Articular chondrocytes and synoviocytes in culture: influence of antioxidants on lipid peroxidation and proliferation

AIMS: Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. METHODS: Human liver microsomal incubations, chemical inhibitors, specific antibodies, and different cytochrome P450 expression systems were used. RESULTS: Km and Vmax values determined in human liver microsomes were lower for the demethylation (61 +/- 21 microM, 159 +/- 42 pmol min(-1) mg protein(-1) mean +/- s.d.; n = 4), than for the N-oxidation of CLZ (308 +/- 1.5 microM, 456 +/- 167 pmol min(-1) mg protein(-1); n = 3). Formation of DCLZ was inhibited by fluvoxamine (53 +/- 28% at 10 microM), triacetyloleandomycin (33 +/- 15% at 10 microM), and ketoconazole (51 +/- 28% at 2 microM) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34 +/- 16% at 10 microM) and ketoconazole (51 +/- 13% at 2 microM), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. Km and Vmax values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. CONCLUSIONS: It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.     

Asymptomatic hypertension in the ED

(R)-(+)-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 (CYP or P450) 2A6. Menthofuran caused a time- and concentration-dependent loss of CYP2A6 activity. The inactivation of CYP2A6 was characterized by a Ki of 2.5 microM and a kinact of 0.22 min-1 for human liver microsomes and a Ki of 0.84 microM and a kinact of 0.25 min-1 for purified expressed CYP2A6. Addition of various nucleophiles, a chelator of iron, or scavengers of reactive oxygen species or extensive dialysis failed to protect CYP2A6 from inactivation. An antibody to metallothionein conjugates of a suspected reactive metabolite of menthofuran was used to detect reactive menthofuran metabolite adducts with CYP2A6. These adducts were formed only in the presence of NADPH-P450 reductase and NADPH. Glutathione, methoxylamine, and semicarbazide did not prevent adduction of reactive menthofuran metabolites to CYP2A6, however. The menthofuran metabolite formation/CYP2A6 inactivation partition ratio was determined to be 3.5 +/- 0.6 nmol/nmol of P450. Menthofuran was unable to inactivate CYP1A2, CYP2D6, CYP2E1, or CYP3A4 in a time- and concentration-dependent manner.     

Kinetics of induction of DNA adducts, cell proliferation and gene mutations in the liver of MutaMice treated with 5,9-dimethyldibenzo[c,g]carbazole

Biotransformation of the M1-muscarinic agonist Lu 25-109 (5-(2-ethyl-2H-tetrazol-5-yl)-1-methyl-1,2,3,6-tetrahydropyridine) , in development for the treatment of Alzheimer's disease, was investigated to obtain information on the identity of human hepatic cytochrome P-450 enzymes involved in its metabolism. The identification of these P-450s was accomplished through studies using 1) simple regression analysis with 14 phenotyped human liver samples, 2) selective chemical inhibitors, and 3) microsomes containing cDNA-expressed enzymes. The production of some metabolites is enhanced in vitro when the pH of the incubation media is shifted from pH 7.4 to 8.5. The metabolite production in human liver microsomes was NADPH-dependent, suggesting that the metabolism of Lu 25-109 in human liver microsomes is primarily P-450-dependent. Lu 25-109 was metabolized by human liver microsomes to Lu 31-126 (de-ethyl Lu 25-109) mainly by CYP2D6; to Lu 29-297 [3-(2-ethyltetrazol-5-yl)-1-methyl-pyridinium] and Lu 25-077 (demethyl Lu 25-109) mainly by CYP1A2, CYP2A6, CYP2C19, and CYP3A4; and to Lu 32-181 (Lu 25-109 N-oxide) by CYP1A2 and possibly by CYP2C19. One metabolite, Lu 32-181 (N-oxide), could be reduced back to Lu 25-109, a reaction not inhibited by the applied cytochrome P-450 inhibitors. This study did not indicate any involvement of FMO3 or MAO in the in vitro metabolism of Lu 25-109 in human liver microsomes.     

An investigation of the formation of cytotoxic, genotoxic, protein-reactive and stable metabolites from naphthalene by human liver microsomes

Chemically reactive epoxide metabolites have been implicated in various forms of drug and chemical toxicity. Naphthalene, which is metabolized to a 1,2-epoxide, has been used as a model compound in this study in order to investigate the effects of perturbation of detoxication mechanisms on the in vitro toxicity of epoxides in the presence of human liver microsomes. Naphthalene (100 microM) was metabolized to cytotoxic, protein-reactive and stable, but not genotoxic, metabolites by human liver microsomes. The metabolism-dependent cytotoxicity and covalent binding to protein of naphthalene were significantly higher in the presence of phenobarbitone-induced mouse liver microsomes than with human liver microsomes. The ratio of trans-1,2-dihydrodiol to 1-naphthol was 8.6 and 0.4 with the human and the induced mouse microsomes, respectively. The metabolism-dependent toxicity of naphthalene toward human peripheral mononuclear leucocytes was not affected by the glutathione transferase mu status of the co-incubated cells. Trichloropropene oxide (TCPO; 30 microM), an epoxide hydrolase inhibitor, increased the human liver microsomal-dependent cytotoxicity (19.6 +/- 0.9% vs 28.7 +/- 1.0%; P = 0.02) and covalent binding to protein (1.4 +/- 0.3% vs 2.8 +/- 0.2%; P = 0.03) of naphthalene (100 microM), and reversed the 1,2-dihydrodiol to 1-naphthol ratio from 6.6 (without TCPO) to 2.6, 0.6 and 0.1 at TCPO concentrations of 30, 100 and 500 microM, respectively. Increasing the human liver microsomal protein concentration reduced the cytotoxicity of naphthalene, while increasing its covalent binding to protein and the formation of the 1,2-dihydrodiol metabolite. Co-incubation with glutathione (5 mM) reduced the cytotoxicity and covalent binding to protein of naphthalene by 68 and 64%, respectively. Covalent binding to protein was also inhibited by gestodene, while stable metabolite formation was reduced by gestodene (250 microM) and enoxacin (250 microM). The study demonstrates that human liver cytochrome P450 enzymes metabolize naphthalene to a cytotoxic and protein-reactive, but not genotoxic, metabolite which is probably an epoxide. This is rapidly detoxified by microsomal epoxide hydrolase, the efficiency of which can be readily determined by measurement of the ratio of the stable metabolites, naphthalene 1,2-dihydrodiol and 1-naphthol.     

Effect of tamoxifen feeding on metabolic activation of tamoxifen by the liver of the rhesus monkey: does liver accumulation of inhibitory metabolites protect from tamoxifen-dependent genotoxicity and cancer?

Tamoxifen induces hepatocellular carcinomas in rats and is converted by rat hepatic cytochrome P450 enzymes into reactive metabolites capable of forming adducts with nucleic acids, proteins and chromosomal aberrations. In rats tamoxifen has also been shown to induce liver cytochrome P450 enzymes, to stimulate its own metabolism leading to greater covalent binding and to induce a higher degree of unscheduled DNA synthesis. This suggests that, at least in the rat, a sensitive species, tamoxifen may contribute significantly to its genotoxic and carcinogenic potential, by assisting its own metabolic activation. We have now investigated the effect of feeding tamoxifen to male and female Rhesus monkeys. A marked induction of the hepatic cytochrome(s) P450 is found in the monkey but, in spite of this, the in vitro metabolism of 7-ethoxyresorufin by microsomes from treated animals is markedly inhibited and so is the dealkylation of two other 7-alkoxyresorufin substrates. Evidence is presented for the accumulation in the liver of monkeys treated with tamoxifen of a powerful inhibitor of drug metabolism, and the inhibitor is identified as a metabolite of tamoxifen, its N,N-didesmethyl derivative. The level of 32P-postlabelled DNA adducts was considerably higher in rats given tamoxifen than in similarly treated monkeys. Also, whereas rats responded to tamoxifen treatment with a marked increase in covalent binding to microsomal protein, in the monkeys, where accumulation of the inhibitory metabolite in the microsomal fraction was also seen, covalent binding was not greater with microsomes from treated animals than in the corresponding controls. N,N-Didesmethyl-tamoxifen, added in vitro to human and rat microsomes, reduced significantly the extent of covalent binding, suggesting that the accumulation of the metabolite observed in the liver of primates may discourage the cytochrome P450-dependent conversion of tamoxifen into reactive derivatives and in this way protect against the formation of adducts. This mechanism may also contribute to protecting the primate against tamoxifen- induced liver cancer.     

Ropivacaine, a new amide-type local anesthetic agent, is metabolized by cytochromes P450 1A and 3A in human liver microsomes

Ropivacaine is a new amide-type local anesthetic agent. Unlike bupivacaine and mepivacaine, two structurally similar local anesthetic compounds, ropivacaine is exclusively the S-(-)-enantiomer. Ropivacaine is predominantly eliminated by extensive metabolism in the liver, with only 1% of the dose being excreted unchanged in the urine of humans. Four of the metabolites formed in human liver microsomes were identified as 3-OH-ropivacaine, 4-OH-ropivacaine, 2-OH-methyl-ropivacaine, and 2',6'-pipecoloxylidide (PPX). The enzymes involved in the human metabolism of ropivacaine have not been identified. To ascertain which forms of cytochrome P450 are involved, ropivacaine was incubated with human microsomes from 10 different livers having different cytochrome P450 activities. A strong correlation was found between the formation of 3-OH-ropivacaine and CYP1A (r = 0.87-0.89) and between the formation of 4-OH-ropivacaine, 2-OH-ropivacaine, and PPX and CYP3A (r = 0.97-1). Incubation of ropivacaine and human liver microsomes in the presence of alpha-naphthoflavone or furafylline, inhibitors of CYP1A, decreased the formation of 3-OH-ropivacaine by about 85%, without affecting the formation of the other metabolites. The formation of 4-OH-ropivacaine, 2-OH-methyl-ropivacaine, and PPX was markedly inhibited in the presence of troleandomycin, an inhibitor of CYP3A. Microsomes from cells expressing CYP1A2 formed 3-OH-ropivacaine, whereas 4-OH-ropivacaine, 2-OH-methyl-ropivacaine, and PPX were formed in microsomes from cells expressing CYP3A4. Inhibitors of CYP2C (sulfaphenazole), CYP2D6 (quinidine), and 2E1 (diethyldithiocarbamate) did not inhibit the formation of any metabolite from ropivacaine. In conclusion, CYP1A catalyzes the formation of 3-OH-ropivacaine, the main metabolite formed in vivo, whereas the formation of 4-OH-ropivacaine, 2-OH-methyl-ropivacaine, and PPX was catalyzed by CYP3A.     

Imipramine-induced inactivation of a cytochrome P450 2D enzyme in rat liver microsomes: in relation to covalent binding of its reactive intermediate

Preincubation of microsomes from male Wistar rats with imipramine (IMI) in the presence of NADPH caused a time-dependent loss of bunitrolol 4-hydroxylase activity, indicating that the CYP2D enzyme is inactivated during IMI metabolism, which has also been observed after in vivo administration of IMI. A similar effect was obtained when desipramine, an N-demethylated metabolite of IMI, was used as an inhibitor, whereas 2-hydroxy-IMI had no effect on the activity. Thus, it seems likely that the inactivation of the CYP2D enzyme is related to 2-hydroxylation process of IMI. Incubation of microsomes with [3H]IMI in the presence of NADPH resulted in covalent binding of a 3H-labeled material to microsomal protein. Formation rates of the reactive metabolites covalently bound to protein followed Michaelis-Menten kinetics, and the K(m) value (1.1 microM) was close to that for microsomal IMI 2-hydroxylation. The metabolism-dependent covalent binding of [3H]IMI was lower in Dark Agouti rats, which is an animal model of CYP2D deficiency, than in Wistar rats. The binding was inhibited by propranolol and quinidine, a substrate and an inhibitor of CYP2D, respectively, and by an antibody against CYP2D. Similar strain difference (Dark Agouti < Wistar) and inhibitory effects by the compounds and the antibody were observed in IMI 2-hydroxylase but not in N-demethylase activity. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of microsomal protein incubated with [3H]IMI and NADPH showed that the binding was prominent at the molecular mass of approximately 50 kDa, which would be consistent with the P450 protein being a target for the binding. Furthermore, proteins to which [3H]IMI metabolites covalently bound were immunoprecipitated with the anti-CYP2D antibody. These results suggest that IMI is biotransformed into a chemically reactive metabolite (probably arene-oxide) through its 2-hydroxylation step by the CYP2D enzyme in rat liver microsomes, and the metabolite binds covalently to the enzyme itself, resulting in the inactivation.     

Metabolism of puerarin and daidzin by human intestinal bacteria and their relation to in vitro cytotoxicity

The steady state plasma concentrations of clozapine and its two major metabolites, norclozapine and clozapine N-oxide, were compared in patients with schizophrenia treated with clozapine in combination with phenobarbital (n=7), and in control patients treated with clozapine alone (n=15). Patients were matched for sex, age, body weight, and antipsychotic dosage. Patients comedicated with phenobarbital had significantly lower plasma clozapine levels than those of the controls (232+/-104 versus 356+/-138 ng/ml; mean, SD, p < 0.05). Plasma norclozapine levels did not differ between the two groups (195+/-91 versus 172+/-61 ng/ml, NS), whereas clozapine N-oxide levels were significantly higher in the phenobarbital group (115+/-49 versus 53+/-31 ng/ml, p < 0.01). Norclozapine/clozapine and clozapine N-oxide/ clozapine ratios were also significantly higher (p < 0.001) in patients comedicated with phenobarbital. These findings suggest that phenobarbital stimulates the metabolism of clozapine, probably by inducing its N-oxidation and demethylation pathways.     

Reductive activation of 1,1-dichloro-1-fluoroethane (HCFC-141b) by phenobarbital- and pyridine-induced rat liver microsomal cytochrome P450

1. During anaerobic reductive incubation of liver microsomes, from either the pyridine- or phenobarbital-treated rat, with 1,1-dichloro-1-fluoroethane (HCFC-141b) in the presence of a NADPH-regenerating system, a time- and dose-dependent formation of reactive metabolites was detected as indicated by a depletion of added exogenous glutathione. 2. A statistically significant, dose-dependent loss of both cytochrome P450 and microsomal haem was also observed under these experimental conditions. Furthermore, a statistically significant decrease of p-nitrophenol hydroxylase and pentoxyresorufin O-depentylase activity was measured in microsomes from the pyridine- and phenobarbital-induced rat, respectively indicating that both P4502E1 and P4502B undergo substrate-dependent inactivation. 3. Both reactive metabolite formation and P450 inactivation were almost completely inhibited by previous bubbling of the incubation mixture with carbon monoxide, indicating that interaction of the substrate with a free and reduced P450 haem iron is required for substrate bioactivation and enzyme loss. 4. The presence in the incubation mixture of the spin-trap N-t-butyl-alpha-phenylnitrone (PBN) and the carbene trap 2,3-dimethyl-2-butene (DMB) largely prevented both glutathione depletion and P450 loss. This suggests that free radical and carbene intermediates formed by the metabolic activation of the substrate are involved in the inactivation of P450 and the loss of its prosthetic haem group.     

Suicide inactivation of cytochrome P450 by midchain and terminal acetylenes. A mechanistic study of inactivation of a plant lauric acid omega-hydroxylase

Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid omega-hydroxylase (omega-LAH), with [1-(14)C]11-dodecynoic acid (11-DDYA) generates a major metabolite characterized as 1,12-dodecandioic acid. In addition to time- and concentration-dependent inactivation of lauric acid and 11-DDYA oxidation, irreversible binding of 11-DDYA (200 pmol of 11-DDYA bound/mg of microsomal protein) at a saturating concentration of 11-DDYA was observed. SDS-polyacrylamide gel electrophoresis analysis showed that 30% of the label was associated with several protein bands of about 53 kDa. The presence of beta-mercaptoethanol in the incubate reduces 1,12-dodecandioic acid formation and leads to a polar metabolite resulting from the interaction of oxidized 11-DDYA with the nucleophile. Although the alkylation of proteins was reduced, the lauric acid omega-hydroxylase activity was not restored, suggesting an active site-directed inactivation mechanism. Similar results were obtained when reconstituted mixtures of cytochrome P450 from family CYP4A from rabbit liver were incubated with 11-DDYA. In contrast, both 11- and 10-DDYA resulted in covalent labeling of the cytochrome P450 2B4 protein and irreversible inhibition of activity. These results demonstrate that acetylenic analogues of substrate are efficient mechanism-based inhibitors and that a correlation between the position of the acetylenic bond in the inhibitor and the regiochemistry of cytochromes P450 oxygenation is essential for enzyme inactivation.     

Platelet-activating factor levels in term and preterm human milk

Tolterodine, a new muscarinic receptor antagonist, is metabolized via two pathways: oxidation of the 5-methyl group and dealkylation of the nitrogen. In an attempt to identify the specific cytochrome P450 enzymes involved in the metabolic pathway, tolterodine was incubated with microsomes from 10 different human liver samples where various cytochrome P450 activities had been rank ordered. Strong correlation was found between the formation of the 5-hydroxymethyl metabolite of tolterodine (5-HM) and CYP2D6 activity (r2, 0.87), as well as between the formation of N-dealkylated tolterodine and CYP3A activity (r2, 0.97). When tolterodine was incubated with human liver microsomes in the presence of compounds known to interact with different P450 isoforms, quinidine was found to be the strongest inhibitor of the formation of 5-HM. Ketoconazole and troleandomycin were found to be the strongest inhibitors of the formation of N-dealkylated tolterodine. A weak inhibitory effect on the formation of N-dealkylated tolterodine was found with sulfaphenazole, whereas tranylcypromine did not inhibit the formation of this metabolite. Microsomes from cells overexpressing CYP2D6 formed 5-HM, whereas N-dealkylated tolterodine was formed by microsomes expressing CYP2C9, -2C19, and -3A4. The Km for formation of N-dealkylated tolterodine by CYP3A4 was similar to that obtained in human liver microsomes and higher for CYP2C9 and -2C19. We conclude from these studies that the formation of 5-HM is catalyzed by CYP2D6 and that the formation of N-dealkylated tolterodine is predominantly catalyzed by CYP3A isoenzymes in human liver microsomes.     

Alternative cyclosporine metabolic pathways and toxicity

There are some indications from clinical studies (41,43) for aberrant cyclosporine metabolism resulting in formation of potentially toxic metabolites. When the activity of cytochrome P450 3A enzymes is low, more substrate is available for hypothetical alternative pathways of cyclosporine. There are several reasons for low P450 3A activity in a liver graft such as inter-individual genetic variability (43,49,84), cold ischemia and reperfusion damage, changes of the P450 activity during cholestasis (85) or other liver diseases (86), the influence of cytokines (87) and drug interactions such as inhibition or enzyme induction (88). Furthermore, low concentrations of cytochrome P450 3A influence the cyclosporine blood trough concentrations. The P450 3A concentration as estimated by the erythromycin breath test can be used to calculate the initial cyclosporine dose required to obtain cyclosporine blood trough concentrations in the therapeutic window (89). In vitro such alternative pathways comprising 3-methylcholanthrene-inducible (44,46,47) and/or ethinyl estradiol-inducible cytochrome P450 enzymes (48) could be identified and resulted in production of cyclized cyclosporine metabolites. The exact identification of the P450 enzymes involved requires metabolism of cyclosporine using reconstituted purified enzymes or single P450 enzymes expressed in cell lines. In addition, it remains to be clarified whether cyclosporine itself or its metabolite AM1 is the substrate for cyclization. Because cyclized metabolites have a low affinity to cyclophilin (58,59) they are mainly found in plasma. When more cyclized metabolites are formed primarily the concentration of cyclosporine metabolites in plasma increases. The free fraction of cyclosporine at 37 degrees C was found to be 1%-1.5% (90,91) of the cyclosporine concentration in blood. To date, nothing is known about the free fraction of cyclosporine metabolites. Because distribution characteristics of the cyclized metabolites in blood and urine are different from those of cyclosporine, it can be speculated that the free fraction of the cyclized metabolites is higher than that of cyclosporine. This might be reflected by a higher renal clearance resulting in relatively higher concentrations in urine compared with blood (61; Figure 3). If this is the case, a shift in the metabolite pattern with increased concentrations of cyclized metabolites will lead to an overproportional increase of the free fraction of cyclosporine metabolites. Although it is tempting to assume that cyclization is the alternative pathway explaining cyclosporine toxicity in patients with low concentrations of P450 3A enzymes in the liver (Figure 6), this has not yet been proven and will require not only quantification of P450 3A but of the complete P450 enzyme pattern in the liver in combination with characterization of the cyclosporine metabolite pattern by HPLC with special respect to the cyclized metabolites AM1c and AM1c9. Also, it is still unclear whether or not the cyclized metabolites contribute to cyclosporine toxicity. At least, it is unlikely that they are involved in covalent binding to macromolecules in the liver and kidney (44,71). In a clinical study using an HPLC method which allowed the specific quantification of 16 cyclosporine metabolites it was shown that the blood trough concentrations of the cyclized metabolite AM1c9 is elevated during early nephrotoxicity in liver graft recipients (82) and it was shown in an in vitro model that AM1c9 increases endothelin production and therefore might have a negative effect on renal hemodynamics.(ABSTRACT TRUNCATED)     

Kinetic characterization of CYP2E1 inhibition in vivo and in vitro by the chloroethylenes

Trans- and cis-1,2-dichloroethylene (DCE) isomers inhibit their own metabolism in vivo by inactivation of the metabolizing enzyme, presumably the cytochrome P450 isoform, CYP2E1. In this study, we examined cytochrome P450 isoform-specific inhibition by three chloroethylenes, cis-DCE, trans-DCE, and trichloroethylene (TCE), and evaluated several kinetic mechanisms of enzyme inhibition with physiological models of inhibition. Trans-DCE was more potent than cis-DCE, and both were much more effective than TCE in inhibiting CYP2E1. The kinetics of in vitro loss of p-nitrophenol hydroxylase (pNP-OH) activity (a marker of CYP2E1) in microsomal incubations and of the in vivo gas uptake results were most consistent with a mechanism in which inhibition of the metabolizing enzyme (CYP2E1) was presumed to be related to interaction of a reactive DCE metabolite with remaining substrate-bound, active CYP2E1. The kinetics of inhibition by TCE, a weak inhibitor in vitro, were very different from that of the dichloroethylenes. With TCE, parent compound concentrations influenced enzyme loss. Trans-DCE was a more potent inhibitor of CYP2E1 than cis-DCE based on both in vivo and in vitro studies. Quantitative differences in the inhibitory properties of the 1,2-DCE isomers may be due to the different stability of epoxides formed from bioactivation by CYP2E1. Epoxide intermediates of DCE metabolism, reacting by water addition, would yield dialdehyde, a potent cross-linking reagent.     

Terfenadine metabolism in human liver. In vitro inhibition by macrolide antibiotics and azole antifungals

To determine whether the clinical adverse interactions of terfenadine with azole antifungals and macrolide antibiotics may be related to inhibition of terfenadine biotransformation, an in vitro system was developed to follow the metabolism of terfenadine by rat liver S9 or human liver microsomes. When test compounds were coincubated with terfenadine, the metabolites formed and unchanged terfenadine was quantitatively analyzed by HPLC. Five metabolites of terfenadine were formed by rat liver S9: predominantly alcohol metabolite (III), with four minor metabolites--azacyclonol (I), acid metabolite (II), an unidentified metabolite (IV), and a new ketone metabolite (V). By human liver microsomes, two major metabolites were formed: azacyclonol (I) and alcohol metabolite (III). Ketoconazole, fluconazole, itraconazole, erythromycin, clarithromycin, and troleandomycin potently inhibited terfenadine metabolism by human liver (IC50 = 4-10 microM), but inhibition by rat liver was weaker (IC50 = 87-218 microM) and 18% maximally for troleandomycin. Other CYP3A substrates (cyclosporin A, naringenin, and midazolam) also demonstrated potent inhibition of terfenadine biotransformation in human liver microsomes (IC50 = 17-24 microM). Substrates of other P450 families [sparteine (CYP2D6), caffeine (CYP1A), and diclofenac (CYP2C)] only very weakly inhibited terfenadine metabolism. Dixon plot analyses for human liver revealed competitive/reversible inhibition by the azole antifungals and macrolide antibiotics of azacyclonol and alcohol metabolite formations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of clozapine by cDNA-expressed human cytochrome P450 enzymes

The metabolism of clozapine was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6, and 2E1. CYP1A2, 3A4, 2C9, 2C19, and 2D6 were able to N-demethylate clozapine. N-Oxide formation was exclusively catalyzed by CYP3A4. CYP2E1 did not metabolize clozapine. With regard to quantitative relationships, CYP1A2, 2C9, 2C19, and 2D6 displayed KM values ranging from 13 to 25 microM, whereas CYP3A4 had a 5-10 times higher KM value. CYP2C19 and 2D6 had the highest Vmax values (149-366 mol/hr/mol CYP). Taking into account the typical relative distribution of amounts of CYP enzymes in the liver, a simulation study suggested that at therapeutic concentrations CYP2C19 and CYP3A4 each accounted for about 35% of the metabolism. At toxic concentrations, the relative importance of CYP3A4 increased.     

The metabolism and DNA binding of the cooked-food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in precision-cut rat liver slices

Precision-cut liver slices prepared from Aroclor 1254 pretreated male rats were used to investigate the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The acetyltransferase and sulfotransferase inhibitors, pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), and the cytochrome P450 inhibitor, alpha-naphthoflavone (ANF), were used to modulate PhIP metabolism and DNA and protein adduct formation. PCP and DCNP had similar effects on the formation of some PhIP metabolites. PCP and DCNP decreased the formation of 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate) and 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP)-glucuronide to 10% and 55% of controls, respectively. 2-Amino-1-methyl-4'-hydroxy-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) was increased by 50% relative to control levels due to PCP and DCNP treatment. PCP and DCNP had different effects on the formation of other PhIP metabolites. Metabolite formation as percent of control for the uncharacterized metabolite, 'Peak A', was 50% and 100% in incubations with PCP and DCNP, respectively. Formation of 4'-hydroxy-PhIP-glucuronide was decreased to 10% of controls with PCP and increased to 147% of controls with DCNP. PCP and DCNP had no effect on the formation of an unidentified metabolite, 'Peak B'. ANF decreased metabolite formation by 60-95%. None of the enzyme inhibitors had a statistically significant effect on PhIP-DNA binding. Covalent binding of PhIP to protein was slightly decreased in incubations containing DCNP or PCP. The lack of significant changes in covalent binding to either DNA or protein suggests that additional pathways may be important in PhIP bioactivation in rat liver slices. With ANF, there was a significant decrease (35%) in protein binding. These observations on the effects of PCP, DCNP and ANF on PhIP metabolism as well as on covalent binding of PhIP to tissue macromolecules are in close agreement with what was reported earlier in hepatocytes. This indicates that tissue slices from various target tissues for tumorigenesis will be a useful in vitro tool for future studies on heterocyclic amine metabolism. This study provides another important example of the utility of precision-cut tissue slices to investigate xenobiotic metabolism and toxicity.     

Induction of cytochromes P450 2B and 2E1 in rat liver by isomeric picoline N-oxides

Pyridine derivatives are widely used solvents and precursors for the synthesis of chemicals of industrial importance. Oxidized metabolites have been implicated in the observed toxicity of pyridines and are known to induce drug-metabolizing enzymes in rat liver. In this study the three isomeric picoline (methylpyridine) N-oxides, as major oxidized metabolites of 2-, 3- and 4-picoline, were evaluated as inducers of cytochrome P450 (CYP) enzymes in rat liver. After a single dose of 100 mg/kg 24 h before sacrifice the 3- and 4-isomers were effective inducers of microsomal substrate oxidations associated with the phenobarbital-inducible CYPs 2B; upregulation of CYP2B protein was confirmed by immunoblotting. In contrast, the 2-isomer did not increase CYP2B protein or activity in rat liver but CYP2E1 protein expression was upregulated by the isomers to 160-200% of control. The three chemicals increased aniline 4-hydroxylation activity in rat liver, which is consistent with induction of CYPs 2B or 2E1 and 4-nitrophenol 2-hydroxylation activity was increased in microsomal fractions from 3- and 4-picoline N-oxide-treated rats. The activities of several other CYPs were also determined and CYP1A-dependent 7-ethylresorufin O-deethylation was increased (to approximately 6- and 2-fold of control) by the 3- and 4-isomer, respectively, whereas the activity of CYP3A-mediated androstenedione 6beta-hydroxylation was decreased by the agents--most notably by the 2-isomer. During NADPH-supported oxidation of CCl4, lipid peroxidation was increased in microsomes from 3- and 4-picoline N-oxide-pretreated rats and was modulated in vitro by the CYP2B inhibitor orphenadrine, but not by the CYP2E1 inhibitor 4-methylpyrazole. These findings establish that particular isomers of picoline N-oxide rapidly upregulate CYP2B or, to a lesser extent, CYP2E1 and implicate CYP2B in the enhanced lipid peroxidation observed in microsomes from rats treated with 3- and 4-picoline N-oxides. Such induction process may contribute to the hepatotoxicity of pyridines by enhancing the capacity for microsomal lipid peroxidation.     

In vivo murine studies on the biochemical mechanism of acetaminophen cataractogenicity

C57BL/6 and DBA/2 mice are, respectively, susceptible and resistant both to the induction of aryl hydrocarbon hydroxylase (cytochrome P450 1A1, or CYP1A1) and to the cataractogenicity of acetaminophen, which may involve its bioactivation to a toxic reactive intermediate, catalysed by P450 and (or) prostaglandin H synthase (PHS). Following induction of P450 using beta-naphthoflavone, the cataractogenicity of acetaminophen (400 mg/kg ip) in C57BL/6 mice was reduced by pretreatment with the P450 inhibitors SKF 525A and metyrapone, the glutathione precursor N-acetylcysteine, the antioxidant vitamin E, and the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (p < 0.05). Acetaminophen (200 mg/kg) cataractogenicity was enhanced by pretreatment with the glutathione depletor diethyl maleate (DEM) and the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) (p < 0.05). No significant effect on acetaminophen cataractogenicity was observed using the PHS cyclooxygenase inhibitors aspirin or naproxen, or the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Accordingly, acetaminophen cataractogenicity in C57BL/6 mice does not appear to be dependent upon bioactivation by PHS. In DBA/2 mice treated with beta-naphthoflavone, a high dose of acetaminophen (750 mg/kg ip) was not cataractogenic, even after pretreatment with DEM, BSO, or BCNU. The resistance of DBA/2 mice to acetaminophen cataractogenesis, despite concomitant pretreatments with an inducer of P450 and several agents that interfere with glutathione-dependent detoxifying pathways, suggests differences in this strain involving cytoprotective pathways subsequent to acetaminophen bioactivation and detoxification of the cataractogenic reactive intermediate. These results indicate that acetaminophen cataractogenicity in C57BL/6 mice results from P450-catalysed bioactivation of acetaminophen to a reactive intermediate, possibly a benzoquinone imine and (or) a free radical, the toxicity of which is reduced by glutathione-dependent reactions.     

Ethanol interactions with other cytochrome P450 substrates including drugs, xenobiotics, and carcinogens

Chronic ethanol abuse is associated with increased activity of the microsomal ethanol-oxidizing system. This effect is due primarily to induction by ethanol of a specific cytochrome P450 (CYP2E1) responsible for enhanced oxidation of ethanol and other P450 substrates and, consequently, for metabolic tolerance to these substances. Furthermore, cytochrome 450 induction increases the activation of numerous xenobiotics to toxic metabolites and of chemical carcinogens to reactive metabolites, thereby accelerating their adverse effects. Microsomal enzyme induction has been associated with increased reactive oxygen species production and enhanced lipid peroxidation, as well as with decreased enzymatic and nonenzymatic scavenger activity, providing another possible explanation for ethanol-mediated toxicity. Yet another effect of chronic alcohol abuse is chronic immune system activation, which is the mechanism underlying alcohol-related liver disease. The metabolism of steroids and vitamins is catalyzed by P450 and is altered in chronic alcoholics. This article reviews recent advances in the understanding of ethanol interactions with drugs, toxic agents, and carcinogens, as well as with steroids and vitamins.     

In vitro evaluation of the antigenotoxic activity of a strain of L. bulgaricus isolated from commercial yogurt

Sequential oxidations at the arylamine moiety of the procainamide molecule leading to the formation of N-hydroxyprocainamide and its nitroso derivative may be responsible for lupus erythematosus observed in patients treated with the drug. The objective of the present study was to characterize major cytochrome P450 isozyme(s) involved in the N-hydroxylation of procainamide. Firstly, incubations were performed with microsomes from either lymphoblastoid cells or yeast transfected with cDNA encoding for specific human cytochrome P450 isozymes. Experiments performed with these enzyme expression systems indicated that the highest formation rate of N-hydroxyprocainamide was observed in the presence of CYP2D6 enriched microsomes. Additional experiments demonstrated that the formation rate of N-hydroxyprocainamide by CYP2D6 enriched microsomes was decreased from 45 +/- 4% to 93 +/- 1% by quinidine at concentrations ranging from 30 nM to 100 microM (all p < 0.05 vs control) and by approximately 75% by antibodies directed against CYP2D6. Secondly, incubations were performed with microsomes prepared from 15 human liver samples. Using this approach, an excellent correlation was observed between the formation rate of N-hydroxyprocainamide and dextromethorphan O-demethylase activity (CYP2D6; r = 0.9305; p < 0.0001). In contrast, no correlation could be established between N-hydroxyprocainamide formation rate and caffeine N3-demethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), S-mephenytoin N-demethylase (CYP2B6), tolbutamide methlhydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), chlorzoxazone 6-hydroxylase (CYP2E1), dextromethorphan N-demethylase (CYP3A4), testosterone 6 beta-hydroxylase (CYP3A4/5) or lauric acid 12-hydroxylase (CYP4A11) activities. Furthermore, formation rate of N-hydroxyprocainamide was decreased in a concentration-dependent manner by quinidine (300 nM to 100 microM) and by antibodies directed against CYP2D6 but not by furafylline 20 microM (CYP1A2), ketoconazole 1 microM (CYP3A4), sulfaphenazole 10 microM (CYP2C9) or antibodies directed against CYP1A1/1A2, CYP2C, CYP2A6, CYP2E1 or CYP3A4/3A5. In conclusion, the results obtained in the present study demonstrate that CYP2D6 is the major human cytochrome P450 isozyme involved in the formation of the reactive metabolite of procainamide, namely N-hydroxyprocainamide.