Vesicular transport of newly synthesized cytochromes P4501A to the outside of rat hepatocyte plasma membranes.

Anti-cytochrome P450 (CYP)1A2 autoantibodies are found in dihydralazine-induced hepatitis, and CYPs2B and 2C have been shown to follow vesicular flow to the plasma membrane (PM). However, it is unknown whether other CYPs follow this route, whether NADPH-CYP reductase is present on the hepatocyte surface, and whether autoimmune hepatitis-inducing drugs increase PM CYPs. In this study, we determined the transmembrane topology and transport of CYPs1A in rat hepatocytes. In cultured hepatocytes, colchicine and other vesicular transport inhibitors decreased PM CYPs1A assessed by flow cytometry. Colchicine administration also decreased PM CYPs1A in vivo. Pulse chase experiments with [(35)S]methionine showed that only the newly synthesized CYP molecules are transferred to the PM, whereas microsomal CYP1A2 was stably radiolabeled for several hours. In contrast, radiolabeled CYP1A2 reached the PM and disappeared from the PM with half-lives of less than 30 min. Confocal microscopy, biotinylation, and coimmunoprecipitation experiments showed that PM CYPs1A and CYP reductase are present on the cell surface, and that the reductase is closely associated with PM CYPs. Exposure of whole cells to an anti-CYP1A1/2 antibody at 4 degrees C, before five washes and PM preparation, abolished PM CYPs1A-supported monooxygenase activity, indicating that PM CYPs are mostly located on the external surface. Dihydralazine and other CYPs1A inducers increased PM CYPs1A. In conclusion, newly synthesized CYPs1A follow vesicular flow to the outside of the PM, and NADPH-CYP reductase also is located on the hepatocyte surface. Dihydralazine administration increases PM CYP1A2, its autoimmune target.

Human microsomal epoxide hydrolase is the target of germander-induced autoantibodies on the surface of human hepatocytes.

Germander, a plant used in folk medicine, caused an epidemic of cytolytic hepatitis in France. In about half of these patients, a rechallenge caused early recurrence, suggesting an immunoallergic type of hepatitis. Teucrin A (TA) was found responsible for the hepatotoxicity via metabolic activation by CYP3A. In this study, we describe the presence of anti-microsomal epoxide hydrolase (EH) autoantibodies in the sera of patients who drank germander teas for a long period of time. By Western blotting and immunocytochemistry, human microsomal EH was shown to be present in purified plasma membranes of both human hepatocytes and transformed spheroplasts and to be exposed on the cell surface where affinity-purified germander autoantibodies recognized it as their autoantigen. Immunoprecipitation of EH activity by germander-induced autoantibodies confirmed this finding. These autoantibodies were not immunoinhibitory. The plasma membrane-located EH was catalytically competent and may act as target for reactive metabolites from TA. To test this hypothesis CYP3A4 and EH were expressed with human cytochrome P450 reductase and cytochrome b(5) in a "humanized" yeast strain. In the absence of EH only one metabolite was formed. In the presence of EH, two additional metabolites were formed, and a time-dependent inactivation of EH was detected, suggesting that a reactive oxide derived from TA could alkylate the enzyme and trigger an immune response. Antibodies were found to recognize TA-alkylated EH. Recognition of EH present at the surface of human hepatocytes could suggest an (auto)antibody participation in an immune cell destruction.

Yeast expressed cytochrome P450 2D6 (CYP2D6) exposed on the external face of plasma membrane is functionally competent.

CYP2D6, a xenobiotic metabolizing cytochrome P450 (P450), was found to be present in significant amount on the outer face of cell plasma membrane in addition to the regular microsomal location. Present work demonstrates that this external P450 is catalytically competent and that activity is supported by NADPH-P450 reductase present on the inner face of plasma membrane. Purified plasma membranes from yeast expressing CYP2D6 sustained NADPH- and cumene hydroperoxide-dependent dextromethorphan demethylation and NADPH-cytochrome c activity confirming previous observations in human hepatocytes. CYP2D6 found on the outside of plasma membrane (by differential immuno-inhibition and acidic shift assays on transformed spheroplasts) was catalytically competent at the cell surface for NADPH-supported activities. Anti-yeast P450-reductase antibodies inhibited neither CYP2D6 nor P450-reductase activities upon incubation with intact spheroplasts. In contrast, both activities were inhibited on isolated plasma membrane fragments. This highly suggested a cytosolic-orientation of the plasma membrane P450-reductase. This finding was confirmed by immunostaining in confocal microscopy. Finally, gene deletion of P450-reductase caused a complete loss of plasma membrane NADPH-supported CYP2D6 activity, which suggests that the reductase participates to some degree in the transmembrane electron transfer chain. This work illustrates that the outside-exposed plasma membrane CYP2D6 is active and may play an important metabolic role.

Topology inversion of CYP2D6 in the endoplasmic reticulum is not required for plasma membrane transport.

The presence of CYP2D6 at the surface of isolated rat and human hepatocytes and its recognition by autoantibodies were reported recently. We wondered whether the unexpected outside orientation at the plasma membrane could be related to topological inversion (luminal-oriented form) of cytochrome P450 in the endoplasmic reticulum. To examine the potential role of cDNA polymorphism, a CYP2D6 variant carrying three positive charges at the amino terminus (2D6ext) was constructed and expressed in yeast. Immunoblotting, flow cytometry, and electron microscopy showed that wild-type CYP2D6 expressed in yeast was present on the outer face of the cell plasma membrane in addition to the regular microsomal location. This location reproduces the hepatocyte situation. 2D6ext expressed in yeast and COS7 cells seemed to be partially N-glycosylated and was located at the plasma membrane surface. Nevertheless, the glycosylated form was not enriched in the plasma membranes compared with microsomes. The relationship between CYP2D6 and 2D6ext topologies and catalytic competence was tested. Cumene hydroperoxide-dependent dextromethorphan demethylation was performed on microsomal vesicles after combined proteolysis and immunoinhibition experiments. CYP2D6 activity was completely abolished, whereas the glycosylated and luminal-oriented fraction of 2D6ext remained active. This suggests that a luminal-oriented glycosylated form is not involved in cytochrome P450 transport to the plasma membrane. Yeast thus reproduces the unusual CYP2D6 plasma membrane location and orientation, which do not require sequence alteration, glycosylation, or even an inverted endoluminal orientation.

Cytochrome P4502B follows a vesicular route to the plasma membrane in cultured rat hepatocytes.

BACKGROUND/AIMS: Autoantibodies against cytochrome P450 are found in some forms of autoimmune hepatitis. Cytochrome P450 is synthesized and mainly located in the endoplasmic reticulum but may also be expressed on the plasma membrane of hepatocytes. Vesicles migrate from the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane along microtubules. We determined the route followed by cytochrome P4502B to reach the plasma membrane. METHODS: Rat hepatocytes were cultured for 2 hours after plating with various inhibitors of cellular trafficking. Detached, uncut, nonpermeabilized hepatocytes were then exposed to a monoclonal antibody specific for cytochrome P4502B and studied by flow cytometry and confocal microscopy. RESULTS: The plasma membrane expression of cytochrome P4502B was markedly decreased after 2 hours of culture with cycloheximide (an inhibitor of protein synthesis), caffeine at 20 degrees C (conditions that decrease vesicular transport from the endoplasmic reticulum to the Golgi apparatus), brefeldin A (which redistributes Golgi components back to the endoplasmic reticulum), monensin (an inhibitor of Golgi functions), and colchicine, vinblastine, or nocodazole (three microtubule inhibitors). CONCLUSIONS: Part of cytochrome P4502B follows a microtubule-dependent vesicular route from the endoplasmic reticulum to the plasma membrane in cultured rat hepatocytes.

Hepatotoxicity of germander in mice.

BACKGROUND/AIMS: An epidemic of hepatitis due to germander teas or capsules recently occurred in France. The aim of the present study was to show the hepatotoxicity of germander and determine its mechanism in mice. METHODS: A germander tea lyophilisate and a fraction that isolated and concentrated 10-fold the furano neo-clerodane diterpenoids of the lyophilisate were prepared. RESULTS: (1) Intragastric administration of the lyophilisate (1.25 g/kg) or the furano neo-clerodane diterpenoid fraction (0.125 mg/kg) produced similar midzonal liver cell necrosis at 24 hours in mice. (2) Toxicity was prevented by pretreatment with a single dose of troleandomycin (a specific inhibitor of cytochromes P4503A) and enhanced by pretreatment with dexamethasone or clotrimazole (two inducers of cytochromes P4503A). (3) Toxicity was attenuated by pretreatment with butylated hydroxyanisole or clofibrate (two inducers of microsomal epoxide hydrolase) and markedly increased by phorone-induced glutathione depletion. CONCLUSIONS: We conclude that germander constituents (probably its furano neo-clerodane diterpenoids) are transformed by cytochromes P450 (particularly P4503A) into hepatotoxic metabolites. The metabolites (probably epoxides) are partly inactivated by glutathione and probably epoxide hydrolase.

Administration of high doses of human recombinant interleukin-2 decreases the expression of several cytochromes P-450 in the rat.

Human recombinant interleukin-2 (IL-2) administration is being tested in patients with advanced cancer. Its effects on the expression of cytochromes P-450 were determined in rats. IL-2 administration (1-25 x 10(6) U/kg i.v. twice daily for 1 to 4 days) resulted in a time- and dose-dependent decrease in cytochrome P-450 measured by the absorbance of its Fe(++)-CO complex. After 25 x 10(6) U/kg twice daily for 4 days, cytochrome P-450 decreased 44%; immunoreactive cytochrome P-450 1A1 decreased nonsignificantly (22%); but cytochrome P-450 1A2 decreased 68%; 2B1/2, 50%; 2C11, 75%; 2D1, 36%; and 3A, 70%. Aminopyrine N-demethylase activity decreased 53%, ethoxycoumarin O-deethylase 64%, benzo(a)pyrene hydroxylase 71%, ethoxyresorufin O-deethylase 42%, pentoxyresorufin O-dealkylase 81% and erythromycin N-demethylase 56%. In rats treated with 3-methylcholanthrene for 4 days, IL-2 coadministration (25 x 10(6) U/kg i.v. twice daily for 4 days) did not decrease significantly immunoreactive cytochrome P-450 1A1 and 1A2, whereas cytochromes P-450 2B1/2, 2C11 and 3A decreased 39, 54 and 67%, respectively. In rats treated with phenobarbital for 4 days, IL-2 coadministration decreased immunoreactive cytochromes P-450 2B1/2 29%, whereas cytochromes P-450 1A2, 2C11 and 3A decreased 38, 63 and 67%, respectively. We conclude that administration of high doses of IL-2 decreases the expression of several cytochromes P-450 in rats. Microsomal enzyme inducers appear to limit the effects of IL-2 on the induced forms of cytochromes P-450. Because much lower doses are used in humans, their potential effects on drug metabolism cannot be assessed from present results.

Metabolic activation of the nitroaromatic antiandrogen flutamide by rat and human cytochromes P-450, including forms belonging to the 3A and 1A subfamilies.

The in vitro metabolic activation of flutamide, a nitroaromatic antiandrogen which produces hepatitis in a few recipients, was first studied with male rat liver microsomes. There was no electron spin resonance evidence for the reduction of flutamide by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase into a nitro anion free radical. In contrast, flutamide was oxidatively transformed by cytochrome P-450 into reactive metabolite(s) that covalently bound to microsomal proteins. Covalent binding required oxygen and NADPH, and was decreased by the nucleophile glutathione and by the cytochrome P-450 inhibitors SKF 525-A, piperonyl butoxide and troleandomycin (an inhibitor of the cytochrome P-450 3A subfamily). Covalent binding was increased markedly by pretreatment with dexamethasone (an inducer of the cytochrome P-450 3A subfamily) and moderately by pretreatment with beta-naphthoflavone (an inducer of the 1A family). Covalent binding was immunoinhibited markedly by anticytochrome P-450 3A immunoglobulin G and moderately by anticytochrome P-450 1A immunoglobulin G. Covalent binding was much lower with liver microsomes from female rats (not expressing P-450 3A2). Covalent binding of flutamide also occurred with human liver microsomes (where it was inhibited by troleandomycin), and with yeast microsomes expressing human liver cytochromes P-450 1A1, 1A2 or 3A4. We concluded that flutamide was oxidatively transformed into chemically reactive metabolite(s) by rat and human cytochromes P-450, including forms belonging to the 3A and 1A subfamilies.

Cytochromes P-450 in human hepatocyte plasma membrane: recognition by several autoantibodies.

BACKGROUND: Anti-cytochrome P-450 autoantibodies are present in several forms of autoimmune hepatitis. The possibility that cytochromes P-450 are present in the plasma membrane of human hepatocytes was examined. METHODS: (1) Plasma membranes with microsomal contamination < 1%, as judged from the activities of glucose-6-phosphatase and NADH-cytochrome c reductase, were prepared. (2) After exposure of uncut, fixed hepatocytes to antibodies, immunofluorescence and immunoperoxidase studies were performed. RESULTS: (1) The specific content of cytochrome P-450 in plasma membrane was 9% of that in microsomes. Plasma membranes showed NADPH-cytochrome c reductase and mono-oxygenase activities; immunoblots showed the presence of cytochromes P-450 1A2, 2C, 2D6, 2E1, and 3A4; cytochromes P-450 1A2, 2D6, and 2C were also recognized by anti-liver microsome and anti-liver/kidney microsome type 1 and type 2 autoantibodies, respectively. (2) Immunofluorescence and immunoperoxidase labeling of the plasma membrane was observed with the three auto-antibodies and with anti-cytochrome P-450 1A2, 2C, 2E1, or 3A4. CONCLUSIONS: It is concluded that cytochromes P-450 are present and functional in the plasma membrane of human hepatocytes and that anti-cytochrome P-450 autoantibodies recognize epitopes expressed on the outer surface.

Nilutamide inhibits mephenytoin 4-hydroxylation in untreated male rats and in human liver microsomes.

1. The effects of nilutamide (an anti-androgen with a hydantoin moiety) on the 4-hydroxylation of mephenytoin were studied in rat liver microsomes. Nilutamide, at a concentration expected in human liver (100 microM) during prolonged administration of nilutamide, inhibited by 40% mephenytoin (0.3 mM) 4-hydroxylase activity in liver microsomes from untreated male rats, but not in microsomes from untreated female rats, or in microsomes from dexamethasone-treated male or female rats. 2. Administration to male rats of nilutamide, in doses (20 mg/kg i.p. twice daily) known to reproduce plasma concentrations observed in human therapeutics, decreased by 60% the 24 h urinary excretion of 4-hydroxymephenytoin after administration of mephenytoin (15 mg/kg oral). 3. Nilutamide (100 microM) markedly inhibited mephenytoin 4-hydroxylase activity in human liver microsomes. Inhibition kinetics were consistent with mixed inhibition. It is concluded that nilutamide inhibits mephenytoin 4-hydroxylase activity in untreated male rats and in human liver microsomes. It is suggested that inhibition is likely to occur in vivo in humans receiving therapeutic doses of nilutamide.

Lipid peroxidation and protective enzymes during myocardial infarction.

Fasting blood taken from 34 patients with myocardial infarction, 19 with unstable angina and 40 healthy controls, was analysed for malondialdehyde and erythrocyte detoxification enzymes, superoxide dismutase and glutathione peroxidase. Malondialdehyde concentration was raised in the patients with myocardial infarction during the initial 48 h after an attack, and correlated with the severity of the attack. 12 days after the infarct, malondialdehyde concentrations were lower but still raised. Superoxide dismutase activity was below normal during the initial 48 h post infarct and raised twelve days after. Glutathione peroxidase was reduced after 12 days. Similar, but less marked changes were seen in the patients unstable angina.

The evolution of oxidative stress indicators in the course of myocardial ischemia.

Two studies were carried out in patients suffering from Unstable Angina (UA) and Myocardial Infarction (MI). The first study investigated the variations of the Malondialdehyde (MDA) rate at 1st, 5th, 12th day of treatment in 27 patients (15 UA and 12 MI), compared to 15 controls. This rate varied in a different way, with a first peak and a rapid decrease in UA, where it regularly decreases in MI. The second study focused on the variations of MDA, Superoxide Dismutase (SOD), Glutathion Peroxydase (GPX) rates at 2nd, 12th days in 53 patients (19 UA and 34 MI), compared to 35 controls. Here again, the rate of MDA was high on day 2 and decreased on day 12. The rate of GPX showed similar evolution while the SOD rate had an opposite evolution. These two studies confirm the evidence of oxidative stress in acute coronary deficiency.

Inhibition of rat liver estrogen 2/4-hydroxylase activity by troleandomycin: comparison with erythromycin and roxithromycin.

Administration of troleandomycin (0.5 mmol.kg-1 p.o. daily for 5 days) decreased by 61% and 36%, respectively, the estradiol and ethinylestradiol 2/4-hydroxylase activities of hepatic microsomes from male Sprague-Dawley rats killed 2 hr after the last dose. This decrease did not appear to be due to the in vivo formation of the inactive cytochrome P-450 p Fe(II)-metabolite complex, since disruption of this complex with potassium ferricyanide did not increase estrogen hydroxylase activities. Troleandomycin administration, however, essentially suppressed cytochrome P-450 UT-A (one of the P-450 forms involved in the hydroxylation of estrogens) and resulted in the appearance of cytochrome P-450 forms whose estradiol hydroxylase activity was inhibitable by troleandomycin in vitro. Similarly, troleandomycin (2 mM) inhibited by 60% estradiol and ethinylestradiol 2/4-hydroxylase activities in microsomes from dexamethasone-treated rats, although it had no inhibitory effect in microsomes from control rats. In contrast, erythromycin and roxithromycin (2 mM) exerted no inhibitory effect, even in microsomes from dexamethasone-treated rats. In vivo, these macrolides (0.5 mmol.kg-1 p.o. daily for 5 days) decreased moderately cytochrome P-450 UT-A levels and estradiol 2/4-hydroxylase activity, and did not modify ethinylestradiol 2/4-hydroxylase activity. We conclude that the administration of troleandomycin, but not that of erythromycin or roxithromycin, decreases ethinylestradiol 2/4-hydroxylase activity in male rat liver microsomes, as a possible consequence of decreased cytochrome P-450 UT-A levels and of the induction of glucocorticoid-responsive P-450 forms whose ethinylestradiol hydroxylase activity is inhibitable by troleandomycin.

Metabolic activation of the new tricyclic antidepressant tianeptine by human liver cytochrome P450.

Incubation of [14C]tianeptine (0.5 mM) with human liver microsomes and a NADPH-generating system resulted in the in vitro covalent binding of a tianeptine metabolite to microsomal proteins. This covalent binding required oxygen and NADPH. It was decreased by piperonyl butoxide (4 mM) by 81%, and SKF 525-A (4 mM) by 87%, two relatively non-specific inhibitors of cytochrome P450, and by glutathione (4 mM) by 70%, a nucleophile. Covalent binding was decreased by 54% in the presence of troleandomycin (0.1 mM), a specific inhibitor of the glucocorticoid-inducible cytochrome P450 IIIA3, but remained unchanged in the presence of quinidine (0.1 mM) or dextromethorphan (0.1 mM), two inhibitors of cytochrome P450 IID6. Preincubation with IgG antibodies directed against cytochrome P450 IIIA3 decreased covalent binding by 65% whereas either preimmune IgG or IgG antibodies directed against P450 IA1, an isoenzyme inducible by polycyclic aromatic compounds, exhibited no significant inhibitory effect. We conclude that tianeptine is activated by human liver cytochrome P450 into a reactive metabolite. This activation is mediated in part by glucocorticoid-inducible isoenzymes but not by P450 IID6 (the isoenzyme which oxidizes debrisoquine) nor by P450 IA1 (an isoenzyme inducible by polycyclic aromatic compounds). The predictive value of this study regarding possible idiosyncratic and immunoallergic reactions in humans remains unknown.

Presence of functional cytochrome P-450 on isolated rat hepatocyte plasma membrane.

Antibodies against cytochrome P-450 are found in some children with autoimmune hepatitis (antiliver/kidney microsome 1) and in patients with ticrynafen hepatitis (antiliver/kidney microsome 2). For an immune reaction against cytochrome P-450 to possibly destroy the hepatocytes, one must assume that cytochrome P-450 is present on the plasma membrane surface of hepatocytes. In a first series of experiments, plasma membranes were prepared with a technique based on the electrostatic attachment of isolated hepatocytes to polyethyleneimine-coated beads. After vortexing, beads were coated with a very pure plasma membrane fraction. Microsomal contamination, judged from the specific activities of glucose-6-phosphatase or NADH-cytochrome c reductase, was less than 1%. Nevertheless, the specific content (per milligram of protein) of CO-binding cytochrome P-450 was 20% of that in microsomes; the specific benzo(a)pyrene hydroxylase activity was 25%, and ethoxycoumarin deethylase 11%. Immunoblots showed the presence of cytochromes P-450 UT-A, UT-H, PB-B, ISF-G and PCN-E, the last three isoenzymes being inducible by, respectively, phenobarbital, 3-methylcholanthrene and dexamethasone. In a second series of experiments, nonpermeabilized isolated hepatocytes from untreated rats were incubated with anticytochrome P-450 antibodies. Immunofluorescence and immunoperoxidase staining confirmed the presence of cytochromes P-450 UT-A, PB-B and ISF-G on the membrane. In a last series of experiments, human antiliver-kidney microsomal 1 antibodies were found to react specifically with rat liver plasma membrane cytochrome P-450 UT-H (IID subfamily). We conclude that several cytochrome P-450 isoenzymes are present, active and inducible on the plasma membrane surface of hepatocytes. It is therefore conceivable that immunization against plasma membrane cytochrome P-450 might lead to the immunological destruction of hepatocytes in some patients.

Metabolic activation of the antidepressant tianeptine. I. Cytochrome P-450-mediated in vitro covalent binding.

Incubation under air of [14C]tianeptine (0.5 mM) with a NADPH-generating system and hamster, mouse or rat liver microsomes resulted in the in vitro covalent binding of [14C]tianeptine metabolites to microsomal proteins. Covalent binding to hamster liver microsomes required NADPH and oxygen; it was decreased in the presence of the cytochrome P-450 inhibitors, carbon monoxide, piperonyl butoxide (4 mM), and SKF 525-A (4 mM) or in the presence of the nucleophile, glutathione (1 or 4 mM). In vitro covalent binding to hamster liver microsomes was not decreased in the presence of quinidine (1 microM), and was similar with microsomes from either female Dark Agouti, or female Sprague-Dawley rats. In contrast, in vitro covalent binding to hamster liver microsomes was decreased in the presence of troleandomycin (0.25 mM), while covalent binding was increased with microsomes from either hamsters, mice or rats pretreated with dexamethasone. Preincubation with IgG antibodies directed against rabbit liver glucocorticoid-inducible cytochrome P-450 3c(P-450 IIIA4) decreased in vitro covalent binding by 53 and 89%, respectively, with microsomes from control hamsters and dexamethasone-pretreated hamsters, and by 60 and 81%, respectively, with microsomes from control and dexamethasone-pretreated rats. We conclude that tianeptine is activated by hamster, mouse and rat liver cytochrome P-450 into a reactive metabolite. Metabolic activation is mediated in part by glucocorticoid-inducible isoenzymes but not by the isoenzyme metabolizing debrisoquine. In vivo studies are reported in the accompanying paper.

Metabolic activation of the antidepressant tianeptine. II. In vivo covalent binding and toxicological studies at sublethal doses.

Administration of [14C]tianeptine (0.5 mmol/kg i.p.) to non-pretreated hamsters resulted in the in vivo covalent binding of [14C]tianeptine metabolites to liver, lung and kidney proteins; this very high dose (360-fold the human therapeutic dose) depleted hepatic glutathione by 60%, and increased SGPT activity 5-fold. Lower doses (0.25 and 0.125 mmol/kg) depleted hepatic glutathione to a lesser extent and did not increase SGPT activity. Pretreatment of hamsters with piperonyl butoxide decreased in vivo covalent binding to liver proteins, and prevented the increase in SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). In contrast, pretreatment of hamsters with dexamethasone increased in vivo covalent binding to liver proteins, and increased SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). Nevertheless, liver cell necrosis was histologically absent 24 hr after the administration of tianeptine (0.5 mmol/kg i.p.) to non-pretreated or dexamethasone-pretreated hamsters. In vivo covalent binding to liver proteins also occurred in mice and rats, being increased by 100% in dexamethasone-pretreated animals. In vivo covalent binding to liver proteins was similar in untreated female Dark Agouti rats and in female Sprague-Dawley rats. These results show that tianeptine is transformed in vivo by cytochrome P-450, including glucocorticoid-inducible isoenzymes, into chemically reactive metabolites that covalently bind to tissue proteins. The metabolites, however, exhibit no direct hepatotoxic potential in hamsters below the sublethal dose of 0.5 mmol/kg i.p. The predictive value of this study regarding possible idiosyncratic and immunoallergic reactions in humans remains unknown.

Genetic predisposition to drug hepatotoxicity: role in hepatitis caused by amineptine, a tricyclic antidepressant.

Amineptine-induced immunoallergic hepatitis is unpredictable. It may be related to its oxidation into a reactive metabolite acting as hapten. We have looked for a possible genetic predisposition involving drug oxidation capacity and/or cell defense mechanisms in nine patients with previous amineptine hepatitis. Drug oxidation capacity was assessed using dextromethorphan, a test compound recently proposed as a substitute for debrisoquine. The eight patients tested had the extensive metabolizer phenotype. The susceptibility to amineptine metabolites was studied by an in vitro test assessing the destruction of the patients' lymphocytes by reactive metabolites generated from amineptine by a standardized oxidation microsomal system. Lymphocyte death increased with the dose of amineptine (1 to 2.5 mM); it was increased by preincubation with trichloropropene oxide, but was absent when amineptine was omitted or when the oxidation system was not operating. Mean lymphocyte death was twice higher in the nine patients with amineptine hepatitis than in 17 healthy controls. In contrast, when the test was performed with acetaminophen (3 to 10 mM), lymphocyte death was similar in controls and in patients. Basal epoxide hydrolase activity toward benzo[a]pyrene-4,5-oxide and glutathione concentration was similar in lymphocytes from controls and patients. Family studies showed an increased susceptibility to amineptine metabolites in lymphocytes from several first-degree relatives of two patients. These results show that amineptine hepatitis occurs in patients with extensive dextromethorphan oxidation capacity but with an increased susceptibility to amineptine reactive metabolites, probably related to a genetic deficiency in a cell defense mechanism.