Jacqueline Loeper

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.

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.

Enrichment of cholesterol in microdissected Alzheimer's disease senile plaques as assessed by mass spectrometry

Extensive knowledge of the protein components of the senile plaques, one of the hallmark lesions of Alzheimers disease, has been acquired over the years, but their lipid composition remains poorly known. Evidence suggests that cholesterol contributes to the pathogenesis of Alzheimers disease. However, its presence within senile plaques has never been ascertained with analytic methods. Senile plaques were microdissected from sections of the isocortex in three Braak VI Alzheimers disease cases and compared with a similar number of samples from the adjoining neuropil, free of amyloid-β peptide (Aβ) deposit. Two cases were apoε4/apoε3, and one case was apoε3/apoε3. A known quantity of 13C-labeled cholesterol was added to the samples as a standard. After hexane extraction, cholesterol content was analyzed by liquid chromatography coupled with electrospray ionization mass spectrometry. The mean concentration of free cholesterol was 4.25 ± 0.1 attomoles/µm3 in the senile plaques and 2.2 ± 0.49 attomoles/µm3 in the neuropil (t = 4.41, P < 0.0009). The quantity of free cholesterol per senile plaque (67 ± 16 femtomol) is similar to the published quantity of Aβ peptide. The highly significant increase in the cholesterol concentration, associated with the increased risk of Alzheimers disease linked to the apoε4 allele, suggests new pathogenetic mechanisms.

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.

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.

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.

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.

Sequence-independent construction of ordered combinatorial libraries with predefined crossover points.

Combinatorial libraries coding for mosaic enzymes with predefined crossover points constitute useful tools to address and model structure-function relationships and for functional optimization of enzymes based on multivariate statistics. The presented method, called sequence-independent generation of a chimera-ordered library (SIGNAL), allows easy shuffling of any predefined amino acid segment between two or more proteins. This method is particularly well adapted to the exchange of protein structural modules. The procedure could also be well suited to generate ordered combinatorial libraries independent of sequence similarities in a robotized manner. Sequence segments to be recombined are first extracted by PCR from a single-stranded template coding for an enzyme of interest using a biotin-avidin-based method. This technique allows the reduction of parental template contamination in the final library. Specific PCR primers allow amplification of two complementary mosaic DNA fragments, overlapping in the region to be exchanged. Fragments are finally reassembled using a fusion PCR. The process is illustrated via the construction of a set of mosaic CYP2B enzymes using this highly modular approach.

Lipidomic characterization of postmortem amyloid plaques isolated by laser capture microdissection

Background: Recent evidence suggests a role for APP processing in the regulation of apoE and cholesterol endocytosis. We have previously demonstrated that the entire cholesterol biosynthesis pathway is upregulated in Tg2576 mouse brain. Tg2576 mice over-express the APPswe mutation and accumulate the BACE1 cleavage products, C99 and Ab. Methods: It is well known that impairment of cholesterol endocytosis increases cholesterol biosynthesis gene expression. We therefore determined whether apoE endocytosis was dysregulated in Tg2576. Results: We found that extracellular cholesterol levels and apoE protein levels were significantly increased in young Tg2576 brain, implying that apoE lipoprotein particles are not being endocytosed and degraded in vivo. By directly examining the effect of APPswe overexpression on lipoprotein endocytosis in vitro we found that primary neurons from Tg2576 showed a dramatic deficit in lipoprotein endocytosis relative to wild type (WT) neurons. Given that Tg2576 cultures massively accumulate both C99 and Ab we wished to determine which cleavage product mediated the inhibitory effects on endocytosis. To directly test this, we treated WT neurons with a g-secretase inhibitor to increase intracellular C99, but reduce Ab levels. Inhibition of g-secretase led to a dose dependant inhibition of lipoprotein endocytosis. This inhibition could be blocked when co-dosed with a b-secretase inhibitor. Importantly, Ab was inhibited > 95 % by the g-secretase inhibitor alone, implying that it was the accumulation of C99 that inhibits lipoprotein endocytosis. Conclusions: These experimental results suggest a role for the amyloidogenic processing of APP in regulating apoE lipoprotein endocytosis and provide further evidence in support of the growing association between Alzheimer’s disease and cholesterol metabolism. Moreover they further establish a biological link between APP, g-secretase and apoE, the major genetic determinants of Alzheimer’s disease.