2. The composition of P2 was characteristic of the individual tissue. CRABP had modest quantitative affects on P2 composition, but did not affect P2 qualitatively. Retinoids bound to CRABP, isolated from a testis microsomal incubation, consisted of 50% retinoic acid, 32% P1 and 17% P2, suggesting that CRABP may bind retinoic acid metabolites in vivo. The effect of CRABP on the rate of metabolism was retinoid specific. Two major components of P2, 4-hydroxy-retinoic acid and 4-oxo-retinoic acid, when bound to CRABP were metabolized slowly, if at all, by testis microsomes, in contrast to CRABP-bound retinoic acid which had an elimination t1/2 of 40 min. Unbound retinoic acid, 4-hydroxy-retinoic acid, and 4-oxo-retinoic acid had elimination t1/2 values of 35, 40, and 9 min, respectively. Reduced metabolism of CRABP-bound C4-derivatized retinoids suggests pathways of retinoic acid metabolism besides the one initiated by C4-hydroxylation. This was corroborated by identification of 18-hydroxy-retinoic acid as a testis, lung, and liver microsomal retinoic acid metabolite. Ketoconazole inhibited the metabolism by testis microsomes of free and CRABP-bound retinoic acid with IC50 values of 2 and 0.7 microM, respectively, denoting catalysis by cytochrome P-450. These results indicate that cloistering retinoic acid in CRABP, while permitting metabolism, may operate throughout CRABP-expressing tissues as a mechanism of controlling the concentrations of free retinoic acid.

Animals↗
PubMedDate not supplied

Monoacetyldapsone inhibition of dapsone N-hydroxylation by human and rat liver microsomes.

Dapsone (DDS) is metabolized by N-hydroxylation and N-acetylation to DDS hydroxylamine (DDS-NOH) and monoacetyldapsone (MAD), respectively. The activities of these two alternative and independent reactions vary widely between individuals and show an inverse relationship during chronic DDS therapy. Toxicity observed during DDS therapy has been attributed to DDS-NOH. The observation of reduced toxicity in rapid acetylators, who are also poor hydroxylators, therefore, raised the possibility that MAD may be inhibiting DDS-NOH formation. This hypothesis was tested in human and rat liver microsomes. Human liver microsomes hydroxylated DDS with a lower affinity (KM 2-fold greater) and lower maximal catalytic activity (Vmax 12-fold lower) than that of the rat. The relative catalytic activity (Vmax/KM) was 22-fold higher in rat compared with human liver microsomes. Furthermore, MAD was a potent inhibitor of DDS N-hydroxylation by rat liver microsomes (52% inhibition at 0.01 mM MAD) compared with human liver microsomes (23% inhibition at 0.4 mM MAD). Human, but not rat, liver microsomes deacetylated MAD to DDS by an NADPH independent mechanism. These results show that substantial differences exist in DDS N-hydroxylase between rats and humans, with respect to substrate affinity, enzyme activity, and susceptibility to inhibition, such that information obtained from the rat should not be extrapolated to humans. We conclude that MAD is a potent inhibitor of DDS-NOH formation in rat liver microsomes. The degree of inhibition in human microsomes, however, suggest that MAD is unlikely to be a significant modulator of enzyme activity in vivo.

Animals↗
PubMed1996

Glutathione-dependent protection against lipid peroxidation in sheep liver microsomes.

In the present studies we have described a glutathione-dependent system in sheep liver microsomes that protects against membrane lipid peroxidation initiated by either Fe+2/NADPH or Fe+2/ascorbate. Glutathione protected against lipid peroxidation in microsomes containing a wide range of alpha-tocopherol levels (0.02-0.11 microgram/mg protein). The addition of glutathione disulfide alone had no effect on microsomal lipid peroxidation, however, it prolonged the protection afforded by glutathione, particularly in assays containing Fe+2/NADPH. Whereas the glutathione-dependent protection was very labile, with loss of activity demonstrated in microsomes stored at 4 degrees C for 24 hours, the combined effect of glutathione and glutathione disulfide was not affected by storage. The glutathione S-transferase inhibitors, bromosulphothalein and S-hexylglutathione, reversed the protection observed with glutathione, indicating a possible role for microsomal glutathione S-transferases in this protection, but not that observed by the combination of glutathione and glutathione disulfide. In general, our findings support previous results observed with rat liver microsomes and suggest that microsomal glutathione S-transferases may be involved in the glutathione-dependent protection in sheep liver microsomes.

Animals↗
PubMed1996

Influence of dolichol on microsomal membrane functions.

Microsomal membranes from rat liver were extracted with n-pentane in order to remove the lipid products of the mevalonate pathway, dolichol, ubiquinone and cholesterol. Dolichol and cholesterol were subsequently reincorporated into these extracted membranes. Electron microscopic examination demonstrated that extraction did not alter the vesicular membrane structure of the microsomes. The extracted vesicles were permeable to uncharged molecules in the same manner as control microsomes but had an increased permeability for charged molecules. Enzyme denaturation was not observed. The contraction of extracted vesicles was greatly increased when the incubation medium was supplemented with non-penetrating compounds such as polyethylene glycol and was much greater than that of control microsomes. When extracted membranes were reconstituted with dolichol or cholesterol, the original lower degree of contraction was reestablished. The effects of dolichol reincorporation on a number of microsomal enzyme activities were investigated and some limited changes were observed. These results demonstrate that extraction of microsomes with n-pentane and subsequent reincorporation of dolichol is an effective approach for investigating the functions of this lipid. Dolichol and cholesterol both affect microsomal membrane fluidity, but only cholesterol modifies the activities of certain integral microsomal membrane enzymes to a larger extent.

Animals↗
PubMed1997

Prediction of in vivo hepatic metabolic clearance of YM796 from in vitro data by use of human liver microsomes and recombinant P-450 isozymes.

The metabolic rate of (S)-(-)-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro [4,5] decane-L-tartarate monohydrate (YM796), an antidementia agent, was determined by use of 12 different human liver microsomal samples. The metabolism of YM796 was shown to consist of three components; one high-affinity (Km1 = 1.67 microM), one low-affinity (Km2 = 654 microM) and a nonsaturable component. Good correlations were observed between the individual CYP3A4 content in 12 different human liver microsomal samples and kinetic parameters such as CL(int, all), the high-affinity component clearance (Vmax1/Km1) and the low-affinity component clearance (Vmax2/Km2). Anti-human CYP3A4/5 antibodies inhibited the metabolism of YM796 at 1 microM by up to 75%. In addition, ketoconazole, an inhibitor of CYP3A4, inhibited YM796 metabolism by >90%. The metabolic clearance of YM796 in each of the 12 human liver microsomal samples was successfully predicted from the kinetic parameters obtained with the recombinant microsomes by taking into consideration the CYP3A4 content in each microsomal sample. Based on the CL(int, all) estimated from the in vitro experiments, the area under the plasma concentration-time curve after oral administration (AUC(oral)) of YM796 was also predicted by taking into account the hepatic blood flow rate (Qh), the unbound fraction of YM796 in human plasma (f(p)) and the fraction absorbed from the gut. In addition, AUC(oral) was determined in six healthy male volunteers. The predicted AUC(oral) was similar to the observed value in vivo, which suggests that the in vitro metabolism data obtained with human liver microsomes are useful for quantitatively predicting human liver metabolism in vivo and that recombinant microsomes are also available when the particular isozyme is almost completely responsible for the metabolism of the drug, the variation in P-450 content of human liver is known and the experimental conditions such as the amount of CYP reductase and cytochrome b5 are carefully optimized to mimic the activity found in native microsomes, as for YM796.

Area Under Curve↗
PubMed1997

Different contributions of cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole by human liver microsomes: effects of contents of these two forms in individual human samples.

Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Correlation coefficients between omeprazole 5-hydroxylation activities (when determined at a substrate concentration of 10 microM) and S-mephenytoin 4'-hydroxylation and testosterone 6beta-hydroxylation activities were found to be 0.64 and 0.67, respectively, in liver microsomes of 84 human samples examined. Omeprazole sulfoxidation activities in these human samples were correlated with testosterone 6beta-hydroxylation activities (r = 0. 86). Omeprazole 5-hydroxylation by liver microsomes of a human sample that contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, although a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive toward fluvoxamine and anti-CYP2C9 antibodies. Sulfaphenazole (at 100 microM) did not affect the omeprazole 5-hydroxylation and sulfoxidation catalyzed by human liver microsomes. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. These results suggest that contributions of CYP2C19 and CYP3A4 in the omeprazole 5-hydroxylation depend upon the ratio of these two P450 levels in human liver microsomes. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal CYP2C19 and CYP3A4 enzymes. Finally, when recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined by analysis of high-performance liquid chromatography. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 microM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver.

Animals↗
PubMed1997

The In vitro hepatic metabolism of quinine in mice, rats and dogs: comparison with human liver microsomes.

The major metabolic pathway of quinine in the human has been shown to be 3-hydroxylation mediated mainly by human cytochrome P450 (CYP) 3A4. In this extended in vitro study, quinine 3-hydroxylation was further investigated using microsomes from mouse, rat, dog and human livers and was compared among them in terms of the in vitro enzyme-kinetic parameters and quinine-drug interaction screenings. In all species, 3-hydroxyquinine was the principal metabolite of quinine. There was intra- and interspecies variability among all the kinetic parameters, and dogs exhibited a closer resemblance to humans in terms of the mean kinetic data. Ketoconazole and troleandomycin inhibited the 3-hydroxylation of quinine in all species. Both alpha-naphthoflavone and diazepam showed an interspecies difference in quinine 3-hydroxylation: a trend toward an activation in dog and human, and a significant inhibition in mouse and rat, liver microsomes. Antisera raised against rat CYP3A2 strongly inhibited quinine 3-hydroxylation by about 96, 84 and 92% with mouse, rat and dog liver microsomes, respectively, but neither anti-rat 2C11 and 2E1 antisera did so with rat liver microsomes. Primaquine, doxycycline and tetracycline substantially inhibited the formation of 3-hydroxyquinine in rat, dog and human species, but proguanil had no such effect in any species. Chloroquine inhibited quinine 3-hydroxylation with rat and dog liver microsomes but not with human liver microsomes. There was a significant correlation (r = 0.986, P < .001) between the CYP3A contents and the formation rates of 3-hydroxyquinine in eight human liver microsomal samples. It is concluded that 3-hydroxyquinine is a main metabolite of quinine and that CYP3A/Cyp3a is a principal isoform involved in this metabolic pathway in the respective (rat, dog and human/mouse) species tested. The dog and possibly the rat may be qualitatively and quantitatively suitable animal models for exploring the quinine 3-hydroxylase activity and for screening quinine-drug interactions in vitro, at certain inconsistency with the human liver microsomal data.

Animals↗
PubMed1998

Isozyme-selective metabolism of ethyl carbamate by cytochrome P450 (CYP2E1) and carboxylesterase (hydrolase A) enzymes in murine liver microsomes.

Cytochrome P450 and carboxylesterase enzymes have been implicated in the metabolism of the carcinogen ethyl carbamate (EC). In this study, we have used a murine liver microsomal system to investigate the relative contributions of P450 and carboxylesterase isozymes to hepatic metabolism of EC. N-Nitrosodimethylamine (NDMA) demethylation and p-nitrophenyl acetate (PNA) hydrolysis were used as catalytic markers of CYP2E1 and carboxylesterase enzymes, respectively. Incubation of liver microsomes with EC (1 mM) produced slight but significant decreases in NDMA demethylation and PNA hydrolysis activities. Incubation of microsomes with paraoxon (PAX), a general carboxylesterase inhibitor, or phenylmethylsulfonyl fluoride (PMSF), a specific inhibitor of hydrolase A, produced decreases of 85 and 45%, respectively, in carboxylesterase activities; neither of the inhibitors elicited alterations in levels of NDMA demethylation. Reaction of microsomes with either PAX or PMSF and then with EC exacerbated the reduction (285%) of NDMA demethylation, and this loss corresponded to decreases in immunodetectable CYP2E1 content. The reduction in PNA hydrolysis activity induced by PAX, PMSF, or EC correlated with decreased immunodetectable hydrolase A in liver microsomes; however, reaction with PAX and not PMSF or EC resulted in loss of immunoreactivity for hydrolase B. These data correlated with levels of covalent binding of [ethyl-14C]EC to liver microsomes, which were significantly elevated in incubations conducted with PAX or PMSF. Antibody inhibition of the CYP2E1 enzyme significantly reduced levels of binding to microsomal proteins, compared with control levels. These results are consistent with the premise that EC is metabolized by CYP2E1 and hydrolase A in liver microsomes of mice.

Animals↗
PubMed1998

Microsomal acetaldehyde oxidation is negligible in the presence of ethanol.

The microsomal ethanol oxidizing system (MEOS), inducible by ethanol and acetone, oxidizes ethanol to acetaldehyde, which causes many toxic effects associated with excess ethanol. Recent studies reported that rat liver microsomes also oxidize acetaldehyde, thereby challenging the validity of the assessment of MEOS activity by measuring acetaldehyde production and suggesting that MEOS activity results in the accumulation not of acetaldehyde but, rather, of its less toxic metabolite, acetate. To address these issues, we compared both metabolic rates of ethanol and acetaldehyde and the effect of ethanol on the acetaldehyde metabolism. Liver microsomes were prepared from Sprague-Dawley rats induced either with acetone for 3 days or ethanol for 3 weeks. NADPH-dependent acetaldehyde (300 microM) metabolism was measured in two ways: (1) by detection of acetaldehyde disappearance by headspace gas chromatography, and (2) by assessment of acetaldehyde oxidation by liquid scintillation counting of acetate formed from [1,2-14C]acetaldehyde. Ethanol (50 mM) oxidation was measured by gas chromatography. In acetone- and ethanol-induced rat liver microsomes, the acetaldehyde disappearance (p < 0.0001) and oxidation (p < 0.0001) rates were both significantly increased. The rates of acetaldehyde oxidation paralleled those of p-nitrophenol hydroxylation (r = 0.974, p < 0.0001), with a Km of 82+/-14 microM and a Vmax of 4.8+/-0.5 nmol/min/mg protein in acetone-induced microsomes. Acetaldehyde disappearance in acetone-induced microsomes and acetaldehyde oxidation in acetone-induced and ethanol-induced microsomes were significantly lower than the corresponding ethanol oxidation, with rates (nmol/min/mg protein) of 4.6+/-0.6 versus 9.0+/-0.8 (p < 0.005), 4.4+/-0.3 versus 9.1+/-0.5 (p < 0.0005), and 14.0+/-0.9 versus 19.5+/-1.8 (p < 0.05), respectively. The presence of 50 mM ethanol decreased this metabolism to 0.9+/-0.3 (p < 0.005), 0.5+/-0.1 (p < 0.001), and 1.8+/-0.3 (p < 0.001), resulting in rates of acetaldehyde metabolism of only 9.8+/-3.2%, 6.0+/-0.5%, and 9.5+/-1.2% (respectively) of those of ethanol oxidation. In conclusion, rat liver microsomes oxidize acetaldehyde at much lower rates than ethanol, and this acetaldehyde metabolism is strikingly inhibited by ethanol. Accordingly, acetaldehyde formation provides an accurate assessment of MEOS activity. Furthermore, because acetaldehyde production vastly exceeds its oxidation, the net result of MEOS activity is the accumulation of this toxic metabolite.

Acetaldehyde↗