Overexpression of gastric leptin precedes adipocyte leptin during high-fat diet and is linked to 5HT-containing enterochromaffin cells.

OBJECTIVES: In obesity, while hyperleptinemia highly correlates with excess fat mass, the status of gastric leptin remains unknown. Here, we investigated the expression of leptin in stomach biopsies of obese humans and analyzed the temporal changes of gastric leptin expression in response to diet-induced obesity and its impact on 5-hydroxytryptamine (5HT)-producing cells. METHODS: Enterochromaffin (EC) cells and expression of leptin, PAX4 (critical factor for EC specification), tryptophane hydroxylase-1 (TPH1, the peripheral rate-limiting enzyme for 5HT) and 5HT were examined by immunofluorescence, quantitative real-time PCR, radioimmunoassay, respectively, in stomach and duodenum biopsies from 19 obese and 14 normo-weighed individuals, and in mucosa scrapings from C57Bl6/J diet-induced obese mice, leptin-deficient ob/ob mice and intestine-specific leptin receptor isoform B-deficient mice. RESULTS: Gastric mucosa of obese subjects displays an increased expression of leptin (LEP mRNA by fivefold and protein by twofold, P<0.01), TPH1 ((1.75-2.73, 95% confidence interval (CI)) vs (0.38-0.67, 95% CI); P<0.01) and PAX4 ((1.33-2.11, 95%CI) vs (0.62-0.81, 95% CI); P<0.01) as compared with normo-weighed individuals. In diet-induced obese mice, the overexpressions of gastric leptin, antral Pax4, Tph1 and increased EC cell number occurred before the onset of obesity and hyperleptinemia (reflect of adipocyte leptin production). In addition, leptin deficiency was associated with reduced Pax4 mRNA, whereas oral leptin treatment enhanced both Tph1 and Pax4 mRNA. Finally, mice with an intestine-specific deletion of leptin signaling exhibit significant decrease in duodenal mucosa 5HT content. CONCLUSIONS: These data demonstrate that gastric leptin is upregulated in obese individuals. RESULTS from high-fat diet mice showed that overexpression of gastric leptin that is linked to gut '5HT pathway' occurred before the onset of obesity and expansion of fat mass. This may be relevant in the pathophysiology of obesity.

In vitro study of lovastatin interactions with amiodarone and with carbon tetrachloride in isolated rat hepatocytes.

AIM: To investigate the interactions at a metabolic level between lovastatin, amiodarone and carbon tetrachloride in isolated rat hepatocytes. METHODS: For cell isolation two-step collagenase liver perfusion was performed. Lovastatin was administered alone in increasing concentrations (1 mumol/L, 3 mumol/L, 5 mumol/L and 10 mumol/L) and in combination with CCl(4) (86 mumol/L). The cells were also pretreated with 14 mumol/L amiodarone and then the other two compounds were added. RESULTS: Lovastatin promoted concentration-dependent significant toxicity estimated by decrease in cell viability and GSH level by 45% and 84%, respectively. LDH-activity increased by 114% and TBARS content by 90%. CCl(4)induced the expected severe damage on the examined parameters. CCl(4) induced toxicity was attenuated after lovastatin pretreatment, which was expressed in less increased values of LDH activity and TBARS levels, as well as in less decreased cell viability and GSH concentrations. However, the pretreatment of hepatocytes with amiodarone abolished the protective effect of lovastatin. CONCLUSION: We suggest that the observed cytoprotective effect was due to interactions between lovastatin, CCl(4) and amiodarone at a metabolic level.

Toxicity of alpidem, a peripheral benzodiazepine receptor ligand, but not zolpidem, in rat hepatocytes: role of mitochondrial permeability transition and metabolic activation.

Whereas alpidem is hepatotoxic, zolpidem is not. Despite closely related chemical structures, alpidem, but not zolpidem, is a peripheral benzodiazepine receptor (PBR) ligand, and is also more lipophilic than zolpidem. We compared their effects in isolated rat liver mitochondria and rat hepatocytes. Zolpidem did not affect calcium-induced mitochondrial permeability transition (MPT) in mitochondria, caused little glutathione depletion in hepatocytes, and was not toxic, even at 500 microM. At 250 to 500 microM, alpidem prevented calcium-induced MPT in isolated mitochondria, but caused severe glutathione depletion in hepatocytes that was increased by 3-methylcholanthrene, a cytochrome P4501A inducer, and decreased by cystine, a glutathione precursor. Although cell calcium increased, mitochondrial cytochrome c did not translocate to the cytosol and cells died of necrosis. Cell death was prevented by cystine, but not cyclosporin A, an MPT inhibitor. At low concentrations (25-50 microM), in contrast, alpidem accelerated calcium-induced MPT in mitochondria. It did not deplete glutathione in hepatocytes, but nevertheless caused some cell death that was prevented by cyclosporin A, but not by cystine. Alpidem (10 microM) also increased the toxicity of tumor necrosis factor-alpha (1 ng/ml) in hepatocytes. In conclusion, low concentrations of alpidem increase both calcium-induced MPT in mitochondria, and TNF-alpha toxicity in cells, like other PBR ligands. Like other lipophilic protonatable amines, however, alpidem inhibits calcium-induced MPT at high concentrations. At these high concentrations, toxicity involves cytochrome P4501A-mediated metabolic activation, glutathione depletion, and increased cell calcium, without MPT involvement. In contrast, zolpidem has no mitochondrial effects, causes little glutathione depletion, and is not toxic.

Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation.

BACKGROUND/AIMS: Although sublingual buprenorphine is safely used as a substitution drug in heroin addicts, large overdoses or intravenous misuse may cause hepatitis. Buprenorphine is N-dealkylated to norbuprenorphine by CYP3A. METHODS: We investigated the mitochondrial effects and metabolic activation of buprenorphine in isolated rat liver mitochondria and microsomes, and its toxicity in isolated rat hepatocytes and treated mice. RESULTS: Whereas norbuprenorphine had few mitochondrial effects, buprenorphine (25-200 microM) concentrated in mitochondria, collapsed the membrane potential, inhibited beta-oxidation, and both uncoupled and inhibited respiration in rat liver mitochondria. Both buprenorphine and norbuprenorphine (200 microM) underwent CYP3A-mediated covalent binding to rat liver microsomal proteins and both caused moderate glutathione depletion and increased cell calcium in isolated rat hepatocytes, but only buprenorphine also depleted cell adenosine triphosphate (ATP) and caused necrotic cell death. Four hours after buprenorphine administration to mice (100 nmol/g body weight), hepatic glutathione was unchanged, while ATP was decreased and serum transaminase increased. This transaminase increase was attenuated by a CYP3A inducer and aggravated by a CYP3A inhibitor. CONCLUSIONS: Both buprenorphine and norbuprenorphine undergo metabolic activation, but only buprenorphine impairs mitochondrial respiration and ATP formation. The hepatotoxicity of high concentrations or doses of buprenorphine is mainly related to its mitochondrial effects.

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.

Interleukin-2 overexpresses c-myc and down-regulates cytochrome P-450 in rat hepatocytes.

The interaction of interleukin-2 (IL-2) with its receptor (IL-2R) decreases cytochrome P-450 (CYP) expression in rat hepatocytes. Because IL-2 increases c-Myc in lymphocytes and because c-myc overexpression represses several genes, we postulated that the IL-2/IL-2R interaction may increase c-Myc and thereby down-regulate CYP in hepatocytes. Cultured rat hepatocytes were exposed for 24 h to IL-2 (350 U/ml) and other agents. IL-2 increased c-myc mRNA and protein but decreased total CYP and the mRNAs and proteins of CYP2C11 and CYP3A. The IL-2-mediated c-myc overexpression and CYP down-regulation were prevented by 1) genistein (a tyrosine kinase inhibitor that blocks the initial transduction of the IL-2R signal), 2) retinoic acid, butyric acid, or dimethyl sulfoxide (three agents that block c-myc transcription), or 3) an antisense c-myc oligonucleotide (which may cause rapid degradation of the c-myc transcript). It is concluded that IL-2 causes the overexpression of c-myc and the down-regulation of CYPs in rat hepatocytes. Block of c-myc overexpression, at three different levels with five different agents, prevents CYP down-regulation, suggesting that c-myc overexpression may directly or indirectly repress CYP in 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.

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.

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.

Suicide inactivation of cytochrome P-450 by methoxsalen. Evidence for the covalent binding of a reactive intermediate to the protein moiety.

Incubation of rat liver microsomes with [3H]methoxsalen and NADPH resulted in the covalent binding of a methoxsalen intermediate to proteins comigrating with cytochromes P-450 UT-A, PB-B/D, ISF-G and PCN-E. Binding was increased by pretreatments with phenobarbital, beta-naphthoflavone (beta NF) and dexamethasone. Such pretreatments also increased the loss of CO-binding capacity either after administration of methoxsalen, or after incubation of hepatic microsomes with methoxsalen and NADPH. Immunoprecipitation of the methoxsalen metabolite-protein adducts in phenobarbital-induced microsomes was moderate with anti-UT-A antibodies, but marked with anti-PB-B/D and anti-PCN-E antibodies. Immunoprecipitation was observed also with anti-ISF-G (anti-beta NF-B) antibodies in beta NF-induced microsomes. Methoxsalen (0.25 mM) inhibited markedly the benzphetamine demethylase activity of phenobarbital-induced microsomes and the erythromycin demethylase activity of dexamethasone-induced microsomes. Whereas methoxsalen itself did not produce any binding spectrum, in contrast either in vivo administration of methoxsalen or incubation in vitro with methoxsalen and NADPH resulted in a low-to-high spin conversion of cytochrome P-450 as suggested by the appearance of a spectrum analogous to a type I binding spectrum. This low-to-high spin conversion was apparently due to a methoxsalen intermediate (probably, covalently bound to the protein and preventing partial sixth ligation of the iron). We conclude that suicide inactivation of cytochrome P-450 by methoxsalen is related to the covalent binding of a methoxsalen intermediate to the protein moiety of several cytochrome P-450 isoenzymes (including UT-A, PB-B/D, PCN-E as well as ISF-G and/or beta NF-B).

Effects of clarithromycin on cytochrome P-450. Comparison with other macrolides.

Repeated administration of clarithromycin (0.5 mmol.kg-1 p.o. daily for 5 days) to rats increased markedly the same cytochrome P-450 isoenzyme (P-450p) as that induced by troleandomycin. Clarithromycin, however, did not form cytochrome P-450 Fe(II)-metabolite complexes in vitro with microsomes from clarithromycin-treated rats or in vivo after repeated doses of clarithromycin. Nevertheless, clarithromycin formed cytochrome P-450 Fe(II)-metabolite complexes with microsomes from dexamethasone-treated rats in vitro, or after administration to dexamethasone-treated rats in vivo. Similar effects were observed with roxithromycin. In contrast, erythromycin and troleandomycin formed metabolic complexes when given alone, whereas josamycin, midecamycin and spiramycin did not form complexes, even in dexamethasone-treated rats. We conclude that clarithromycin and roxithromycin induce cytochrome P-450p, but do not form complexes with this isoenzyme, although they do form complexes with other glucocorticoid-inducible isoenzymes. We propose that macrolides may be classified into three groups, those forming complexes when given alone (e.g., erythromycin and troleandomycin), those forming complexes only in glucocorticoid-pretreated rats (clarithromycin and roxithromycin) and those not forming complexes (josamycin, midecamycin and spiramycin).

Presence of covalently bound metabolites on rat hepatocyte plasma membrane proteins after administration of isaxonine, a drug leading to immunoallergic hepatitis in man.

Isaxonine and several other drugs transformed by cytochrome P-450 into reactive metabolites apparently lead to immunoallergic hepatitis in man. Protein epitopes modified by the covalent binding of the metabolites have been proposed as possible targets for the immune response. The purpose of this work was to determine whether covalently bound metabolites are indeed present on hepatocyte plasma membrane proteins. In a first series of experiments, rats were killed 15 or 60 min after administration of [2-14C]isaxonine (0.2 mmol.kg-1 i.p.), and various fractions were prepared from isolated hepatocytes; microsomal contamination of the plasma membrane fraction was 1.2% or less. At 60 min, the amount of isaxonine metabolite covalently bound per mg of protein was similar in plasma membranes (0.42 nmole metabolite.mg protein-1) and in microsomes (0.38); both values were decreased by about 70% in rats pretreated with piperonyl butoxide, an inhibitor of cytochrome P-450. At 15 min, however, covalent binding to plasma membrane proteins (0.06 nmole metabolite.mg protein-1) was only half of that to microsomal proteins (0.12). In a second series of experiments, [2-14C] isaxonine (0.1 mM) was incubated with NADPH, hepatic microsomes and plasma membranes. The reactive isaxonine metabolite became bound extensively to microsomal proteins, but not to plasma membrane proteins. These results show that administration of isaxonine leads to the presence of isaxonine adducts on the proteins of the hepatocyte plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Methoxsalen decreases the metabolic activation and prevents the hepatotoxicity and nephrotoxicity of chloroform in mice.

The effects of methoxsalen, a potent inhibitor of cytochrome P-450, on the hepatotoxicity and nephrotoxicity of chloroform have been determined in mice. Hepatic and renal monooxygenase activities and the in vitro covalent binding of chloroform metabolites to hepatic and renal microsomal proteins were decreased by 20-70% in microsomes from mice killed 2 hr after the administration of methoxsalen (250 mumol.kg-1ip) alone. Administration of methoxsalen (250 mumol.kg-1ip), 30 min before [14C]chloroform (1 ml.kg-1ip), did not modify blood levels of [14C]chloroform (and metabolites) but decreased the in vivo covalent binding of [14C]chloroform metabolites to hepatic and renal proteins 4 hr after the administration of [14C]chloroform. This pretreatment markedly decreased serum glutamic pyruvic transaminase activity, blood urea nitrogen, glucosuria, liver and kidney lesions, and mortality 24 hr after the administration of chloroform (0.125-1.5 ml.kg-1ip). Other cytochrome P-450 inhibitors (SKF 525-A or piperonyl butoxide), given at the same molar dose (250 mumol.kg-1ip), exerted no protective effect. Pretreatment with methoxsalen appears to decrease the metabolic activation of chloroform and essentially prevents its hepatotoxicity and nephrotoxicity in mice. Methoxsalen may have use as a tool to determine the role of metabolic activation by cytochrome P-450 in the hepatotoxicity and nephrotoxicity of drugs and chemicals.

The drug methoxsalen, a suicide substrate for cytochrome P-450, decreases the metabolic activation, and prevents the hepatotoxicity, of carbon tetrachloride in mice.

Methoxsalen, a potent suicide inhibitor of cytochrome P-450 that can be used in humans, might be of value for the prevention of hepatitis in subjects with carbon tetrachloride poisoning. As a preliminary step, we have determined its effects on the hepatotoxicity of carbon tetrachloride in mice. Several monooxygenase activities, the in vitro covalent binding of carbon tetrachloride metabolites to microsomal proteins, and in vitro microsomal lipid peroxidation initiated by carbon tetrachloride metabolites were decreased by 60-90% in microsomes from mice killed 2 hr after the administration of methoxsalen (250 mumol X kg-1); microsomal lipid peroxidation mediated by endogenous iron and NADPH was not modified. Administration of methoxsalen (250 mumol X kg-1) 30 min before carbon tetrachloride (0.1 ml X kg-1) decreased both the in vivo formation of conjugated dienes in microsomal lipids and the in vivo covalent binding of carbon tetrachloride metabolites to lipids and proteins. This pretreatment completely prevented the hepatotoxicity of carbon tetrachloride. Other cytochrome P-450 inhibitors (cimetidine, SKF 525-A or piperonyl butoxide) given at this low molar dose (250 mumol X kg-1) exerted no protective effect. Methoxsalen (500 mumol X kg-1) was also effective, but only partially, when given 30 min after carbon tetrachloride (0.025 ml X kg-1). We conclude that pretreatment with methoxsalen decreases the metabolic activation of carbon tetrachloride, and completely prevents its hepatotoxicity in mice. Post-treatment with methoxsalen must be given early and is only partially effective in mice.

Inactivation of human liver cytochrome P-450 by the drug methoxsalen and other psoralen derivatives.

The effects of psoralen derivatives on cytochrome P-450 have been studied in human liver microsomes. CO-binding cytochrome P-450 was decreased by 33% after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 microM methoxsalen (8-methoxypsoralen). No destruction of cytochrome P-450 was observed when either NADPH or methoxsalen was omitted. A similar (27%) decrease in CO-binding required a 100-times higher concentration of allylisopropylacetamide (2 mM). The activities of 7-ethoxycoumarin deethylase and benzo(a)pyrene hydroxylase were decreased by about 50% in the presence of 12.5 microM methoxsalen. At this low concentration, neither cimetidine nor SKF 525-A or piperonyl butoxide had any significant inhibitory effect. Monooxygenase activities were also decreased in the presence of 12.5 microM bergapten (5-methoxypsoralen) or 12.5 microM psoralen, but not with 12.5 microM trioxsalen (trimethylpsoralen). CO-binding cytochrome P-450 was not decreased after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 microM trioxsalen. We conclude that methoxsalen is an extremely potent suicide inhibitor of cytochrome P-450 in human liver microsomes. Bergapten and psoralen are also inhibitory whereas trioxsalen has little effects. In the latter derivative, a methyl group is attached on the furan ring and may hinder its metabolic activation and the inactivation of cytochrome P-450.

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

Incubation of [14C]amineptine (1 mM) with hamster liver microsomes resulted in the irreversible binding of an amineptine metabolite to microsomal proteins. Covalent binding measured in the presence of various concentrations of amineptine (0.0625-1 mM) followed Michaelis-Menten kinetics. Pretreatment with phenobarbital increased not only the Vmax, but also the Km, for this binding. Covalent binding required NADPH and molecular oxygen and was decreased when the incubation was made in the presence of inhibitors of cytochrome P-450 such as piperonyl butoxide (4 mM), SKF 525-A (4 mM) or carbon monoxide (80:20 CO-O2 atmosphere). In contrast, binding was increased when microsomes from untreated hamsters were incubated in the presence of 0.5 mM 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrolase. Metabolic activation also occurred in kidney microsomes. In vitro covalent binding to kidney microsomal proteins required NADPH and was decreased by piperonyl butoxide (4 mM) but was not increased by pretreatment with phenobarbital. We conclude that amineptine is activated by hamster liver and kidney microsomes into a chemically reactive metabolite that covalently binds to microsomal proteins.

Metabolic activation of the tricyclic antidepressant amineptine--II. Protective role of glutathione against in vitro and in vivo covalent binding.

Incubation of [11-14C]amineptine (1 mM) with an NADPH-generating system and hamster liver microsomes resulted in the in vitro covalent binding of an amineptine metabolite to microsomal proteins; this binding was decreased by 41-71% in the presence of cysteine, lysine, glycine or glutathione (0.5 mM). An inverse relationship was found between the concentration of glutathione in the incubation mixture (0.25-4 mM) and the extent of covalent binding in vitro, which became undetectable at concentrations of glutathione of 2 mM and higher. Administration of [11-14C]amineptine (300 mg/kg-1 i.p.) to hamsters pretreated with phorone (500 mg/kg i.p.) resulted in the in vivo covalent binding of an amineptine metabolite to hepatic proteins. This binding was increased by phenobarbital-pretreatment and decreased by piperonyl butoxide-pretreatment. After various doses of phorone (150-500 mg/kg), an inverse relationship was found between hepatic glutathione content and in vivo covalent binding. Administration of amineptine alone (300 mg/kg i.p.) depleted hepatic glutathione by 16% only; in these animals, in vivo covalent binding was undetectable from background. Amineptine (300 mg/kg i.p.) did not produce hepatic necrosis, even in hamsters pretreated with phorone and/or phenobarbital. We conclude that physiologic concentrations of glutathione essentially prevent the in vivo covalent binding of an amineptine metabolite to hepatic proteins, and that this binding does not produce liver cell necrosis in hamsters.