Metabolism of ethyl methyl sulphide and sulphoxide in rat microsomal fractions

1. Gas chromatographic (GC) methods for the analysis of ethyl methyl sulphide (EMS) and its corresponding sulphoxide (EMSO) and sulphone (EMSO2) in rat microsomes and aspects of the in vitro metabolism of EMS and EMSO are described. 2. EMS and the internal standard (dimethyl sulphide) were extracted by a headspace procedure and separated satisfactorily using a column packed with 4% Carbowax 20 M/0.8% KOH on Carbopack B. EMSO, EMSO2 and the internal standard n-propyl sulphone were separated satisfactorily using a 2-m column packed with 10% Carbowax 20 M on Chromosorb W. 3. Under the optimum conditions (incubation of 10 min and microsomal protein content of approximately 4 mg/ml), 10% of the initial EMS concentration (2.5 mM) was converted to the corresponding sulphoxide in rat liver microsomal incubations. However, < 0.1% of the sulphone was detected when rat liver microsomes were incubated with EMS. Similarly, 2.5% of the initial EMSO concentration (2.5 mM) was converted to the corresponding sulphone by rat liver microsomes (approximately 4 mg/ml protein) during an incubation of 30 min. However, no EMS was detected after incubation with EMSO under these conditions. 4. The estimated apparent Vmax and Km for the sulphoxidation of EMS were 3.8+/-0.02 nmol/mg protein/min and 1.9+/-0.10 mM respectively. Vmax1, Vmax2 and Km1 and Km2 for the S-oxidation of EMSO were 0.5+/-0.01 and 0.2+/-0.01 nmol/mg protein/min and 0.7+/-0.02 and 0.1+/-0.00 mM respectively. 5. Studies with selective inducers and inhibitors of microsomal monooxygenases indicated that the sulphoxidation of EMS is mediated mainly by FMO, whereas both FMO and cytochrome P450 are involved in the S-oxidation of EMSO.     

Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of human liver and kidney microsomes and mammalian enzymes

The stereoselective sulfoxidation of the pharmacologically active metabolite of sulindac, sulindac sulfide, was characterized in human liver, kidney, and cDNA-expressed enzymes. Kinetic parameter estimates (pH = 7.4) for sulindac sulfoxide formation in human liver microsomes (N = 4) for R- and S-sulindac sulfoxide were V(max) = 1.5 +/- 0.50 nmol/min/mg, K(m) = 15 +/- 5.1 microM; and V(max) = 1.1 +/- 0.36 nmol/min/mg, K(m) = 16 +/- 6.1 microM, respectively. Kidney microsomes (N = 3) produced parameter estimates (pH = 7.4) of V(max) = 0.9 +/- 0.29 nmol/min/mg, K(m) = 15 +/- 2.9 microM; V(max) = 0.5 +/- 0.21 nmol/min/mg, K(m) = 22 +/- 1.9 microM for R- and S-sulindac sulfoxide, respectively. In human liver and flavin-containing monooxygenase 3 (FMO3) the V(max) for R-sulindac sulfoxide increased 60-70% at pH = 8.5, but for S-sulindac sulfoxide was unchanged. In fourteen liver microsomal preparations, significant correlations occurred between R-sulindac sulfoxide formation and either immunoquantified FMO or nicotine N-oxidation (r = 0.88 and 0.83; P < 0.01). The R- and S-sulindac sulfoxide formation rate also correlated significantly (r = 0.85 and 0.75; P < 0.01) with immunoquantified FMO in thirteen kidney microsomal samples. Mild heat deactivation of microsomes reduced activity by 30-60%, and a loss in stereoselectivity was observed. Methimazole was a potent and nonstereoselective inhibitor of sulfoxidation in liver and kidney microsomes. n-Octylamine and membrane solubilization with lubrol were potent and selective inhibitors of S-sulindac sulfoxide formation. cDNA-expressed CYPs failed to appreciably sulfoxidate sulindac sulfide, and CYP inhibitors were ineffective in suppressing catalytic activity. Purified mini-pig liver FMO1, rabbit lung FMO2, and human cDNA-expressed FMO3 efficiently oxidized sulindac sulfide with a high degree of stereoselectivity towards the R-isomer, but FMO5 lacked catalytic activity. The biotransformation of the sulfide to the sulfoxide is catalyzed predominately by FMOs and may prove to be useful in characterizing FMO activity.     

The flavin-containing monooxygenase of mouse kidney. A comparison with the liver enzyme

Flavin-containing monooxygenase (FMO; EC 1.14.13.8) was purified from mouse kidney microsomes and compared to that isolated from mouse liver microsomes. The purified enzymes from kidney and liver appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 58,000 daltons. On wide range (pH 3.5 to 9.0) isoelectric focusing, FMOs from kidney and liver resolved as a single band with an isoelectric point of 8.2. The enzymes from both kidney and liver have a pH optimum of 9.2. Thiobenzamide-S-oxidation catalyzed by both enzymes was sensitive to inhibition by the competitive inhibitors thiourea and methimazole. At an n-octylamine concentration of 3 mM, thiobenzamide-S-oxidation by the kidney FMO was increased by 122% and that by the liver FMO by 148%. Km and Vmax values were determined and compared between the two tissue enzymes for xenobiotic substrates containing nucleophilic nitrogen, sulfur or phosphorus atoms. In general, for most FMO substrates, Km and Vmax values were similar between kidney and liver FMO with only a few exceptions. The Km and Vmax values for fenthion for kidney were only half of those observed for liver FMO. Fonofos was unusual in having a low Km as well as a low Vmax for both tissue enzymes. Anti-sera developed to the FMO purified from kidney and liver showed cross-reactivity with each purified enzyme as well as with a protein with the same molecular weight as the purified FMO present in both kidney and liver microsomes. These bands showed equal intensity based on an equivalent amount of protein. Analysis of kidney and liver FMO by proteolytic digestion followed by visualization of peptides by silver staining or immunoblotting showed only minor differences between the enzymes of the two tissues. The amino acid composition of both mouse kidney and liver FMO was low in methionine and histidine and rich in aspartate/asparagine, glutamate/glutamine, leucine, valine and glycine. Edman degradation of the purified mouse kidney and liver FMO provided a single amino acid sequence of the NH2-terminus. This sequence matched exactly with the cDNA-deduced sequence reported for the pig and rabbit liver beginning with the fifth amino acid and contained the highly conserved FAD-binding domain Gly-X-Gly-X-X-Gly, commonly found in a number of other FAD-binding proteins. These studies indicate that the renal and hepatic forms of FMO from mouse are similar enzymes that are immunologically related and show only a few minor differences.     

Reduction of L-methionine selenoxide to seleno-L-methionine by endogenous thiols, ascorbic acid, or methimazole

Seleno-L-methionine (SeMet) can be oxidized to L-methionine selenoxide (MetSeO) by flavin-containing monooxygenase 3 (FMO3) and rat liver microsomes in the presence of NADPH. MetSeO can be reduced by GSH to yield SeMet and GSSG. In the present study, the potential reduction of MetSeO to SeMet by other cellular components and antioxidants was investigated. Besides GSH, other thiols (L-cysteine, or N-acetyl-L-cysteine) and antioxidants (ascorbic acid and methimazole) also reduced MetSeO to SeMet. This reduction is unique to MetSeO since methionine sulfoxide was not reduced to methionine under similar conditions. The MetSeO reduction by thiols was instaneous and much faster than the reduction by ascorbic acid or methimazole. However, only one molar equivalent of ascorbic acid or methimazole was needed to complete the reduction, as opposed to two molar equivalents of thiols. Whereas the disulfides produced by the reactions of MetSeO with thiols are chemically stable, methimazole disulfide readily decomposed at pH 7.4, 37 degrees C to yield methimazole, methimazole-sulfenic acid, methimazole sulfinic acid, methimazole S-sulfonate, 1-methylimidazole (MI) and sulfite anion. Collectively, the results demonstrate reduction of MetSeO to SeMet by multiple endogenous thiols, ascorbic acid, and methimazole. Thus, oxidation of SeMet to MetSeO may result in depletion of endogenous thiols and antioxidant molecules. Furthermore, the novel reduction of MetSeO by methimazole provides clear evidence that methimazole should not be used as an alternative FMO substrate when studying FMO-mediated oxidation of SeMet.     

Reduction of l-methionine selenoxide to seleno-l-methionine by endogenous thiols, ascorbic acid, or methimazole

Seleno-l-methionine (SeMet) can be oxidized to l-methionine selenoxide (MetSeO) by flavin-containing monooxygenase 3 (FMO3) and rat liver microsomes in the presence of NADPH. MetSeO can be reduced by GSH to yield SeMet and GSSG. In the present study, the potential reduction of MetSeO to SeMet by other cellular components and antioxidants was investigated. Besides GSH, other thiols (l-cysteine, or N-acetyl-l-cysteine) and antioxidants (ascorbic acid and methimazole) also reduced MetSeO to SeMet. This reduction is unique to MetSeO since methionine sulfoxide was not reduced to methionine under similar conditions. The MetSeO reduction by thiols was instaneous and much faster than the reduction by ascorbic acid or methimazole. However, only one molar equivalent of ascorbic acid or methimazole was needed to complete the reduction, as opposed to two molar equivalents of thiols. Whereas the disulfides produced by the reactions of MetSeO with thiols are chemically stable, methimazole disulfide readily decomposed at pH 7.4, 37 °C to yield methimazole, methimazole-sulfenic acid, methimazole sulfinic acid, methimazole S-sulfonate, 1-methylimidazole (MI) and sulfite anion. Collectively, the results demonstrate reduction of MetSeO to SeMet by multiple endogenous thiols, ascorbic acid, and methimazole. Thus, oxidation of SeMet to MetSeO may result in depletion of endogenous thiols and antioxidant molecules. Furthermore, the novel reduction of MetSeO by methimazole provides clear evidence that methimazole should not be used as an alternative FMO substrate when studying FMO-mediated oxidation of SeMet.     

Biphasic kinetics of imipramine N-oxidation in rat brain microsomes

Imipramine (IMI) N-oxidase activity in brain microsomes from rats of both sexes was determined by high performance liquid chromatography, and compared with the results in rat liver microsomes. Brain and liver microsomal IMI N-oxidation was sensitive to thermal inactivation and had an optimal pH at around 9.0. IMI N-oxidase activity (15.54 pmol/min/mg protein) in brain microsomes was about one-hundredth that of liver microsomes (2.08 nmol/min/mg protein) at a substrate concentration of 5 mM. IMI N-oxidase activities in both brain and liver microsomes displayed biphasic kinetics that associated a low Km-low Vmax element with a high Km-high Vmax component. Furthermore, a significant sex difference was observed in Vmax values (male>female) in both phases, but Km values were similar between male and female rats, resulting in a significant sex difference (male>female) in intrinsic clearance values (Vmax/Km) of the low-Km and the high-Km phases.     

Hepatic microsomal metabolism of indole to indoxyl, a precursor of indoxyl sulfate.

The aim of our study was to determine which microsomal cytochrome P450 isozyme(s) were responsible for the microsomal oxidation of indole to indoxyl, an important intermediate in the information of the uremic toxin indoxyl sulfate. Indole was incubated together with an NADPH-generating system and rat liver microsomes. Formation of indigo, an auto-oxidation product of indoxyl, was used to determine the indole-3-hydroxylation activity. Apparent Km and Vmax values of 0.85 mM and 1152 pmol min(-1) mg(-1) were calculated for the formation of indoxyl from indole using rat liver microsomes. The effects of various potential inducers and inhibitors on the metabolism of indole to indoxyl by rat liver microsomes were studied to elucidate the enzymes responsible for metabolism. Studies with general and isozyme-specific P450 inhibitors demostrated that P450 enzymes and not FMO are responsible for the formation of indoxyl. In the induction studies, rate of indoxyl formation in the microsomes from untreated vs induced rats correlated nearly exactly with the CYP2E1 activity (4-nitrophenol 2-hydroxylation). These results suggests that CYP2E1 is the major isoform for the microsomal oxidation of indole to indoxyl.     

Diethyl ether as a substrate for acetone/ethanol-inducible cytochrome P-450 and as an inducer for cytochrome(s) P-450

The ability of diethyl ether to serve as a substrate for microsomal and purified cytochrome P-450 (P-450) and as an inducer for rat hepatic microsomal monooxygenase activities was examined. Microsomal oxidation of ether to acetaldehyde, as monitored by high pressure liquid chromatography, was elevated 3- to 5-fold by treatment of rats with acetone or ethanol, 1.5- to 2-fold by treatment with ether, and only slightly by phenobarbital treatment. Ether also induced N-nitrosodimethylamine demethylase by up to 2-fold and 7-pentoxyresorufin dealkylation by up to 10-fold. These trends agreed with immunoblot experiments in which ether was a weak inducer of the P-450 isozyme IIE1 (encoded by the rat gene P450IIE1), but a stronger inducer of IIB1. A monoclonal antibody against IIE1 inhibited the deethylation by 78% in microsomes from acetone-treated rats and by 45% in controls. N-Nitrosodimethylamine, as well as common inhibitors of IIE1 such as hexane, benzene, pyrazole, and phenylethylamine, strongly inhibited ether deethylation. Using microsomes from acetone-induced rats, the apparent Km for deethylation was 13.4 +/- 2.4 microM and the Vmax was 8.2 +/- 0.2 (nmol of acetaldehyde/min/nmol of P-450). The Km for the controls was 71.3 +/- 9.5 microM. The rates of deethylation at 1 mM ether by purified, reconstituted IIE1 and IIB1 were 4.2 and 0.42 (nmol of acetaldehyde/min/nmol of P-450), respectively. Cytochrome b5 stimulated the rate due to IIE1 apparently by a decrease in the Km. These findings, along with previous work showing marked inhibition by ether of IIE1-dependent reactions, strongly support a major role for this isozyme in ether metabolism.     

Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes

The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the Vmax/Km values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25 nM-1 min-1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a Vmax value of 6.96 nmol min-1 nmol-1 P450 and apparent Km value of 0.09 mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the Km values for the formation of fenchone, 6-exo- hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03 mM, and Vmax values were 2.94, 6.1 and 13.8 nmol min-1 nmol-1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.     

Identification of cytochrome P4503A as the major subfamily responsible for the metabolism of roquinimex in man

1. Roquinimex, a novel immunomodulator, is metabolized in liver microsomes from mouse and rat via cytochrome P450s to four hydroxylated and two demethylated metabolites (R1-6). The study investigated which cytochrome P450 enzyme(s) is responsible for the metabolism of roquinimex in man. 2. Enzyme kinetic analysis demonstrated an apparent Km = 1.28-7.00 mM and Vmax = 50-159 pmol x mg(-1) microsomal protein x min(-1) for the primary metabolites in human liver microsomes. The sum of Cl(int) for the primary pathways was 0.167 microl x mg(-1) microsomal protein x min(-1). 3. A correlation between the formation rate of R1-6 and 6beta-hydroxylation of testosterone was obtained within a panel of liver microsomes from 11 individuals (r2 = 0.72-0.97). Furthermore, significant inhibition (>90%) of roquinimex primary metabolism was demonstrated by ketoconazole and troleandomycin, specific inhibitors of CYP3A4 as well as with anti-CYP3A4 antibodies. Moreover, a similar metabolite pattern was produced from roquinimex by incubation with cDNA-expressed CYP3A4 as by human liver microsomes. 4. In conclusion, these data indicate a major role for CYP3A4 in the formation of roquinimex primary metabolites in human liver microsomes.     

Glucuronidation of codeine and morphine in human liver and kidney microsomes: effect of inhibitors

Glucuronidation of codeine was detected and compared with that of morphine in microsomes from human livers and kidneys. Vmax values for codeine-6-glucuronide (C6G) were 0.54 +/- 0.24 and 0.74 +/- 0.35 nmol/mg/min. in the livers and 0.10 and 0.13 nmol/mg/min. in the kidney, respectively, when codeine and UDP-glucuronic acid (UDPGA) were incubated with microsomal preparation. The corresponding Km values were 2.21 +/- 0.68 and 1.41 +/- 0.36 mM in the livers and 6.69 and 4.12 mM in the kidney. The average codeine glucuronyltransferase (GT) activity was 14-fold lower in the six kidneys than in the 11 livers. Higher GT activities were observed in liver microsomes from patients who had been exposed to enzyme inducers. Rates of glucuronide formation from morphine correlated significantly with those from codeine in both human liver and kidney microsomes. Morphine, amitriptyline, diazepam, probenecid and chloramphenicol inhibited codeine glucuronidation with Ki values of 3.6, 0.13, 0.18, 1.7 and 0.27 mM, respectively.     

The formation of procainamide hydroxylamine by rat and human liver microsomes

A method is described, using HPLC and electrochemical detection, which permits the direct quantitation of procainamide hydroxylamine. Procainamide hydroxylamine was formed from procainamide by hepatic microsomes from both rat and human, with rat microsomes showing higher apparent formation rates. The apparent Km for formation of procainamide hydroxylamine was 0.044 mM for rat liver microsomes, with an apparent Vmax of 2.81 nmol/min/mg of protein. Estimates of Km from three human microsomal samples were 6.29, 2.89, and 6.88 mM. Vmax estimates were 0.31, 0.74, and 0.74 nmol/min/mg of protein, respectively, roughly an order of magnitude less than that observed for the rat. Microsomal formation in both species was inhibited by boiling the microsomes, eliminating NADPH from the incubation system, by preincubation with SKF 525A, cimetidine, or n-octylamine, or by gassing the microsomal incubation mixture with carbon monoxide. These observations suggest that procainamide hydroxylamine formation is cytochrome P-450 mediated. Procainamide hydroxylamine could not be detected in the blood of rats treated with a single dose of procainamide, 100 mg/kg, po. One potential reason for the inability to detect this metabolite in blood is indicated by the rapid disappearance in vitro of procainamide hydroxylamine added to whole blood. Most of this disappearance appears to be due to an interaction with hemoglobin.     

Inhibitory effect of stiripentol on carbamazepine and saquinavir metabolism in human

AIMS: To characterize the in vitro and in vivo inhibitory effect of stiripentol, a new anticonvulsant, on the metabolism of carbamazepine and saquinavir, which are substrates of CYP3A4. METHODS: Human liver microsomes and cDNA-expressed CYP enzymes were used for the in vitro experiments. Pharmacokinetic data from epileptic children and healthy adults were used for the carbamazepine and saquinavir in vivo studies, respectively. RESULTS: Carbamazepine biotransformation to its 10,11-epoxide by human liver microsomes (Vmax = 10.3 nmol min(-1) nmol(-1) P450, apparent Km = 362 microm), cDNA-expressed CYP3A4 (Vmax = 1.17 nmol min(-1) nmol(-1) P450, apparent Km = 119 microm) and CYP2C8 (Vmax = 0.669 nmol min(-1) nmol(-1) P450, apparent Km = 757 microm) was inhibited by stiripentol (IC50 14, 5.1, 37 microM and apparent Ki 3.7, 2.5, 35 microm, respectively). Saquinavir biotransformation to its major metabolite M7 by human liver microsomes (Vmax = 5.7 nmol min(-1) nmol(-1) P450, apparent Km = 0.79 microm) was inhibited by stiripentol (IC50 163 microM, apparent Ki 86 microm). In epileptic children treated with carbamazepine and stiripentol, the plasma concentration ratio of carbamazepine epoxide/carbamazepine was decreased by 65%. The in vivo apparent Ki for stiripentol ranged from 10.5 to 41.4 microm. The pharmacokinetics of saquinavir was not modified by stiripentol in healthy adults. The 95% confidence intervals for the difference for Cmax and AUC of saquinavir between the placebo and stiripentol phase were (-39.8, 39.8) and (-33.2, 112), respectively. CONCLUSIONS: These results showed that stiripentol was a weak inhibitor of saquinavir metabolism both in vitro and in vivo. In contrast, stiripentol is a potent inhibitor of carbamazepine 10,11-epoxide formation in vitro and in vivo in epileptic patients.     

In vitro evaluation of potential in vivo probes for human flavin-containing monooxygenase (FMO): metabolism of benzydamine and caffeine by FMO and P450 isoforms

AIMS To determine the FMO and P450 isoform selectivity for metabolism of benzydamine and caffeine, two potential in vivo probes for human FMO. METHODS Metabolic incubations were conducted at physiological pH using substrate concentrations of 0.01-10 mM with either recombinant human FMOs, P450s or human liver microsomes serving as the enzyme source. Products of caffeine and benzydamine metabolism were analysed by reversed-phase h.p.l.c. with u.v. and fluorescence detection. RESULTS CYP1A2, but none of the human FMOs, catalysed metabolism of caffeine. In contrast, benzydamine was a substrate for human FMO1, FMO3, FMO4 and FMO5. Apparent Km values for benzydamine N-oxygenation were 60 +/- 8 microM, 80 +/- 8 microM, > 3 mM and > 2 mM, for FMO1, FMO3, FMO4 and FMO5, respectively. The corresponding Vmax values were 46 +/- 2 min-1, 36 +/- 2 min-1, < 75 min-1 and < 1 min-1. Small quantities of benzydamine N-oxide were also formed by CYPs 1A1, 1A2, 2C19, 2D6 and 3A4. CONCLUSIONS: FMO1 and FMO3 catalyse benzydamine N-oxygenation with the highest efficiency. However, it is likely that the metabolic capacity of hepatic FMO3 is a much greater contributor to plasma levels of the N-oxide metabolite in vivo than is extrahepatic FMO1. Therefore, benzydamine, but not caffeine, is a potential in vivo probe for human FMO3.     

Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: evidence for selectivity towards CYP3A4

1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (+/- TSEM) Km and Vmax = 4.6 +/- 0.3 microM and 20.0 +/- 3.8 pmol/min/mg protein, respectively. 4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of lO microM (i.e. around twice Km). Good correlations (r2 = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms. 5. While 10O microM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. The metabolism of 10 microM BFBFC in human liver microsomes was markedly inhibited by 5-50 microM troleandomycin and 0.2-5 microM ketoconazole, but stimulated by 0.2-10 microM alpha-naphthoflavone. The metabolism of 10 microM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4. 7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed Km and Vmax = 70 microM and 3.39 nmol/min/mg protein, respectively. 8. While 80 microM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYPIA2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.     

Use of thiocarbamides as selective substrate probes for isoforms of flavin-containing monooxygenases.

The oxidation of thiourea, phenylthiourea, 1,3-diphenylthiourea, 1,3-bis-(3,4-dichlorophenyl)-2-thiourea and 1,1-dibenzyl-3-phenyl-2-thiourea was measured in reactions catalyzed by purified pig liver flavin-containing monooxygenase (FMO-1) and by microsomal fractions isolated from pig, guinea pig, chicken, rat and rabbit tissues. The reactions, followed by measuring substrate-dependent thiocholine oxidation [Guo and Ziegler, Anal Biochem 198: 143-148, 1991], were carried out in the presence of 2 mM 1-benzylimidazole to minimize potential interference from reactions other than those catalyzed by isoforms of the flavin-containing monooxygenase (FMO). While at saturating substrate concentrations the Vmax for purified FMO-1 catalyzed oxidation of all five thiocarbamides was essentially constant, velocities for the microsomal catalyzed reactions varied not only with tissue and species but also with the van der Waals' surface area of the thiocarbamide. Rat liver, rat kidney and rabbit liver microsomes failed to catalyze detectable oxidation of thiocarbamides larger than 1,3-diphenylthiourea and lung microsomes from a female rabbit only accepted substrates smaller than 1,3-diphenylthiourea. On the other hand, liver microsomes from chickens, pigs and guinea pigs catalyzed the oxidation of larger thiocarbamides, but the rates decreased with increasing substrate size and chicken liver microsomes showed no detectable activity with the largest thiocarbamide tested. To define more precisely the parameters affecting thiocarbamide substrate specificity of microsomal preparations, activities present in detergent extracts of guinea pig liver microsomes were separated into three distinct fractions. The substrate specificities of these partially purified fractions were different and consistent with the difference observed with microsomal catalyzed reactions. This strongly suggests that thiocarbamides that differ in size may be useful probes for measuring the number of activities of FMO isoforms in crude tissue preparations.     

Glutathione conjugation of trichloroethylene in human liver and kidney: kinetics and individual variation.

Isolated human hepatocytes exhibited time-, trichloroethylene (Tri) concentration-, and cell concentration-dependent formation of S-(1, 2-dichlorovinyl)glutathione (DCVG) in incubations in sealed flasks with 25 to 10,000 ppm Tri in the headspace, corresponding to 0.011 to 4.4 mM in hepatocytes. Maximal formation of DCVG (22.5 +/- 8.3 nmol/120 min per 10(6) cells) occurred with 500 ppm Tri. Time-, protein concentration-, and both Tri and GSH concentration-dependent formation of DCVG were observed in liver and kidney subcellular fractions. Two kinetically distinct systems were observed in both cytosol and microsomes from pooled liver samples, whereas only one system was observed in subcellular fractions from pooled kidney samples. Liver cytosol exhibited apparent Km values (microM Tri) of 333 and 22.7 and Vmax values (nmol DCVG formed/min per mg protein) of 8.77 and 4.27; liver microsomes exhibited apparent Km values of 250 and 29.4 and Vmax values of 3.10 and 1.42; kidney cytosol and microsomes exhibited apparent Km values of 26.3 and 167, respectively, and Vmax values of 0.81 and 6.29, respectively. DCVG formation in samples of liver cytosol and microsomes from 20 individual donors exhibited a 6.5-fold variation in microsomes but only a 2.4-fold variation in cytosol. In coincubations of pooled liver cytosol and microsomes, addition of an NADPH-regenerating system produced marked inhibition of DCVG formation, but addition of GSH had no effect on cytochrome P-450-catalyzed formation of chloral hydrate. These results indicate that both human kidney and liver have significant capacity to catalyze DCVG formation, indicating that the initial step of the GSH-dependent pathway is not limiting in the formation of nephrotoxic and nephrocarcinogenic metabolites.     

Metabolism of acrylonitrile to 2-cyanoethylene oxide in F-344 rat liver microsomes, lung microsomes, and lung cells

The metabolism of acrylonitrile to the epoxide, 2-cyanoethylene oxide (ANO) was examined in rat liver microsomes, lung microsomes, and isolated enriched lung cell preparations. GC/high resolution MS was used to quantitate ANO in microsomal and cellular extracts by monitoring the fragment ion C2H3N (m/z 41.0265). The limit of detection was 0.05 pmol of ANO/0.5 microliter of standard solution, microsomal extract, or cellular extract injected onto the column, and the linear range of analysis was 0.05 to 12.5 pmol of ANO. Kinetic parameters of Vmax, V/K, and Km were calculated for microsomal ANO formation. Liver microsomes were quantitatively more active than lung microsomes on a mg of protein basis. The Vmax (pmol of ANO formed/min/mg of protein) was 666.61 for liver and 45.07 for lung microsomes. The V/K (pmol of ANO/min/mg of protein/microM) was 12.83 for liver and 0.02 for lung microsomes. The apparent Km was 51.93 microM and 1853.83 microM for liver and lung microsomes, respectively. When calculated as nmol of ANO formed/min/nmol of microsomal P-450, the Vmax for lung was equivalent to the Vmax for liver. ANO formation in the rat lung was cell specific. The rates of metabolism in the Clara cell-enriched fraction, the alveolar type II cell-enriched fraction, and the cell suspension were 2.55, 0.38, and 0.67 pmol of ANO formed/min/mg of protein, respectively. No metabolism was observed in the endothelial (small) cell-enriched fraction or in the alveolar macrophages. The results suggest that the lung contributes to the metabolism and disposition of inhaled acrylonitrile.     

Benzydamine N-oxidation as an index reaction reflecting FMO activity in human liver microsomes and impact of FMO3 polymorphisms on enzyme activity

AIMS: The role of flavin containing monooxygenases (FMO) on the disposition of many drugs has been insufficiently explored. In vitro and in vivo tests are required to study FMO activity in humans. Benzydamine (BZD) N-oxidation was evaluated as an index reaction for FMO as was the impact of genetic polymorphisms of FMO3 on activity. METHODS: BZD was incubated with human liver microsomes (HLM) and recombinant enzymes. Human liver samples were genotyped using PCR-RFLP. RESULTS: BZD N-oxide formation rates in HLM followed Michaelis-Menten kinetics (mean Km = 64.0 microM, mean Vmax = 6.9 nmol mg-1 protein min-1; n = 35). N-benzylimidazole, a nonspecific CYP inhibitor, and various CYP isoform selective inhibitors did not affect BZD N-oxidation. In contrast, formation of BZD N-oxide was almost abolished by heat treatment of microsomes in the absence of NADPH and strongly inhibited by methimazole, a competitive FMO inhibitor. Recombinant FMO3 and FMO1 (which is not expressed in human liver), but not FMO5, showed BZD N-oxidase activity. Respective Km values for FMO3 and FMO1 were 40.4 microM and 23.6 microM, and respective Vmax values for FMO3 and FMO1 were 29.1 and 40.8 nmol mg-1 protein min-1. Human liver samples (n = 35) were analysed for six known FMO3 polymorphisms. The variants I66M, P135L and E305X were not detected. Samples homozygous for the K158 variant showed significantly reduced Vmax values (median 2.7 nmol mg-1 protein min-1) compared to the carriers of at least one wild type allele (median 6.2 nmol mg-1 protein min-1) (P < 0.05, Mann-Whitney-U-test). The V257M and E308G substitutions had no effect on enzyme activity. CONCLUSIONS: BZD N-oxidation in human liver is mainly catalysed by FMO3 and enzyme activity is affected by FMO3 genotype. BZD may be used as a model substrate for human liver FMO3 activity in vitro and may be further developed as an in vivo probe reflecting FMO3 activity.     

Human liver microsomal metabolism and DNA adduct formation of the tumorigenic pyrrolizidine alkaloid,riddelliine

Riddelliine, a widespread naturally occurring genotoxic pyrrolizidine alkaloid, induced liver tumors in rats and mice in an NTP 2-year carcinogenicity bioassay. We have determined that riddelliine induces liver tumors in rats through a genotoxic mechanism involving the formation of (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), which reacts with DNA to form a set of eight DNA adducts. To determine the relevance to humans of the results obtained in experimental animals, the metabolism of riddelliine was conducted using human liver microsomes. As with rat liver microsomes, DHP and riddelliine N-oxide were major metabolites in incubations conducted with human liver microsomes. The levels of DHP and riddelliine N-oxide were 0.20-0.62 and 0.03-0.15 nmol/min/mg protein, respectively, which are comparable to those obtained from rat liver microsomal metabolism. When metabolism was conducted in the presence of calf thymus DNA, the same set of eight DHP-derived DNA adducts was formed. Both the metabolism pattern and DNA adduct profile were very similar to those obtained from rat liver microsomes. When metabolism was conducted in the presence of the P450 3A4 enzyme inhibitor triacetyleandomycin, the formation of DHP and riddelliine N-oxide was reduced 84 and 92%, respectively. For DHP formation, the Km values were determined to be 0.37 +/- 0.05 and 0.66 +/- 0.08 mM from female rats and female humans; the Vmax values from female rat and human liver microsomal metabolism were 0.48 +/- 0.03 and 1.70 +/- 0.09 nmol/min/mg protein, respectively. These results strongly indicate the mechanistic data on liver tumor induction obtained for riddelliine in laboratory rodents is highly relevant to humans.