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PubMed1984

Hydroxylation of prostaglandin A1 by the microsomes of rabbit intestinal mucosa.

Microsomes from rabbit small intestine mucosa were found to catalyze the hydroxylation of PGA1 in the presence of NADPH. The major product was identified as 20-hydroxy PGA1 by using high performance liquid chromatography and gas chromatography-mass spectrometry, and the minor product was assumed to be 19-hydroxy PGA1. The ratio of the former product to the latter was about 24.1. The specific PGA1 omega-hydroxylase activity of small intestine microsomes was comparable to that of liver microsomes, and was significantly higher than those of microsomes from other tissues such as kidney cortex and lung. Microsomes from rabbit colon mucosa also catalyzed the hydroxylation of PGA1 in the presence of NADPH, with the ratio of omega- to (omega-1)-hydroxy PGA1 formed being 33.0. The PGA1 hydroxylase activities of the microsomes from both small intestine and colon were inhibited markedly by carbon monoxide, indicating the participation of cytochrome P-450. A cytochrome P-450 was solubilized from small intestine microsomes, and purified to a specific content of 10.5 nmol of cytochrome P-450/mg of protein. This cytochrome hydroxylated PGA1 at the omega-position with a turnover rate of 38.2 nmol/min/nmol of cytochrome P-450 in the reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and phosphatidylcholine. It is suggested that this cytochrome P-450 is specialized for the omega-hydroxylation of PGA1 in small intestine microsomes.

Animals↗
PubMed1992

Cytochrome P450 isozymes catalyzing 4-hydroxylation of parkinsonism-related compound 1,2,3,4-tetrahydroisoquinoline in rat liver microsomes.

Microsomal 4-hydroxylase of 1,2,3,4-tetrahydroisoquinoline (TIQ), a possible candidate for causing Parkinson disease, was characterized by using rat hepatic microsomes and purified P450 isozymes. Kinetic analysis revealed that Km and Vmax values (mean +/- SE) for hepatic microsomal TIQ 4-hydroxylase of male Wistar rats were 319.6 +/- 26.8 microM and 12.13 +/- 1.43 pmol.min-1.mg-1 protein, respectively. When TIQ 4-hydroxylase activity was compared in Wistar (an animal model of extensive debrisoquine metabolizers) and Dark Agouti (an animal model of poor debrisoquine metabolizers) rats, significant strain (Wistar greater than Dark Agouti) and sex (male greater than female) differences were observed. The microsomal activity toward TIQ 4-hydroxylation was increased by pretreatment of male Wistar rats with P448 inducers (beta-naphthoflavone and sudan I), but not with phenobarbital. Pretreatment with propranolol, an inhibitor of P450 isozymes belonging to the P450 IID gene subfamily, decreased TIQ 4-hydroxylase activity. P450 BTL, a P450 isozyme belonging to the IID subfamily, showed TIQ 4-hydroxylase activity of 64.1 pmol.min-1.nmol P450(-1), which was 3.2-fold that of microsomes (20.9 pmol.min-1.nmol P450(-1)). Antibody (IgG) against this isozyme suppressed microsomal TIQ 4-hydroxylase activity concentration-dependently. A male-specific P450 ml (P450IIC11) catalyzed this reaction to a much lesser extent (10.0 pmol.min-1.nmol P450(-1)), and its antibody did not affect the microsomal activity. These results suggest that TIQ 4-hydroxylation in hepatic microsomes are catalyzed predominantly by a P450 isozyme (or isozymes) belonging to the IID gene subfamily in non-treated rats and its immunochemically related P450 isozyme (or isozymes), and that a P450 isozyme (or isozymes) belonging to the IA subfamily also participates in TIQ 4-hydroxylation in rats pretreated with P448-inducers.

Amino Acid Sequence↗
PubMed1983

Studies on the role of reactive oxygen species in mediating lipid peroxide formation in epidermal microsomes of rat skin.

The role of superoxide, hydrogen peroxide, and singlet oxygen in mediating nonenzymic and NADPH-supported enzymic lipid peroxidation in skin microsomes was investigated. Incubation of skin microsomes with NADPH and/or Fe+3-ADP or ascorbate resulted in the formation of lipid peroxides. The epidermis was the major target site for microsomal lipid peroxide formation in skin. Enzymic peroxidation of epidermal microsomes required NADPH and was oxygen-dependent. Addition of the nonenzymic catalysts, Fe+3 and ADP, to the enzymic peroxidation system had an additive effect on the generation of lipid peroxide in epidermal microsomes. Epidermal microsomal lipid peroxidation was inhibited by singlet oxygen quenchers such as dimethylfuran, histidine, and beta-carotene. Hydroxyl ion scavengers such as mannitol, benzoate, or ethyl alcohol and the enzymic scavenger of superoxide, superoxide dismutase, were all ineffective in this respect. Addition of EDTA, Mn+2, cytochrome c+3, and catalase to the NADPH-supported enzymic peroxidation system resulted in strong inhibition of lipid peroxide formation in skin. Glutathione or epidermal cytosol added alone or in combination to the NADPH-supported incubation system enhanced peroxidation of microsomal lipids. Vitamin E (alpha-tocopherol) inhibited lipid peroxidation. These results indicate that singlet oxygen may mediate lipid peroxide formation in epidermal microsomes.

Adenosine Diphosphate↗
PubMed1981

Cytochrome P-450 monooxygenase activities in human and rat liver microsomes.

Microsomes were prepared from human livers obtained from renal donors of various ages and both sexes. Their drug-metabolizing capacity was measured and compared to that of rat liver microsomes. The following parameters were investigated: cytochrome P-450, cytochrome B5, NADPH-cytochrome c reductase, epoxide hydrolase, aryl hydrocarbon hydroxylase, benzphetamine N-demethylase, p-nitroanisole-O-demethylase, ethoxycoumarin-O-deethylase, steroid-16 alpha-hydroxylase. In addition, the metabolism of benzo(a)pyrene, progesterone, pregnenolone, testosterone, dehydroepiandrosterone and estradiol was studied in detail in vitro. The inhibitory effect of metyrapone and alpha-naphthoflavone on 7-ethoxycoumarin-O-deethylase was measured. The microsomal proteins of both species were separated by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The following conclusions were drawn from the results obtained. Human liver microsomes can be stored under optimal conditions for the measurement of a large variety of enzymic activities. Human liver microsomes are able to metabolize the various xenobiotics used as substrates with a rate similar to that of female rat liver microsomes. No sex-linked difference in enzymic activity was observed in human microsomes. Significant differences in benzo(a)pyrene and steroid metabolism were registered when human and rat liver microsomes were compared. The monooxygenase activities, the sensitivity to in vitro alpha-naphthoflavone and metyrapone, the results of steroid metabolism, and slab gel electrophoresis are strong indications for multiplicity of human liver cytochrome P-450.

Animals↗
PubMed1986

Metabolism of n-pentane by ethanol-inducible cytochrome P-450 in liver microsomes and reconstituted membranes.

The cytochrome P-450-dependent metabolism of n-pentane was studied by head-space gas chromatographic analysis of incubations with liver microsomes and reconstituted systems from rat and rabbit liver. The alkane was metabolized in liver microsomes from imidazole-treated rabbits at an apparent Vmax of 3 nmol mg-1 min-1 and a Km of 35 microM and in liver microsomes from acetone-treated rats at an apparent Vmax of 10 nmol mg-1 min-1 and a Km of 9 microM. The rate of microsomal n-pentane metabolism was enhanced fivefold by acetone treatment of rats, compared to the rate observed in liver microsomes from control rats. The reaction was inhibited in microsomes by compounds that are known to interact with the acetone and ethanol-inducible form of liver microsomal cytochrome P-450, like acetone, imidazole, ethanol and benzene. Effective inhibition was also accomplished when microsomal incubations were performed in the presence of IgG against this form of cytochrome P-450 from either rat or rabbit liver. In reconstituted membrane vesicles containing NADPH--cytochrome P-450 reductase, ethanol-inducible P-450 from rat liver (P-450j) was a fivefold more efficient catalyst of pentane metabolism than was the corresponding P-450 form from rabbit liver (P-450 LMeb). The rabbit enzyme metabolized the hydrocarbon with an apparent Vmax of 4 nmol nmol-1 min-1 and a Km of 8 microM, By contrast, phenobarbital-inducible P-450 LM2 or 3-methylcholanthrene-inducible P-450 LM4 from rabbit liver were quite ineffective catalysts of n-pentane metabolism. It is concluded that n-pentane constitutes a good substrate for cytochrome P-450 and, in particular, for the ethanol-inducible form of this hemoprotein. It is suggested that n-pentane is an important substrate for P-450 under in vivo conditions and that pentane measurements in expired air as an indicator of lipid peroxidation must be interpreted with caution.

Animals↗
PubMed1997

Visualization of a covalent intermediate between microsomal epoxide hydrolase, but not cholesterol epoxide hydrolase, and their substrates.

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of alpha/beta-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with [1-(14)C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1,1,1-trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluorochalcone-oxide-sensitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-(14)C]cholesterol-5alpha,6alpha-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.

Amino Acid Sequence↗
PubMed1976

Comparison of the actions of acebutolol, practolol and propranolol on calcium transport by heart microsomes and mitochondria.

1 The effects of acebutolol, practolol and propranolol (0,5-3 mM) on calcium uptake, calcium binding and ATPase activities of the rabbit and rat heart microsomal and mitochondrial fractions were investigated. 2 Dose-response and time course experiments revealed that propranolol greatly inhibited microsomal and mitochondrial calcium uptake whereas both acebutolol and practolol showed slight depressant effects. 3 The ATPase activities of microsomal and mitochondrial fractions were decreased by acebutolol, practolol and propranolol; however, the latter agent was more effective than the other two. 4 The inhibitory effects of acebutolol, practolol and propranolol on mitochondria and microsomes were not antagonized by adrenaline. 5 Propranolol decreased calcium binding by the microsomal fraction only, whereas acebutolol and practolol had no effect on microsomal or mitochondrial calcium binding. 6 The sensitivity of the rabbit heart subcellular fractions to the beta-adrenoceptor blocking drugs was similar to that of the rat heart; however, the calcium uptake and ATPase activities of microsomes were more sensitive to propranolol than mitochondria in both species. 7 Perfusion of rat hearts with 0.2-1 mM propranolol decreased contractile force, and microsomal and mitochondrial fractions obtained from these hearts accummulated less calcium in comparison to the control. On the other hand, acebutolol and practolol (0.2-1nM) had no appreciable effects on contractile force or subcellular fractions under similar conditions. 8 The negative inotropic effect of propranolol may partly be due to its inhibitory actions on calcium transport by subcellular organelles of the myocardium; the depressant action of propranolol on calcium transport is unlikely to be due to its beta-adrenoceptor blocking property.

Acebutolol↗
PubMed1988

Microsomal enzyme induction, lipoproteins and atherosclerosis.

The liver is the principal site for the synthesis and elimination of lipoproteins circulating in plasma, and alterations in hepatic function influence plasma lipoprotein levels. High HDL-C/T-C and apo A-I/apo B ratios, which are characteristic of a low coronary risk, have been typical of healthy subjects with high microsomal enzyme activity in the liver. Microsomal enzyme inducing drugs such as phenytoin, phenobarbital and carbamazepine, and also alcohol, influence serum lipid and apoprotein concentrations. The inducers increase the concentrations of hepatic microsomal enzyme and apo A-I mRNA, and also proteins and phospholipids. They similarly increase serum HDL-C and apo A-I levels and the HDL-C/LDL-C ratio, powerful protective factors against coronary heart disease. These parameters parallel hepatic protein and phospholipid concentrations, and microsomal enzyme activity as assessed by liver cytochrome P-450 or antipyrine kinetics. Serum LDL-C levels are inversely proportional to hepatic cytochrome P-450 concentrations. Experimental studies indicate that phenobarbital retards cholesterol accumulation in the arterial wall and the formation of atherosclerotic plaque. A decreased mortality rate from coronary heart disease has been reported for subjects who take enzyme inducers, drugs or alcohol, whereas impairment of hepatic microsomal function may promote atherogenesis. In addition to drugs non-pharmacological factors such as dietary constituents and physical exercise may influence hepatic microsomal function and hence improve the serum lipoprotein profile. These observations, which connect microsomal inducers, liver lipids and proteins, serum lipids and apoproteins characteristic of a low risk of atherosclerotic disease and low incidence of coronary deaths, lead to the conclusion that the activation of liver microsomal function can prevent atherogenesis in man.

Arteriosclerosis↗
PubMed2004

Protective effect of N-acetyl-serotonin on the nonenzymatic lipid peroxidation in rat testicular microsomes and mitochondria.

N-acetyl-serotonin, the immediate precursor of melatonin in the tryptophan metabolic pathway in the pineal gland, has been reported to be an antioxidant. The aim of this study was to test the in vitro protective effect of N-acetyl-serotonin on the ascorbate-Fe(++) induced lipid peroxidation of polyunsaturated fatty acids (PUFAs) located in testis microsomes and mitochondria. We assayed increasing concentrations (0-10 mM) of N-acetyl-serotonin in testis microsomes and (0-1 mM) of N-acetyl-serotonin in testis mitochondria. Control experiments were performed by incubating microsomal and mitochondrial membranes with N-acetyl-serotonin in the absence of lipid peroxidation-inducing drugs. Special attention was paid to the changes produced on the highly PUFAs C20:4 n6 and C22:5 n6. The light emission (chemiluminescence) used as a marker of lipid peroxidation was similar in both organelles when the control and peroxidized groups were compared. N-acetyl-serotonin reduced lipid peroxidation in testicular microsomes or mitochondria for both C20:4 n6 and C22:5 n6. Both long chain PUFAs were protected when N-acetyl-serotonin was incorporated either into microsomes or mitochondria. The N-acetyl-serotonin concentration required to inhibit by approximately 70% lipid peroxidation process was 10 mM in microsomes and between 0.50 and 1 mM in mitochondria. IC 50 values calculated from the inhibition curve of N-acetyl-serotonin on the chemiluminescence rates were higher in microsomes (4.50 mM) than in mitochondria (0.25 mM). In these experimental conditions, N-acetyl-serotonin was about 18 times more potent in testicular mitochondria in inhibiting the oxidative processes than it was in testicular microsomes. These results suggest that the protective role of N-acetyl-serotonin in preserving the long PUFAs may be related to its ability to reduce lipid peroxidation.

Animals↗
PubMed1987

The microsomal ethanol oxidizing system and its interaction with other drugs, carcinogens, and vitamins.

The interaction of ethanol with the oxidative drug-metabolizing enzymes present in liver microsomes results in a number of clinically significant side effects in the alcoholic. Following chronic ethanol consumption, the activity of the microsomal ethanol oxidizing system (MEOS) increases. This enhancement of MEOS activity is primarily due to the induction of a unique microsomal cytochrome P-450 isozyme, which has a high capacity for ethanol oxidation, as shown in reconstituted systems. Normally present in liver microsomes at low levels, this form of cytochrome P-450 increases dramatically after chronic ethanol intake in many species, including baboons. The in-vivo role of cytochrome P-450 in hepatic ethanol oxidation, especially following chronic ethanol consumption, has been conclusively demonstrated in deer-mice lacking liver ADH. Induction of microsomal cytochrome P-450 by ethanol is associated with the enhanced oxidation of other drugs as well, resulting in metabolic tolerance to these agents. There is also increased cytochrome P-450-dependent activation of known hepatotoxins such as carbon tetrachloride and acetaminophen, which may explain the greater susceptibility of alcoholics to the toxicity of industrial solvents and commonplace analgesics. In addition, the ethanol-inducible form of cytochrome P-450 has the highest capacity of all known P-450 isozymes for the activation of dimethylnitrosamine, a potent (and ubiquitous) carcinogen. Moreover, cytochrome P-450-catalyzed oxidation of retinol is accelerated in liver microsomes, which may contribute to the hepatic vitamin A depletion seen in alcoholics. In contrast to chronic ethanol consumption, acute ethanol intake inhibits the metabolism of other drugs via competition for shared microsomal oxidation pathways. Thus, the interplay between ethanol and liver microsomes has a profound impact on the way heavy drinkers respond to drugs, solvents, vitamins, and carcinogens.

Alcohol Dehydrogenase↗
PubMed2004

Trans-3'-hydroxycotinine O- and N-glucuronidations in human liver microsomes.

Trans-3'-hydroxycotinine is a major metabolite of nicotine in humans and is mainly excreted as O-glucuronide in smoker's urine. Incubation of human liver microsomes with UDP-glucuronic acid produces not only trans-3'-hydroxycotinine O-glucuronide but also N-glucuronide. The formation of N-glucuronide exceeds the formation of O-glucuronide in most human liver microsomes, although N-glucuronide has never been detected in human urine. Trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes was significantly correlated with nicotine and cotinine N-glucuronidations, which are catalyzed mainly by UDP-glucuronosyltransferase (UGT)1A4 and was inhibited by imipramine and nicotine, which are substrates of UGT1A4. Recombinant UGT1A4 exhibited substantial trans-3'-hydroxycotinine N-glucuronosyltransferase activity. These results suggest that trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes would be mainly catalyzed by UGT1A4. In the present study, trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes was thoroughly characterized, since trans-3'-hydroxycotinine O-glucuronide is one of the major metabolites of nicotine. The kinetics were fitted to the Michaelis-Menten equation with a K(m) of 10.0 +/- 0.8 mM and a V(max) of 85.8 +/- 3.8 pmol/min/mg. Among 11 recombinant human UGT isoforms expressed in baculovirus-infected insect cells, UGT2B7 exhibited the highest trans-3'-hydroxycotinine O-glucuronosyltransferase activity (1.1 pmol/min/mg) followed by UGT1A9 (0.3 pmol/min/mg), UGT2B15 (0.2 pmol/min/mg), and UGT2B4 (0.2 pmol/min/mg) at a substrate concentration of 1 mM. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7 increased with an increase in the substrate concentration up to 16 mM (10.5 pmol/min/mg). The kinetics by recombinant UGT1A9 were fitted to the Michaelis-Menten equation with K(m) = 1.6 +/- 0.1 mM and V(max) = 0.69 +/- 0.02 pmol/min/mg of protein. Trans-3'-hydroxycotinine O-glucuronosyltransferase activities in 13 human liver microsomes ranged from 2.4 to 12.6 pmol/min/mg and were significantly correlated with valproic acid glucuronidation (r = 0.716, p < 0.01), which is catalyzed by UGT2B7, UGT1A6, and UGT1A9. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity in human liver microsomes was inhibited by imipramine (a substrate of UGT1A4, IC(50) = 55 microM), androstanediol (a substrate of UGT2B15, IC(50) = 169 microM), and propofol (a substrate of UGT1A9, IC(50) = 296 microM). Interestingly, imipramine (IC(50) = 45 microM), androstanediol (IC(50) = 21 microM), and propofol (IC(50) = 41 microM) also inhibited trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7. These findings suggested that trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes is catalyzed by mainly UGT2B7 and, to a minor extent, by UGT1A9.

Animals↗
PubMed2002

Troglitazone glucuronidation in human liver and intestine microsomes: high catalytic activity of UGT1A8 and UGT1A10.

Troglitazone glucuronidation in human liver and intestine microsomes and recombinant UDP-glucuronosyltransferases (UGTs) were thoroughly characterized. All recombinant UGT isoforms in baculovirus-infected insect cells (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) exhibited troglitazone glucuronosyltransferase activity. Especially UGT1A8 and UGT1A10, which are expressed in extrahepatic tissues such as stomach, intestine, and colon, showed high catalytic activity, followed by UGT1A1 and UGT1A9. The kinetics of the troglitazone glucuronidation in the recombinant UGT1A10 and UGT1A1 exhibited an atypical pattern of substrate inhibition when the substrate concentration was over 200 micro M. With a Michaelis-Menten equation at 6 to 200 micro M troglitazone, the K(m) value was 11.1 +/- 5.8 micro M and the V(max) value was 33.6 +/- 3.7 pmol/min/mg protein in recombinant UGT1A10. In recombinant UGT1A1, the K(m) value was 58.3 +/- 29.2 micro M and the V(max) value was 12.3 +/- 2.5 pmol/min/mg protein. The kinetics of the troglitazone glucuronidation in human liver and jejunum microsomes also exhibited an atypical pattern. The K(m) value was 13.5 +/- 2.0 micro M and the V(max) value was 34.8 +/- 1.2 pmol/min/mg for troglitazone glucuronidation in human liver microsomes, and the K(m) value was 8.1 +/- 0.3 micro M and the V(max) was 700.9 +/- 4.3 pmol/min/mg protein in human jejunum microsomes. When the intrinsic clearance was estimated with the in vitro kinetic parameter, microsomal protein content, and weight of tissue, troglitazone glucuronidation in human intestine was 3-fold higher than that in human livers. Interindividual differences in the troglitazone glucuronosyltransferase activity in liver microsomes from 13 humans were at most 2.2-fold. The troglitazone glucuronosyltransferase activity was significantly (r = 0.579, p < 0.05) correlated with the beta-estradiol 3-glucuronosyltransferase activity, which is mainly catalyzed by UGT1A1. The troglitazone glucuronosyltransferase activity in pooled human liver microsomes was strongly inhibited by bilirubin (IC(50) = 1.9 micro M), a typical substrate of UGT1A1. These results suggested that the troglitazone glucuronidation in human liver would be mainly catalyzed by UGT1A1. Interindividual differences in the troglitazone glucuronosyltransferase activity in S-9 samples from five human intestines was 8.2-fold. The troglitazone glucuronosyltransferase activity in human jejunum microsomes was strongly inhibited by emodin (IC(50) = 15.6 micro M), a typical substrate of UGT1A8 and UGT1A10, rather than by bilirubin (IC(50) = 154.0 micro M). Therefore, it is suggested that the troglitazone glucuronidation in human intestine might be mainly catalyzed by UGT1A8 and UGT1A10.

Catalysis↗
PubMed2003

Glucuronidation of etoposide in human liver microsomes is specifically catalyzed by UDP-glucuronosyltransferase 1A1.

A metabolite formed by incubation of human liver microsomes, etoposide, and UDP-glucuronic acid was identified as etoposide glucuronide by liquid chromatography-tandem mass spectrometry analysis. According to the derivatization with trimethylsilylimidazole (Tri-Sil-Z), it was confirmed that the glucuronic acid is linked to an alcoholic hydroxyl group of etoposide and not to a phenolic group. Among nine recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9. UGT1A10, UGT2B7, and UGT2B15), only UGT1A1 exhibited the catalytic activity of etoposide glucuronidation. The enzyme kinetics in pooled human liver microsomes and recombinant UGT1A1 microsomes showed a typical Michaelis-Menten plot. The kinetic parameters of etoposide glucuronidation were K(m) = 439.6 +/- 70.7 microM and V(max) = 255.6 +/- 19.2 pmol/min/mg of protein in human liver microsomes and K(m) = 503.2 +/- 110.2 microM and V(max) = was 266.5 +/- 28.6 pmol/min/mg of protein in recombinant UGT1A1. The etoposide glucuronidation in pooled human liver microsomes was inhibited by bilirubin (IC(50) = 31.7 microM) and estradiol (IC(50) = 34 microM) as typical substrates for UGT1A1. The inhibitory effects of 4-nitrophenol (IC(50) = 121.0 microM) as a typical substrate for UGT1A6 and UGT1A9, imipramine (IC(50) = 393.8 microM) as a typical substrate for UGT1A3 and UGT1A4, and morphine (IC(50) = 109.3 microM) as a typical substrate for UGT2B7 were relatively weak. The interindividual difference in etoposide glucuronidation in 13 human liver microsomes was 78.5-fold (1.4-109.9 pmol/min/mg of protein). The etoposide glucuronidation in 10 to 13 human liver microsomes was significantly correlated with beta-estradiol-3-glucuronidation (r = 0.841, p < 0.01), bilirubin glucuronidation (r = 0.935, p < 0.01), and the immunoquantified UGT1A1 protein content (r = 0.800, p < 0.01). These results demonstrate that etoposide glucuronidation in human liver microsomes is specifically catalyzed by UGT1A1.

Animals↗
PubMed2003

Impact of nonspecific binding to microsomes and phospholipid on the inhibition of cytochrome P4502D6: implications for relating in vitro inhibition data to in vivo drug interactions.

The effects of microsomal concentration on the inhibitory potencies of four compounds--fluoxetine, quinidine, imipramine, and ezlopitant--on heterologously expressed recombinant CYP2D6-catalyzed bufuralol 1'-hydroxylase activity were determined. Increasing microsomal concentration from 0.0088 to 2.0 mg/ml, using additional microsomes not containing cytochrome P450, resulted in a marked increase in IC(50) and K(I) values for fluoxetine, ezlopitant, and imipramine, when inhibition constants were calculated using the nominal concentration of inhibitor added to the incubation mixture. The extent of nonspecific binding of these inhibitors to microsomes was determined using equilibrium dialysis. The extent of binding increased with increasing microsomal concentration. Binding was greatest for ezlopitant, followed by fluoxetine, imipramine, and quinidine. Correcting inhibition constants for the extent of nonspecific binding resulted in greater consistency of these values with differing microsomal protein concentrations. This effect was also studied with added phospholipid. Inhibition constants increased with increasing phospholipid, and nonspecific binding was also observed for these four drugs to phospholipid. This suggests that the phospholipid component of microsomes possesses some or all of the responsibility for nonspecific binding, and its effect on inhibitors of drug-metabolizing enzymes. These findings suggest that inhibition constants for drugs as inhibitors of microsomal drug-metabolizing enzymes, such as cytochrome P450, should be corrected for the extent of nonspecific binding to components of the in vitro matrix. The implications of this on the prediction of drug-drug interactions from in vitro data are discussed.

Benzylamines↗
PubMed2004

Stereo- and regioselectivity account for the diversity of dehydroepiandrosterone (DHEA) metabolites produced by liver microsomal cytochromes P450.

The purpose of this study was to quantify the oxidative metabolism of dehydroepiandrosterone (3beta-hydroxy-androst-5-ene-17-one; DHEA) by liver microsomal fractions from various species and identify the cytochrome P450 (P450) enzymes responsible for production of individual hydroxylated DHEA metabolites. A gas chromatography-mass spectrometry method was developed for identification and quantification of DHEA metabolites. 7alpha-Hydroxy-DHEA was the major oxidative metabolite formed by rat (4.6 nmol/min/mg), hamster (7.4 nmol/min/mg), and pig (0.70 nmol/min/mg) liver microsomal fractions. 16alpha-Hydroxy-DHEA was the next most prevalent metabolite formed by rat (2.6 nmol/min/mg), hamster (0.26 nmol/min/mg), and pig (0.16 nmol/min/mg). Several unidentified metabolites were formed by hamster liver microsomes, and androstenedione was produced only by pig microsomes. Liver microsomal fractions from one human demonstrated that DHEA was oxidatively metabolized at a total rate of 7.8 nmol/min/mg, forming 7alpha-hydroxy-DHEA, 16alpha-hydroxy-DHEA, and a previously unidentified hydroxylated metabolite, 7beta-hydroxy-DHEA. Other human microsomal fractions exhibited much lower rates of metabolism, but with similar metabolite profiles. Recombinant P450s were used to identify the cytochrome P450s responsible for DHEA metabolism in the rat and human. CYP3A4 and CYP3A5 were the cytochromes P450 responsible for production of 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, and 16alpha-hydroxy-DHEA in adult liver microsomes, whereas the fetal/neonatal form CYP3A7 produced 16alpha-hydroxy and 7beta-hydroxy-DHEA. CYP3A23 uniquely formed 7alpha-hydroxy-DHEA, whereas other P450s, CYP2B1, CYP2C11, and CYP2D1, were responsible for 16alpha-hydroxy-DHEA metabolite production in rat liver microsomal fractions. These results indicate that the stereo- and regioselectivity of hydroxylation by different P450s account for the diverse DHEA metabolites formed among various species.

Animals↗
PubMed2002

Stereoselective metabolism of bufuralol racemate and enantiomers in human liver microsomes.

A new HPLC method was developed using a chiral column to efficiently separate four 1"-hydroxybufuralol (1"-OH-BF) diastereomers that are major metabolites of bufuralol (BF). Employing this method, we examined diastereomer selectivity in the formation of 1"-OH-BF from BF racemate or enantiomers in four individual samples of human liver microsomes. Three different human liver microsomes showed a selectivity of 1"R-OH < 1"S-OH for BF enantiomers, which was similar to that of recombinant CYP2D6 expressed in insect cell microsomes, whereas one human liver microsomal fraction yielded a selectivity of 1"R-OH > 1"S-OH for BF enantiomers, which was similar to those of recombinant CYP2C19 expressed in insect cell microsomes. Recombinant CYP1A2 and CYP3A4 showed a selectivity similar to that of CYP2D6, but their BF 1"-hydroxylase activities were much lower than those of CYP2D6. In inhibition studies, quinidine, a known CYP2D6 inhibitor, markedly inhibited BF 1"-hydroxylation in the fractions of human liver microsomes that showed the CYP2D6-type selectivity. Furthermore, omeprazole, a known CYP2C19 inhibitor, efficiently suppressed the formation of 1"-OH-BF diastereomers from BF in the microsomal fraction that showed the CYP2C19-type selectivity. From these results, we concluded that the diastereomer selectivity in the formation of 1"-OH-BF from BF differs between CYP2D6 and CYP2C19, both of which can be determinant enzymes in the diastereoselective 1"-hydroxylation of BF in human liver microsomes.

Adult↗
PubMed2002

O-Dealkylation of fluoxetine in relation to CYP2C19 gene dose and involvement of CYP3A4 in human liver microsomes.

This work evaluated the kinetic behavior of fluoxetine O-dealkylation in human liver microsomes from different CYP2C19 genotypes and identified the isoenzymes of cytochrome P450 involved in this metabolic pathway. The kinetics of the rho-trifluoromethylphenol (TFMP) formation from fluoxetine was determined in human liver microsomes from three homozygous (wt/wt) and three heterozygous (wt/m1) extensive metabolizers (EMs) and three poor metabolizers (PMs) with m1 mutation (m1/m1) with respect to CYP2C19. The formation rate of TFMP was determined by gas chromatograph with electron-capture detection. The kinetics of TFMP formation was best described by the two-enzyme and single-enzyme Michaelis-Menten equation for liver microsomes from CYP2C19 EMs and PMs, respectively. The mean intrinsic clearance (V(max)/K(m)) for the high- and low-affinity component was 25.2 microl/min/nmol and 3.8 microl/min/nmol of cytochrome P450 in the homozygous EMs microsomes and 12.8 microl/min/nmol and 2.9 microl/min/nmol of cytochrome P450 in the heterozygous EMs microsomes, respectively. Omeprazole (a CYP2C19 substrate) at a high concentration and triacetyloleandomycin (a selective inhibitor of CYP3A4) substantially inhibited O-dealkylation of fluoxetine. Furthermore, fluoxetine O-dealkylation was correlated significantly with S-mephenytoin 4'-hydroxylation at a low substrate concentration and midazolam 1'-hydroxylation at a high substrate concentration in liver microsomes of 11 Chinese individuals, respectively. Moreover, there were obvious differences in the O-dealkylation of fluoxetine in liver microsomes from different CYP2C19 genotypes and in microsomal fractions of different human-expressed lymphoblast P450s. The results demonstrated that polymorphic CYP2C19 and CYP3A4 enzymes were the major cytochrome P450 isoforms responsible for fluoxetine O-dealkylation, whereas CYP2C19 catalyzed the high-affinity O-dealkylation of fluoxetine, and its contribution to this metabolic reaction was gene dose-dependent.

Algorithms↗
PubMed1985

The in vitro metabolism of benzo[a]pyrene by polychlorinated and polybrominated biphenyl induced rat hepatic microsomal monooxygenases.

The metabolism of benzo[a]pyrene by halogenated biphenyl-induced rat hepatic microsomal monooxygenases was determined using a high pressure liquid chromatographic assay system. Incubation of benzo[a]pyrene with microsomes from rats pretreated with phenobarbitone or phenobarbitone-type inducers (2,2',4,4',5,5'-hexachlorobiphenyl, 2,2',4,4',6,6'-hexachlorobiphenyl, 2,2',5,5'-tetrachlorobiphenyl, 2,2',4,4',5,5'-hexabromobiphenyl, and 2,2',5,5'-tetrabromobiphenyl) resulted in increased overall metabolism of the hydrocarbon (less than fourfold) into phenolic, quinone, and diol metabolites, with the most striking increase observed in the formation of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene. In contrast, the metabolism of benzo[a]pyrene by microsomes from rats induced with 3-methylcholanthrene or 3,3',4,4'-tetrachlorobiphenyl resulted in a greater than 10-fold increase in overall benzo[a]pyrene metabolism, with the largest increases observed in the formation of the trans-7,8- and -9,10-dihydrodiol metabolites of benzo[a]pyrene. However, in comparison to control and phenobarbitone-induced microsomes, the oxidative conversion of benzo[a]pyrene by microsomes induced with 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl into the 6,12-quinone was substantially inhibited. Previous reports have shown that the commercial halogenated biphenyl mixtures, fireMaster BP-6, and Aroclor 1254 are mixed-type inducers and that microsomes from rats pretreated with these mixtures markedly enhance the overall metabolism of benzo[a]pyrene. Not surprisingly, the metabolism of benzo[a]pyrene by microsomes from rats pretreated with the mixed-type inducers, 2,3,3',4,4'-penta-,2,3,3',4,4',5-hexa-, and 2',3,3',4,4',5-hexa- chlorobiphenyl was also increased and the metabolic profile was similar to that observed with fireMaster BP-6 and Aroclor 1254 induced microsomes.

Animals↗