Human Liver Microsomal Enzymes Research Articles

Inhibitory effects of silibinin on cytochrome P-450 enzymes in human liver microsomes.

Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7-300 microM) at 37 degrees in 0.1 M KH2PO4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC50 values were determined for each substrate at respective K(M) concentrations. Silibinin had little effect (IC50>200 microM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(+)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC50=173 microM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC50=29 microM and 46 microM) and S(-)-warfarin 7-hydroxylation (CYP2C9; IC50=43 microM and 45 microM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (Ki.c=2.3 microM and 2.4 microM). Inhibition was competitive for S(-)-warfarin 7-hydroxylation (Ki,c=18 microM and 19 microM) and mainly non-competitive for denitronifedipine oxidation (Ki,n=9 microM and 12 microM). With therapeutic silibinin peak plasma concentrations of 0.6 microM and biliary concentrations up to 200 microM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.

Selegiline Metabolism and Cytochrome P450 Enzymes:In vitro Study in Human Liver Microsomes*

Abstract:Although being a drug therapeutically used for a long time, the enzymatic metabolism of selegiline has not been adequately studied. In the current work we have studied the cytochrome P450 (CYP)‐catalyzed oxidative metabolism of selegiline to desmethylselegiline and l‐methamphetamine and the effects of selegiline, desmethylselegiline and l‐methamphetamine on hepatic CYP enzymes in human liver microsomes in vitro. The apparent Km values for desmethylselegiline and l‐methamphetamine formation were on an average 149 μM and 293 μM, and the apparent Vmax values, 243 pmol/min./mg and 1351 pmol/min./mg, respectively. Furafylline and ketoconazole, the known reference inhibitors for CYP1A2 and CYP3A4, respectively, inhibited the formation of desmethylselegiline with Ki value of 1.7 μM and 15 μM. Ketoconazole inhibited also the formation of l‐methamphetamine with Ki of 18 μM. Fluvoxamine, an inhibitor of CYP1A2, CYP2C19 and CYP3A4, inhibited the formation of desmethylselegiline and l‐methamphetamine with Ki values of 9 and 25 μM, respectively. On the basis of these results we suggest that CYP1A2 and CYP3A4 contribute to the formation of desmethylselegiline and that CYP3A4 participates in the formation of l‐methamphetamine. In studies with CYP‐specific model activities, both selegiline and desmethylselegiline inhibited the CYP2C19‐mediated S‐mephenytoin 4′‐hydroxylation with average IC50 values of 21 μM and 26 μM, respectively. The Ki for selegiline was determined to be around 7 μM. Selegiline inhibited CYP1A2‐mediated ethoxyresorufin O‐deethylation with a Ki value of 76 μM. Inhibitory potencies of selegiline, desmethylselegiline and l‐methamphetamine towards other CYP‐model activities were much lower. On this basis, selegiline and desmethylselegiline were shown to have a relatively high affinity for CYP2C19, but no evidence about selegiline metabolism by CYP2C19 was obtained.

Selegiline metabolism and cytochrome P450 enzymes: in vitro study in human liver microsomes.

Although being a drug therapeutically used for a long time, the enzymatic metabolism of selegiline has not been adequately studied. In the current work we have studied the cytochrome P450 (CYP)-catalyzed oxidative metabolism of selegiline to desmethylselegiline and 1-methamphetamine and the effects of selegiline, desmethylselegiline and 1-methamphetamine on hepatic CYP enzymes in human liver microsomes in vitro. The apparent Km values for desmethylselegiline and 1-methamphetamine formation were on an average 149 microM and 293 microM, and the apparent Vmax values, 243 pmol/min./mg and 1351 pmol/min./mg, respectively. Furafylline and ketoconazole, the known reference inhibitors for CYP1A2 and CYP3A4, respectively, inhibited the formation of desmethylselegiline with Ki value of 1.7 microM and 15 microM. Ketoconazole inhibited also the formation of 1-methamphetamine with Ki of 18 microM. Fluvoxamine, an inhibitor of CYP1A2, CYP2C19 and CYP3A4, inhibited the formation of desmethylselegiline and 1-methamphetamine with Ki values of 9 and 25 microM, respectively. On the basis of these results we suggest that CYP1A2 and CYP3A4 contribute to the formation of desmethylselegiline and that CYP3A4 participates in the formation of 1-methamphetamine. In studies with CYP-specific model activities, both selegiline and desmethylselegiline inhibited the CYP2C19-mediated S-mephenytoin 4'-hydroxylation with average IC50 values of 21 microM and 26 microM, respectively. The Ki for selegiline was determined to be around 7 microM. Selegiline inhibited CYP1A2-mediated ethoxyresorufin O-deethylation with a Ki value of 76 microM. Inhibitory potencies of selegiline, desmethylselegiline and 1-methamphetamine towards other CYP-model activities were much lower. On this basis, selegiline and desmethylselegiline were shown to have a relatively high affinity for CYP2C19, but no evidence about selegiline metabolism by CYP2C19 was obtained.

CYP2A6: a human coumarin 7-hydroxylase

Coumarin 7-hydroxylation is catalysed by a high-affinity CYP2A6 enzyme in human liver microsomes. CYP2A6 is the only enzyme catalysing this reaction and consequently the formation of 7-hydroxycoumarin can be used as ‘an in vitro and in vivo probe’ for CYP2A6. CYP2A6 is a major contributor to the oxidative metabolism of nicotine and cotinine, and it also contributes, to a larger or smaller extent, to the metabolism of a few pharmaceuticals (e.g. fadrozole), nitrosamines, other carcinogens (e.g. aflatoxin B1) and a number of coumarin-type alkaloids. CYP2A6 may be inducible by antiepileptic drugs and it is decreased in alcohol-induced severe liver cirrhosis. Several mutated or deleted CYP2A6 alleles have been characterized. Although CYP2A6 represent up to 15% of human microsomes P450 proteins, it is still one of the less well characterised cytochrome P450 enzymes.

Inhibitory effects of trospium chloride on cytochrome P450 enzymes in human liver microsomes.

Trospium chloride, an atropine derivative used for the treatment of urge incontinence, was tested for inhibitory effects on human cytochrome P450 enzymes. Metabolic activities were determined in liver microsomes from two donors using the following selective substrates: dextromethorphan (CYP2D6), denitronifedipine (CYP3A4), caffeine (CYP1A2), chlorzoxazone (CYP2E1), S-(+)-mephenytoin (CYP2C19), S-(-)-warfarin (CYP2C9) and coumarin (CYP2A6). Incubations with each substrate were carried out without a possible inhibitor and in the presence of trospium chloride at varying concentrations (37-3000 microM) at 37 degrees in 0.1 M KH2PO4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by high-performance liquid chromatography (HPLC) in all cases except CYP2A6 where direct fluorescence spectroscopy was used. First, trospium chloride IC50 values were determined for each substrate at respective K(M) concentrations. Trospium chloride did not show relevant inhibitory effects on the metabolism of most substrates (IC50 values considerably higher than 1 mM). The only clear inhibition was seen for the CYP2D6-dependent high-affinity O-demethylation of dextromethorphan, where IC50 values of 27 microM and 44 microM were observed. Therefore, additional dextromethorphan concentrations (0.4-2000 microM) were tested. Trospium chloride was a competitive inhibitor of the reaction with Ki values of 20 and 51 microM, respectively. Thus, trospium chloride has negligible inhibitory effects on CYP3A4, CYP1A2, CYP2E1, CYP2C19, CYP2C9 and CYP2A6 activity but is a reasonably potent inhibitor of CYP2D6 in vitro. Compared to therapeutic trospium chloride peak plasma concentrations below 50 nM, the 1000-times higher competitive inhibition constant Ki however suggests that inhibition of CYP2D6 by trospium chloride is without any clinical relevance.

Differences in interaction of HMG-CoA reductase inhibitors with various cytochrome-P450 enzymes in human liver microsomes, and in human and simian hepatocytes

Differences in interaction of HMG-CoA reductase inhibitors with various cytochrome-P450 enzymes in human liver microsomes, and in human and simian hepatocytes

Cytochrome P450 reconstitution systems.

AbstractMultiple forms of cytochrome P450 (CYP) exist in liver mrcrosomes and these P450 forms have been shown to play important roles in the oxidation of structurally diverse xenobrotrc chemicals such as drugs, toxic chemicals, and carcinogens, as well as endobiottc chemicals including steroids, fatty acids, fat-soluble vitamins, and prostaglandins (1). Major cytochrome P450 enzymes in human liver microsomes identified to date mclude CYPlA2,2A6,2B6,2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5 (and 3A7 in fetal livers) (2). CYPlAl and 1 B 1 have also been reported to be involved in the oxidation of drugs, toxic chemicals, and carcinogens in extrahepatic tissues (3). There are large mtermdividual variations in the amounts of each of these cytochrome P450 enzymes, and these variations are considered to be one of the major factors m contributing to differences in susceptibilities of humans towards actions and toxicities of drugs, toxic chemicals, and carcmogens (2). Recently, in order to investigate the consequences for enzymic actrvity of genetic polymorphisms of individual cytochrome P450 enzymes in humans, many investigators have carried out studies with cytochrome P450 proteins purified from heterologous organisms in which native and modified human cytochrome P450 cDNAs have been introduced (4)KeywordsLiver MicrosomeHuman Liver MicrosomeCytochrome P450 EnzymeSodium CholateCytochrome P450 ReductaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Characterization of Inducible Nitric-oxide Synthase by Cytochrome P-450 Substrates and Inhibitors: INHIBITION BY CHLORZOXAZONE

Nitric-oxide synthases (NOS, EC 1.14.13.39) are heme-containing enzymes that catalyze the formation of nitric oxide from L-Arg. General cytochrome P-450 inhibitors and cytochrome P-450 isoform-selective substrates and inhibitors were used to characterize the activity of recombinant human inducible NOS (iNOS). Classical cytochrome P-450 ligands such as the mechanism-based inactivator 1-aminobenzotriazole did not inhibit iNOS. Of a panel of 30 human cytochrome P-450 isoform-selective substrates and inhibitors, only chlorzoxazone, a cytochrome P-450 2E1 (CYP2E1) substrate, showed any significant inhibition of iNOS activity. Chlorzoxazone was not a substrate for iNOS but was a potent competitive inhibitor with respect to L-Arg with Ki = 3.3+/-0.7 microM. The binding of chlorzoxazone to iNOS and human and rat liver microsomal cytochrome P-450 induced a high spin, type I spectra, which was reversed by imidazole. Although the binding of chlorzoxazone to iNOS and its inhibition of iNOS activity suggest some similarity between iNOS and CYP2E1 activity, other CYP2E1 substrates and inhibitors including zoxazolamine were not inhibitors of iNOS. Overall, iNOS activity is distinctly different from the major cytochrome P-450 enzymes in human liver microsomes.

Identification of human liver cytochrome P450 enzymes that metabolize the nonsedating antihistamine loratadine: Formation of descarboethoxyloratadine by CYP3A4 and CYP2D6

[ 3H]Loratadine was incubated with human liver microsomes to determine which cytochrome P450 (CYP) enzymes are responsible for its oxidative metabolism. Specific enzymes were identified by correlation analysis, by inhibition studies (chemical and immunoinhibition), and by incubation with various cDNA-expressed human P450 enzymes. Descarboethoxyloratadine (DCL) was the major metabolite of loratadine detected following incubation with pooled human liver microsomes. Although DCL can theoretically form by hydrolysis, the conversion of loratadine to DCL by human liver microsomes was not inhibited by the esterase inhibitor phenylmethylsulfonyl fluoride (PMSF), and was dependent on NADPH. A high correlation ( r 2 = 0.96, N = 10) was noted between the rate of formation of DCL and testosterone 6β-hydroxylation, a CYP3A-mediated reaction. With the addition of ketoconazole (CYP3A4 inhibitor) to the incubation mixtures, the residual rate of formation of DCL correlated ( r 2 = 0.81) with that for dextromethorphan O-demethylation, a CYP2D6 reaction. Rabbit polyclonal antibodies raised against the rat CYP3A1 enzyme (5 mg IgG/nmol P450) and troleandomycin (0.5 μM), a specific inhibitor of CYP3A4, decreased the formation of DCL by 53 and 75%, respectively, when added to 1.42 μM loratadine microsomal incubations. Quinidine (5 μM), a CYP2D6 inhibitor, inhibited the formation of DCL approximately 20% when added to microsomal incubations of loratadine at concentrations of 7–35 μM. Incubation of loratadine with cDNA-expressed CYP3A4 and CYP2D6 microsomes catalyzed the formation of DCL with formation rates of 135 and 633 pmol/min/nmol P450, respectively. The results indicated that loratadine was metabolized to DCL primarily by the CYP3A4 and CYP2D6 enzymes in human liver microsomes. In the presence of a CYP3 A4 inhibitor, loratadine was metabolized to DCL by the CYP2D6 enzyme. Conformational and electrostatic analysis of loratadine indicated that its structure is consistent with substrate models for the CYP2D6 enzyme.

Human cytochrome P450 mono-oxygenase system is suppressed by propofol

We have studied the effect of propofol on the cytochrome P450-dependent mono-oxygenase system in human liver microsomes by assaying mono-oxygenase activities toward specific cytochrome P450 isoform test substrates, aniline, 7-ethoxycoumarin, benzphetamine and benzo(a) pyrene. Propofol inhibited benzo(a)pyrene hydroxylation to a greater extent than the oxidative metabolism of the other test substrates, even at 0.05 mmol litre-1. The degrees of inhibition of benzphetamine N-demethylation and 7-ethoxy-coumarin O-de-ethylation were similar, while aniline hydroxylation was least affected by propofol. Spectral analysis showed that propofol competed with carbon monoxide for binding to the haem moiety of haemoprotein in the P450 enzyme. The variable inhibition observed may be caused by the differential binding of propofol to P450 isoforms. Propofol 0.05-1.0 mmol litre-1 exhibited a concentration-dependent inhibitory effect on human cytochrome P450 2E1, 2B1 and 1A1. These inhibitory actions of propofol on human liver microsomal enzymes in vitro suggest that potential drug interactions may exist between propofol and other drugs administered clinically.

Identification of Cytochrome P450 2E1 as the Predominant Enzyme Catalyzing Human Liver Microsomal Defluorination of Sevoflurane, Isoflurane, and Methoxyflurane

Renal and hepatic toxicity of the fluorinated ether volatile anesthetics is caused by biotransformation to toxic metabolites. Metabolism also contributes significantly to the elimination pharmacokinetics of some volatile agents. Although innumerable studies have explored anesthetic metabolism in animals, there is little information on human volatile anesthetic metabolism with respect to comparative rates or the identity of the enzymes responsible for defluorination. The first purpose of this investigation was to compare the metabolism of the fluorinated ether anesthetics by human liver microsomes. The second purpose was to test the hypothesis that cytochrome P450 2E1 is the specific P450 isoform responsible for volatile anesthetic defluorination in humans. Microsomes were prepared from human livers. Anesthetic metabolism in microsomal incubations was measured by fluoride production. The strategy for evaluating the role of P450 2E1 in anesthetic defluorination involved three approaches: for a series of 12 human livers, correlation of microsomal defluorination rate with microsomal P450 2E1 content (measured by Western blot analysis), correlation of defluorination rate with microsomal P450 2E1 catalytic activity using marker substrates (para-nitrophenol hydroxylation and chlorzoxazone 6-hydroxylation), and chemical inhibition by P450 isoform-selective inhibitors. The rank order of anesthetic metabolism, assessed by fluoride production at saturating substrate concentrations, was methoxyflurane > sevoflurane > enflurane > isoflurane > desflurane > 0. There was a significant linear correlation of sevoflurane and methoxyflurane defluorination with antigenic P450 2E1 content (r = 0.98 and r = 0.72, respectively), but not with either P450 1A2 or P450 3A3/4. Comparison of anesthetic defluorination with either para-nitrophenol or chlorzoxazone hydroxylation showed a significant correlation for sevoflurane (r = 0.93, r = 0.95) and methoxyflurane (r = 0.78, r = 0.66). Sevoflurane defluorination was also highly correlated with that of enflurane (r = 0.93), which is known to be metabolized by human P450 2E1. Diethyldithiocarbamate, a selective inhibitor of P450 2E1, produced a concentration-dependent inhibition of sevoflurane, methoxyflurane, and isoflurane defluorination. No other isoform-selective inhibitor diminished the defluorination of sevoflurane, whereas methoxyflurane defluorination was inhibited by the selective P450 inhibitors furafylline (P450 1A2), sulfaphenazole (P450 2C9/10), and quinidine (P450 2D6) but to a much lesser extent than by diethyldithiocarbamate. These results demonstrate that cytochrome P450 2E1 is the principal, if not sole human liver microsomal enzyme catalyzing the defluorination of sevoflurane. P450 2E1 is the principal, but not exclusive enzyme responsible for the metabolism of methoxyflurane, which also appears to be catalyzed by P450s 1A2, 2C9/10, and 2D6. The data also suggest that P450 2E1 is responsible for a significant fraction of isoflurane metabolism. Identification of P450 2E1 as the major anesthetic metabolizing enzyme in humans provides a mechanistic understanding of clinical fluorinated ether anesthetic metabolism and toxicity.

A novel UDP-Glc-specific glucosyltransferase catalyzing the biosynthesis of 6-O-glucosides of bile acids in human liver microsomes

Two active site-directed photoaffinity analogs, 5-[beta-32P]azido-UDP-glucuronic acid and 5-[beta-32P]azido-UDP-glucose, were used for the characterization of UDP-sugar-utilizing enzymes in human liver microsomes. Both compounds were recognized by human microsomal proteins: major photolabeled bands of 50-56 kDa were detected. Both photoincorporations were competitively decreased by increasing concentrations of either UDP-Glc or UDP-GlcUA, indicating a high affinity for both nucleotides. The patterns of photoaffinity labeling in the 50-56-kDa range by the two probes were significantly different, indicating the presence of different UDP-GlcUA- and UDP-Glc-specific enzymes of similar molecular mass. The presence of a UDP-Glc-dependent transferase was confirmed by the identification of an enzymatic activity catalyzing the formation of glucosides of the 6 alpha-hydroxylated bile acid hyodeoxycholic acid (3 alpha, 6 alpha-diOH (HDCA)) in the presence of UDP-Glc. The specific activity of 1.5-3.2 nmol/min/mg of protein was similar to that of 6 alpha-glucuronidation of HDCA. The apparent Km for UDP-Glc estimated with HDCA was 280 microM, and the formation of HDCA glucosides was strongly inhibited by UDP-GlcUA (apparent Ki = 7 microM). Evidence is presented that HDCA-specific UDP-glucuronosyltransferase (clone UGT2B4) expressed in V79 cells is not involved in glucosidation of HDCA and is not photolabeled with 5-[beta-32P]azido-UDP-Glc. Rigorous structure identification of the biosynthetic product proved that HDCA was glucosidated at the 6-position. Thus, this UDP-Glc-dependent activity catalyzing the biosynthesis of 6-O-glucosides of 6 alpha-hydroxylated bile acids represents a new pathway in the metabolism of these bile acids.

The NH2 -Terminal Structures of Human and Rat Liver Microsomal NADH-Cytochrome b5 Reductases1

Detergent-solubilized NADH-cytochrome b5 reductase was purified from human liver microsomes. Both the purified enzyme and the membrane-binding domain isolated from the purified enzyme were determined to be modified at the NH2-terminal amino acid, glycine, by myristic acid in an amide form. Myristic acid was identified as a methyl ester by gas chromatography after the acid methanolysis of the purified enzyme and the NH2-terminal peptide. The NH2-terminal structure of the membrane-binding domain was determined to be CH3(CH2)12-CO-Gly-Ala-Gln-Leu-Ser-Thr-Leu-Gly-His-Met-Val-Leu-Phe-Pro-Va l- Trp-Phe-Leu-Tyr-Ser-Leu-Leu-Met-Lys. The sequence from Leu-7 to Lys-24 completely coincided with that deduced from the base sequence of complementary DNA (cDNA) from human placenta (Yubisui, T. et al. (1987) Proc. Natl. Acad. Sci. U.S. 84, 3609-3613). The NH2-terminal structure of the detergent-solubilized enzyme from rat liver microsomes was also analyzed for comparison with that of human liver microsomal enzyme. The NH2-terminal myristic acid and the first 7 amino acids of the membrane-binding domains of human, rat, and steer liver microsomal enzymes are completely conserved, and more than 70% homology was observed over the whole membrane-binding domains, implying the importance of the conserved structure as an anchor of the enzyme to the membrane.

Serum selenium, glutathione peroxidase, lipids, and human liver microsomal enzyme activity

This study evaluated selenium status in relation to lipid peroxidation, liver microsomal function, and serum lipids in humans. Serum selenium concentration, glutathione peroxidase (GSH-Px) activity, liver microsomal enzyme activity, assessed by plasma antipyrine clearance (AP-CL) rate, and serum lipids were determined in 23 healthy subjects in a double-blind placebo-controlled trial of selenium supplementation. The low selenium concentration (74.0±14.2 μg/L, mean±SD) is attributable to the low selenium content of the diet. Subjects with the lowest selenium levels (n=11) had reduced serum GSH-Px activity, AP-CL rate, high-density lipoprotein cholesterol (HDL-C), and total cholesterol (T-C) as compared with subjects with higher selenium concentrations (n=12). Low AP-CL rates were associated with low HDL-C: T-C ratios. Selenium supplementation, 96 μg/d for 2 wk, increased serum selenium, GSH-Px activity, and the HDL-C: T-C ratio. The results suggest that a low serum selenium level is associated with a decrease in liver microsomal enzyme activity and serum HDL-C and T-C concentrations. Selenium supplementation in subjects with low serum selenium may favorably influence relations between serum lipoproteins connected with the development of atherosclerotic vascular disease.

In vitro inhibition of rat and human liver microsomal enzymes by 4-hydroxycoumarin anticoagulants and related compounds

The in vitro inhibition of rat and human liver microsomal mixed-function oxidase by 4-hydroxycoumarin, phenprocoumon, warfarin, acenocoumarin, bishydroxycoumarin, and salicylic acid is reported. Warfarin was used for detailed kinetic analysis. It caused a modified type II spectrum (420 nm maximum), with a K s value of 0.21–0.25 m M. Inhibition of aniline hydroxylation was noncompetitive, with a K i of 11.7 m M; aminopyrine demethylation was competitively inhibited, with a K i of 0.13 m M. I 50 values are reported for the other compounds studied. Warfarin inhibited aminopyrine demethylation to a similar extent in rat and human liver microsomes, whereas inhibition of aniline hydroxylation was more pronounced in hepatic microsomes from rats compared to human. The possible clinical significance of these findings is discussed.