Patrick M. Dansette

Hepatotoxicity of germander in mice

BACKGROUND/AIMSnAn 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.nnnMETHODSnA germander tea lyophilisate and a fraction that isolated and concentrated 10-fold the furano neo-clerodane diterpenoids of the lyophilisate were prepared.nnnRESULTSn(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.nnnCONCLUSIONSnWe 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.

Autoantibodies against Cytochromes P450: Role in Human Diseases

Publisher Summary Autoantibodies directed against several cytochromes P450 (P450) were found in the sera of patients suffering from several types of diseases, all of which seemed to be caused by an abnormal immunological response. This chapter considers two classes of disease: (1) Those for which it has not been possible to identify a xenobiotic as the cause of the disease—namely, idiopathic autoimmune hepatitis, and Addisons disease; and (2) those for which a drug was clearly identified as the causative agent—namely, tienilic acid-induced hepatitis, and dihydralazine-induced hepatitis. For each of these diseases, the chapter presents the clinical aspects, the identification of the P450 involved, the metabolism of the drug if it has been identified, and possible mechanisms leading to the production of these antibodies. The chapter concludes with possible avenues for future research in this field, where many points still remain obscure.

Drug-induced immunotoxicity.

SummaryImmune-related drug responses are one of the most common sources of idiosyncratic toxicity. A number of organs may be the target of such reactions; however, this review concentrates mostly on the liver. Drug-induced hepatitis is generally divided into two categories: acute hepatitis in which the drug or a metabolite destroys a vital target in the cell; immunoallergic hepatitis in which the drug triggers an adverse immune response directed against the liver. Their clinical features are: a) low frequency; b) dose independence; c) typical immune system manifestations such as fever, eosinophilia; d) delay between the initiation of treatment and onset of the disease; e) a shortened delay upon rechallenge; and f) occasional presence of autoantibodies in the serum of patients. Such signs have been found in cases of hepatitis triggered by drugs such as halothane, tienilic acid, dihydralazine and anticonvulsants. They will be taken as examples to demonstrate the recent progress made in determining the mechanisms responsible for the disease. The following mechanisms have been postulated: 1) the drug is first metabolized into a reactive metabolite which binds to the enzyme that generated it; 2) this produces a neoantigen which, once presented to the immune system, might trigger an immune response characterized by 3) the production of antibodies recognizing both the native and/or the modified protein; 4) rechallenge leads to increased neoantigen production, a situation in which the presence of antibodies may induce cytolysis. Toxicity is related to the nature and amount of neoantigen and also to other factors such as the individual immune system. An effort should be made to better understand the precise mechanisms underlying this kind of disease and thereby identify the drugs at risk; and also the neoantigen processes necessary for their introduction into the immune system. An animal model would be useful in this regard.

Hydroxylation and formation of electrophilic metabolites of tienilic acid and its isomer by human liver microsomes: Catalysis by a cytochrome P450 IIC different from that responsible for mephenytoin hydroxylation

Tienilic acid (TA) is metabolized by human liver microsomes in the presence of NADPH with the major formation of 5-hydroxytienilic acid (5-OHTA) which is derived from the hydroxylation of the thiophene ring of TA. Besides this hydroxylation, TA is oxidized into reactive metabolites which covalently bind to microsomal proteins. Oxidation of an isomer of tienilic acid (TAI), bearing the aroyl substituent on position 3 (instead of 2) of the thiophene ring, by human liver microsomes, gives a much higher level of covalent binding to proteins. Both covalent binding of TA and TAI metabolites are almost completely suppressed in the presence of glutathione. These three activities of human liver microsomes (TA 5-hydroxylation, covalent binding of TA and TAI metabolites) seem dependent on the same cytochrome P450 of the IIC subfamily, since (i) antibodies against human liver cytochromes P450 IIC strongly inhibit these three activities, (ii) there is a clear correlation between these activities in various human liver microsomes, and (iii) TA acts as a competitive inhibitor for TAI activation into electrophilic metabolites (Ki approximately equal to 25 microM) and TAI inhibits TA 5-hydroxylation. However cross inhibition experiments indicate that tienilic acid hydroxylation and mephenytoin hydroxylation, a typical reaction of some human liver P450 IIC isoenzymes, are not catalysed by the same member of the P450 IIC subfamily.

Paraoxonase-1 and clopidogrel efficacy

Microsomal Incubations: Human microsomes (pool, 10 mg protein/mL) were obtained from BD-Gentest (Le Pont de Claix, France). Typical incubations were performed in potassium phosphate buffer (0.1M, pH 7.4) containing 2 mM CaCl2, 100 mM KF, microsomes (1 mg protein/mL), 2-oxo-clopidogrel (100 μM) and a reducing agent (20 mM ascorbic acid, or 0.5 mM dithiothreitol, or 5 mM glutathione (GSH)) with or without NADPH generating system (1 mM NADP, 15 mM glucose-6-phosphate, 2 u/mL of glucose-6-phosphate dehydrogenase) at 37°C for 30 min. Reactions were stopped by adding one half volume of CH3CN: CH3COOH (9:1) and proteins were removed by centrifugation at 13000g.

Mechanism of catalysis for the hydration of substituted styrene oxides by hepatic epoxide hydrase

Abstract The effect of aryl substituents on the rate at which epoxide hydrase catalyzes the addition of water to styrene and cis -stilbene oxides has been examined. Plots of log V m for each substrate versus the Hammett σ constants for the substituent suggest that a nucleophilic attack occurs and that a free carbonium ion form of the substrate is not involved at the rate-determining step in the mechanism. For the stilbene oxides, high selectivity for attack by water at the carbon atom with [S] absolute stereochemistry was observed.

Assay and partial purification of epoxide hydrase from rat liver microsomes

Abstract Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [3H]styrene oxide, [3H]naphthalene oxide, [3H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [3H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [3H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.

UPD-glucuronosyltransferase, epoxide hydrolase and gultathione S-transferase

Abstract The activities of UDPglucuronosyltransferase, microsomal epoxide hydrolas and cytosolic glutathione S-transferase were measured in the liver of spontaneously (db/db and ob/ob) or streptozotocin-induced diabetic mice. An important (2–3-fold) increase of most phase II activities was observed instreptozotocin-treated animals, whereas sligher changes were detected in spontaneously diabetic animals. The latter exhibit physico-chemical modifications of liver microsomal membranes, as shown by the temperature-induced variations of epoxide hydrolase activity.

Automated multiple analysis of protein structures: Application to homology modeling of cytochromes P450

A computational strategy for homology modeling, using several protein structures comparison, is described. This strategy implies a formalized definition of structural blocks common to several protein structures, a new program to compare these structures simultaneously, and the use of consensus matrices to improve sequence alignment between the structurally known and target proteins. Applying this method to cytochromes P450 led to the definition of 15 substructures common to P450cam, P450BM3, and P450terp, and to proposing a 3D model of P450eryF. Proteins 28:388–404, 1997

Metabolism and Biological Activity of Benzo[a]pyrene and Its Metabolic Products

The carcinogenicity of benzo[a]pyrene (BP) and its widespread occurrence as an environmental pollutant have prompted numerous studies on the metabolic fate and biological activity of this hydrocarbon. Our laboratories in Nutley and Bethesda have jointly undertaken a systematic study of the metabolic fate and biological activity of BP and its many metabolites. The goal of this research program is to elucidate the molecular events that result in the metabolic activation of BP and to identify the proximate and ultimate carcinogenic forms of this environmental carcinogen. This chapter is a progress report that describes (1) a high-pressure liquid chromatography system for the separation of BP dihydrodiols, BP phenols, and BP quinones; (2) the metabolism of BP by a solubilized and reconstituted cytochrome P448 monooxygenase system in the presence and absence of purified epoxide hydrase; (3) the metabolic activation of BP to mutagenic metabolites by a cytochrome P448 or cytochrome P450 reconstituted monooxygenase system; (4) the effect of epoxide hydrase on the cytochrome P448-mediated activation of BP to mutagenic metabolites; (5) the mutagenic activity of 28 synthetic derivatives of BP; and (6) the carcinogenicity of three BP oxides on mouse skin.