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Patrick M Dansette - One of the best experts on this subject based on the ideXlab platform.
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Identification of liver protein targets modified by Tienilic Acid metabolites using a two-dimensional Western blot-mass spectrometry approach
International Journal of Mass Spectrometry, 2020Co-Authors: Ruth Menque Methogo, Patrick M Dansette, Klaus KlarskovAbstract:International audienceA combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of Tienilic Acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase
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identification of liver protein targets modified by Tienilic Acid metabolites using a two dimensional western blot mass spectrometry approach
International Journal of Mass Spectrometry, 2007Co-Authors: Ruth Menque Methogo, Patrick M Dansette, Klaus KlarskovAbstract:A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of Tienilic Acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.
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kinetics of Tienilic Acid bioactivation and functional generation of drug protein adducts in intact rat hepatocytes
Biochemical Pharmacology, 2005Co-Authors: Pilar M Lopezgarcia, Patrick M Dansette, Julio ColomaAbstract:Abstract Drug-induced autoimmune hepatitis is among the most severe hepatic idiosyncratic adverse drug reactions. Considered multifactorial, the disease combines immunological and metabolic aspects, the latter being to date much better known. As for many other model drugs, studies on Tienilic Acid (TA)-induced hepatitis have evidenced the existence of bioactivation during the hepatic oxidation of the drug, allowing the identification of the neoantigen of anti-LKM2 autoantibodies and the pathway responsible for its formation. However, most of these results are based on the use of microsomal fractions whose relevance to the liver in vivo still needs to be established. In the more complex intact cell environment, several endogenous processes may play a significant role on triggering the reaction and should therefore be considered. In this work we have characterised the kinetics of TA biotransformation in metabolically competent hepatocytes, the influence of TA bioactivation on physiological GSH levels, and the qualitative and quantitative profile of drug–protein conjugates generated in situ, as a function of exposure time. Results confirm that intact hepatocytes reproduce in vitro the metabolic sequence that leads to the functional generation of drug–protein adducts, in conditions that simulate clinical human exposure to TA. Metabolically competent cultured hepatocytes appear as a very promising approach to investigate the early preimmunological events of drug-induced autoimmune hepatitis, adequate to identify the conditions that may modulate the formation and specificity of drug–protein adducts in vivo, to study the hepatic disposition of the TA-protein targets, and to define the specific role of the hepatocyte in the origin of this adverse reaction.
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substrate selectivity of human cytochrome p450 2c9 importance of residues 476 365 and 114 in recognition of diclofenac and sulfaphenazole and in mechanism based inactivation by Tienilic Acid
Archives of Biochemistry and Biophysics, 2003Co-Authors: Armelle Melet, Patrick M Dansette, Maryse Jaouen, Pascale Jean, Nadine Assrir, Maria Pilar Lopezgarcia, Cristina Marquessoares, Marie Agnes Sari, Daniel MansuyAbstract:Abstract A series of six site-directed mutants of CYP 2C9 were constructed with the aim to better define the amino Acid residues that play a critical role in substrate selectivity of CYP 2C9, particularly in three distinctive properties of this enzyme: (i) its selective mechanism-based inactivation by Tienilic Acid (TA), (ii) its high affinity and hydroxylation regioselectivity toward diclofenac, and (iii) its high affinity for the competitive inhibitor sulfaphenazole (SPA). The S365A mutant exhibited kinetic characteristics for the 5-hydroxylation of TA very similar to those of CYP 2C9; however, this mutant did not undergo any detectable mechanism-based inactivation by TA, which indicates that the OH group of Ser 365 could be the nucleophile forming a covalent bond with an electrophilic metabolite of TA in TA-dependent inactivation of CYP 2C9. The F114I mutant was inactive toward the hydroxylation of diclofenac; moreover, detailed analyses of its interaction with a series of SPA derivatives by difference visible spectroscopy showed that the high affinity of SPA to CYP 2C9 ( K s =0.4 μ M) was completely lost when the phenyl substituent of Phe 114 was replaced with the alkyl group of Ile ( K s =190±20 μ M), or when the phenyl substituent of SPA was replaced with a cyclohexyl group ( K s =120±30 μ M). However, this cyclohexyl derivative of SPA interacted well with the F114I mutant ( K s =1.6±0.5 μ M). At the opposite end, the F94L and F110I mutants showed properties very similar to those of CYP 2C9 toward TA and diclofenac. Finally, the F476I mutant exhibited at least three main differences compared to CYP 2C9: (i) big changes in the kcat and Km values for TA and diclofenac hydroxylation, (ii) a 37-fold increase of the Ki value found for the inhibition of CYP 2C9 by SPA, and (iii) a great change in the regioselectivity of diclofenac hydroxylation, the 5-hydroxylation of this substrate by CYP 2C9 F476I exhibiting a kcat of 28 min −1 . These data indicate that Phe 114 plays an important role in recognition of aromatic substrates of CYP 2C9, presumably via Π-stacking interactions. They also provide the first experimental evidence showing that Phe 476 plays a crucial role in substrate recognition and hydroxylation by CYP 2C9.
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use of isotopes and lc ms esi tof for mechanistic studies of Tienilic Acid metabolic activation
Advances in Experimental Medicine and Biology, 2001Co-Authors: Maya Belghazi, Daniel Mansuy, Pascale Jean, Sonia Poli, Jeanmarie Schmitter, Patrick M DansetteAbstract:Tienilic Acid (TA) is a uricosuric drug marketed in 1978 and which caused a number of rare immunoallergic hepatitis. It was withdrawn in US in 1980, in France in 1992. Early batches of Tienilic Acid also contained 0.1–0.5% Tienilic Acid isomer. Tienilic Acid isomer (TAI), has been shown to be metabolised by Cytochrome P450 into a reactive thiophene 1-oxide which either binds to proteins, or can be trapped by sulfur nucleophiles (Valadon et al. 1996). Tienilic Acid is metabolized by human cytochrome P450 2C9 into 5-hydroxyTienilic Acid (a major metabolite representing 70% of the dose excreted in human urine) but it also forms (a) reactive metabolite(s) which binds covalently to CYP 2C9 and it is a mechanism based inhibitor of CYP 2C9. Adding glutathione to incubations decreases the covalent binding, but only to 1 mol/mol P450. However the reactive metabolite of Tienilic Acid is still unknown. Recently Koenigs et al. have shown using ESI-LC-MS that CYP 2C9 binds ≈ 2 mol of TA in absence of GSH and only one in presence of 3 mM GSH (Koenigs et al. 1999).
Daniel Mansuy - One of the best experts on this subject based on the ideXlab platform.
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substrate selectivity of human cytochrome p450 2c9 importance of residues 476 365 and 114 in recognition of diclofenac and sulfaphenazole and in mechanism based inactivation by Tienilic Acid
Archives of Biochemistry and Biophysics, 2003Co-Authors: Armelle Melet, Patrick M Dansette, Maryse Jaouen, Pascale Jean, Nadine Assrir, Maria Pilar Lopezgarcia, Cristina Marquessoares, Marie Agnes Sari, Daniel MansuyAbstract:Abstract A series of six site-directed mutants of CYP 2C9 were constructed with the aim to better define the amino Acid residues that play a critical role in substrate selectivity of CYP 2C9, particularly in three distinctive properties of this enzyme: (i) its selective mechanism-based inactivation by Tienilic Acid (TA), (ii) its high affinity and hydroxylation regioselectivity toward diclofenac, and (iii) its high affinity for the competitive inhibitor sulfaphenazole (SPA). The S365A mutant exhibited kinetic characteristics for the 5-hydroxylation of TA very similar to those of CYP 2C9; however, this mutant did not undergo any detectable mechanism-based inactivation by TA, which indicates that the OH group of Ser 365 could be the nucleophile forming a covalent bond with an electrophilic metabolite of TA in TA-dependent inactivation of CYP 2C9. The F114I mutant was inactive toward the hydroxylation of diclofenac; moreover, detailed analyses of its interaction with a series of SPA derivatives by difference visible spectroscopy showed that the high affinity of SPA to CYP 2C9 ( K s =0.4 μ M) was completely lost when the phenyl substituent of Phe 114 was replaced with the alkyl group of Ile ( K s =190±20 μ M), or when the phenyl substituent of SPA was replaced with a cyclohexyl group ( K s =120±30 μ M). However, this cyclohexyl derivative of SPA interacted well with the F114I mutant ( K s =1.6±0.5 μ M). At the opposite end, the F94L and F110I mutants showed properties very similar to those of CYP 2C9 toward TA and diclofenac. Finally, the F476I mutant exhibited at least three main differences compared to CYP 2C9: (i) big changes in the kcat and Km values for TA and diclofenac hydroxylation, (ii) a 37-fold increase of the Ki value found for the inhibition of CYP 2C9 by SPA, and (iii) a great change in the regioselectivity of diclofenac hydroxylation, the 5-hydroxylation of this substrate by CYP 2C9 F476I exhibiting a kcat of 28 min −1 . These data indicate that Phe 114 plays an important role in recognition of aromatic substrates of CYP 2C9, presumably via Π-stacking interactions. They also provide the first experimental evidence showing that Phe 476 plays a crucial role in substrate recognition and hydroxylation by CYP 2C9.
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use of isotopes and lc ms esi tof for mechanistic studies of Tienilic Acid metabolic activation
Advances in Experimental Medicine and Biology, 2001Co-Authors: Maya Belghazi, Daniel Mansuy, Pascale Jean, Sonia Poli, Jeanmarie Schmitter, Patrick M DansetteAbstract:Tienilic Acid (TA) is a uricosuric drug marketed in 1978 and which caused a number of rare immunoallergic hepatitis. It was withdrawn in US in 1980, in France in 1992. Early batches of Tienilic Acid also contained 0.1–0.5% Tienilic Acid isomer. Tienilic Acid isomer (TAI), has been shown to be metabolised by Cytochrome P450 into a reactive thiophene 1-oxide which either binds to proteins, or can be trapped by sulfur nucleophiles (Valadon et al. 1996). Tienilic Acid is metabolized by human cytochrome P450 2C9 into 5-hydroxyTienilic Acid (a major metabolite representing 70% of the dose excreted in human urine) but it also forms (a) reactive metabolite(s) which binds covalently to CYP 2C9 and it is a mechanism based inhibitor of CYP 2C9. Adding glutathione to incubations decreases the covalent binding, but only to 1 mol/mol P450. However the reactive metabolite of Tienilic Acid is still unknown. Recently Koenigs et al. have shown using ESI-LC-MS that CYP 2C9 binds ≈ 2 mol of TA in absence of GSH and only one in presence of 3 mM GSH (Koenigs et al. 1999).
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opposite behaviors of reactive metabolites of Tienilic Acid and its isomer toward liver proteins use of specific anti Tienilic Acid protein adduct antibodies and the possible relationship with different hepatotoxic effects of the two compounds
Chemical Research in Toxicology, 1999Co-Authors: Eric Bonierbale, Patrick M Dansette, Philippe Valadon, Catherine Pons, Bernard Desfosses, Daniel MansuyAbstract:Tienilic Acid (TA) is responsible for an immune-mediated drug-induced hepatitis in humans, while its isomer (TAI) triggers a direct hepatitis in rats. In this study, we describe an immunological approach developed for studying the specificity of the covalent binding of these two compounds. For this purpose, two different coupling strategies were used to obtain TA−carrier protein conjugates. In the first strategy, the drug was linked through its carboxylic Acid function to amine residues of carrier proteins (BSA−N−TA and casein−N−TA), while in the second strategy, the thiophene ring of TA was attached to proteins through a short 3-thiopropanoyl linker, the corresponding conjugates (BSA−S−5-TA and βLG−S−5-TA) thus preferentially presenting the 2,3-dichlorophenoxyacetic moiety of the drug for antibody recognition. The BSA−S−5-TA conjugate proved to be 30 times more immunogenic than BSA−N−TA. Anti-TA−protein adduct antibodies were obtained after immunization of rabbits with BSA−S−5-TA (1/35000 titer against β...
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oxidation of Tienilic Acid by human yeast expressed cytochromes p 450 2c8 2c9 2c18 and 2c19
FEBS Journal, 1996Co-Authors: Pascale Jean, Patrick M Dansette, Daniel Mansuy, Pilar Lopezgarcia, Joyce L GoldsteinAbstract:Oxidation of Tienilic Acid by human cytochromes P-450 (CYP) 2C9, 2C18, 2C8 and 2C19 was studied using recombinant enzymes expressed in yeast. CYP 2C9 was the best catalyst for 5-hydroxylation of Tienilic Acid (Km= 5 ±1 μM, kcat= 1.7 ± 0.2 min−1), 30-fold more potent in terms of kcat/Km than CYP 2C18 (Km= 150 ± 15 μM, kcat= 1.8 ± 0.2 min−1 and 300-fold more potent than CYP 2C8 (Km= 145 ± 15 μM, kcat= 0.2 ± 0.1 min−1). CYP 2C19 was unable to catalyze this hydroxylation under our experimental conditions. During this study, a marked effect of the ionic strength on the activities (hydroxylations of Tienilic Acid and tolbutamide) of these cytochromes P-450 expressed in the yeast strain 334 was observed. The effect was particularly great in the case of CYP 2C18, with a tenfold decrease of activity upon increasing ionic strength from 0.02 to 0.1. Specific-covalent binding of Tienilic Acid metabolites to cytochrome P-450 (incubations in the presence of 5 mM glutathione) was markedly higher upon Tienilic Acid oxidation by CYP 2C9 than by CYP 2C18 and CYP 2C8. Mechanism-based inactivation of cytochrome P-450 during Tienilic Acid oxidation was observed in the case of CYP 2C9 but was not detectable with CYP 2C18 and CYP 2C8. Tienilic Acid thus appears to be a mechanism-based inhibitor specific for CYP 2C9 in human liver. Experiments performed with human liver microsomes confirmed that Tienilic Acid 5-hydroxylase underwent a time-dependent inactivation (apparent t½= 10 ± 5 min) during 5-hydroxylation of Tienilic Acid.
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NEW BIOLOGICAL REACTIVE INTERMEDIATES Metabolic Activation of Thiophene Derivatives
Advances in Experimental Medicine and Biology, 1996Co-Authors: Daniel Mansuy, Patrick M DansetteAbstract:Metabolic activation of benzene rings as well as that of many aromatic heterocycles such as furans generally involves the intermediate formation of arene oxides as electrophilic reactive metabolites. Much less was known until very recently about the metabolic oxidative activation of thiophene derivatives (Rance, 1989). A first report on the metabolism of thiophene itselfin rats has shown the formation of thiophene-derived mercapturates in urine and proposed the intermediate formation of a thiophene epoxide (Bray et ai., 1971). More recently, it has been shown that the major oxidized metabolites of severaI2-aroylthiophenes, such as Tienilic Acid (Mansuy et ai., 1984) were derived from 5-hydroxylation of their thiophene ring (Neau et ai., 1990). Our interest in the detailed mechanism of the metabolic activation of thiophene derivatives came from a study of the origin of the hepatotoxic effects of a thiophene-containing drug, Tienilic Acid (TA) (Fig. I) and of its isomer TAl (Fig. 2).
Philippe Beaune - One of the best experts on this subject based on the ideXlab platform.
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Tienilic Acid induced autoimmune hepatitis anti liver and kidney microsomal type 2 autoantibodies recognize a three site conformational epitope on cytochrome p4502c9
Molecular Pharmacology, 1996Co-Authors: S Lecoeur, C Andre, Philippe BeauneAbstract:Tienilic Acid-induced hepatitis is characterized by the presence of anti-liver and -kidney microsomal (anti-LKM2) autoantibodies in patient sera. Cytochrome P4502C9(CYP2C9), involved in the metabolism of Tienilic Acid, was shown to be a target for Tienilic Acid-reactive metabolites and for autoantibodies. To further investigate the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, the specificity of anti-LKM2 autoantibodies toward CYP2C9 was first determined, and the antigenic sites on CYP2C9 were localized. By constructing several deletion mutants derived from CYP2C9 cDNA and by probing the corresponding proteins with different anti-LKM2 sera, we defined three regions (amino Acids 314-322, 345-356, and 439-455); they interacted to form a major conformational autoantibody binding site. This binding site was immunoreactive with 100% of sera and allowed removal of the entire reactivity of the sera tested by immunoblotting. Epitope mapping studies have been performed for CYP2D6, CYP17, CYP21A2, and, recently, CYP3A. Those data were compared with the results obtained in the current study with CYP2C9 in an attempt to elucidate one of the mechanisms by which CYP becomes immunogenic.
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specificity of in vitro covalent binding of Tienilic Acid metabolites to human liver microsomes in relationship to the type of hepatotoxicity comparison with two directly hepatotoxic drugs
Chemical Research in Toxicology, 1994Co-Authors: Sylvaine Lecoeur, Eric Bonierbale, Patrick M Dansette, F Ballet, Daniel Mansuy, Philippe Valadon, Dominique Challine, Jeancharles Gautier, Rachel Catinot, Philippe BeauneAbstract:: In order to better understand the first steps leading to drug-induced immunoallergic hepatitis, we studied the target of anti-LKM2 autoantibodies appearing in Tienilic Acid-induced hepatitis, and the target of Tienilic Acid-reactive metabolites. It was identified as cytochrome P450 2C9, (P450 2C9): indeed, anti-LKM2 specifically recognized P450 2C9, but none of the other P450s tested (including other 2C subfamily members, 2C8 and 2C18). Tienilic Acid-reactive metabolite(s) specifically bound to P450 2C9, and experiments with yeast expressing active isolated P450s showed that P450 2C9 was responsible for Tienilic Acid-reactive metabolite(s) production. Results of qualitative and quantitative covalent binding of Tienilic Acid metabolite(s) to human liver microsomes were then compared to those obtained with two drugs leading to direct toxic hepatitis, namely, acetaminophen and chloroform. Kinetic constants (Km and Vmax) were measured, and the covalent binding profile of the metabolites to human liver microsomal proteins was studied. Tienilic Acid had both the lowest Km and the highest covalent binding rate at pharmacological doses. For acetaminophen and chloroform, several microsomal proteins were covalently bound, while covalent binding was highly specific for Tienilic Acid and dihydralazine, another drug leading to immunoallergic hepatitis. Although low numbers of drugs were tested, these results led us to think that there may exist a relationship between the specificity of covalent binding and the type of hepatotoxicity.
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human liver cytochromes p 450 expressed in yeast as tools for reactive metabolite formation studies oxidative activation of Tienilic Acid by cytochromes p 450 2c9 and 2c10
FEBS Journal, 1993Co-Authors: Pilar Lopez M Garcia, Patrick M Dansette, Philippe Valadon, Claudine Amar, Philippe Beaune, Peter F Guengerich, D MansuyAbstract:Human liver cytochromes P-450 (P450) 2C9 and 2C10 expressed in yeast reproduce all the metabolic features of the oxidation of Tienilic Acid (2-aryloxo-thiophene) and its isomer (3-aroyl-thiophene) by human liver microsomes. Microsomes of yeast expressing either P450 2C9 or P450 2C10 catalyze (a) the 5-hydroxylation of Tienilic Acid by NADPH and O2 (Km= 6 μM, Vmax= 2.5 turnover/min), (b) the activation of Tienilic Acid and its isomer into electrophilic metabolites which covalently bind to proteins, and (c) the formation of a mercaptoethanol adduct which results from the trapping of the Tienilic Acid isomer sulfoxide by this thiol. Microsomes of yeast expressing human liver P450 3A4, 1A1 and 1A2 are unable to catalyze these reactions. There is a striking similarity between the quantitative characteristics of the oxidation of Tienilic Acid (and its isomer) by yeast-expressed P450 2C9 (or 2C10) and by human liver microsomes: (a) analogous Km values (around 10 μM) for Tienilic Acid 5-hydroxylation, (b) a strong inhibition of Tienilic Acid oxidation by human sera containing anti-(liver kidney microsomes type 2) (anti-LKM2) antibodies, and (c) almost identical relative ratios of Tienilic Acid metabolic activation/5-hydroxylation and of Tienilic Acid activation/the activation of its isomer with both systems. Rates of oxidation of Tienilic Acid (and its isomer) by yeast microsomes are 6–8-fold higher than those found in human liver microsomes, which would be in agreement with the previously reported amount of P450 2C9 in human liver. These results not only suggest the important role of P450 2C9 in the oxidative metabolism of Tienilic Acid in human liver, but also indicate that the 5-hydroxylation reaction could be a useful marker for P450 2C9 activity and underline the interest of human liver P450s expressed in yeast as tools for studying the formation of reactive metabolites.
D Mansuy - One of the best experts on this subject based on the ideXlab platform.
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antigenic targets in Tienilic Acid hepatitis both cytochrome p450 2c11 and 2c11 Tienilic Acid adducts are transported to the plasma membrane of rat hepatocytes and recognized by human sera
Journal of Clinical Investigation, 1996Co-Authors: Marieanne Robin, Michel Maratrat, F Le P Breton, Eric Bonierbale, Patrick M Dansette, F Ballet, D Mansuy, Dominique PessayreAbstract:Patients with Tienilic Acid hepatitis exhibit autoantibodies that recognize unalkylated cytochrome P450 2C9 in humans but recognize 2C11 in rats. Our aim was to determine whether the immune reaction is also directed against neoantigens. Rats were treated with Tienilic Acid and hepatocytes were isolated. Immunoprecipitation, immunoblotting, and flow cytometry experiments were performed with an anti-Tienilic Acid or an anti-cytochrome P450 2C11 antibody. Cytochrome P450 2C11 was the main microsomal or plasma membrane protein that was alkylated by Tienilic Acid. Inhibitors of vesicular transport decreased flow cytometric recognition of both unalkylated and Tienilic Acid-alkylated cytochrome P450 2C11 on the plasma membrane of cultured hepatocytes. Tienilic Acid hepatitis sera that were preadsorbed on microsomes from untreated rats (to remove autoantibodies), poorly recognized untreated hepatocytes in flow cytometry experiments, but better recognized Tienilic Acid-treated hepatocytes. This recognition was decreased by adsorption with Tienilic Acid or by preexposure to the anti-Tienilic Acid or the anti-cytochrome P450 2C11 antibody. We conclude that cytochrome P450 2C11 is alkylated by Tienilic Acid and follows a vesicular route to the plasma membrane. Tienilic Acid hepatitis sera contain antibodies against this Tienilic Acid adduct, in addition to the previously described anticytochrome P450 autoantibodies.
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human liver cytochromes p 450 expressed in yeast as tools for reactive metabolite formation studies oxidative activation of Tienilic Acid by cytochromes p 450 2c9 and 2c10
FEBS Journal, 1993Co-Authors: Pilar Lopez M Garcia, Patrick M Dansette, Philippe Valadon, Claudine Amar, Philippe Beaune, Peter F Guengerich, D MansuyAbstract:Human liver cytochromes P-450 (P450) 2C9 and 2C10 expressed in yeast reproduce all the metabolic features of the oxidation of Tienilic Acid (2-aryloxo-thiophene) and its isomer (3-aroyl-thiophene) by human liver microsomes. Microsomes of yeast expressing either P450 2C9 or P450 2C10 catalyze (a) the 5-hydroxylation of Tienilic Acid by NADPH and O2 (Km= 6 μM, Vmax= 2.5 turnover/min), (b) the activation of Tienilic Acid and its isomer into electrophilic metabolites which covalently bind to proteins, and (c) the formation of a mercaptoethanol adduct which results from the trapping of the Tienilic Acid isomer sulfoxide by this thiol. Microsomes of yeast expressing human liver P450 3A4, 1A1 and 1A2 are unable to catalyze these reactions. There is a striking similarity between the quantitative characteristics of the oxidation of Tienilic Acid (and its isomer) by yeast-expressed P450 2C9 (or 2C10) and by human liver microsomes: (a) analogous Km values (around 10 μM) for Tienilic Acid 5-hydroxylation, (b) a strong inhibition of Tienilic Acid oxidation by human sera containing anti-(liver kidney microsomes type 2) (anti-LKM2) antibodies, and (c) almost identical relative ratios of Tienilic Acid metabolic activation/5-hydroxylation and of Tienilic Acid activation/the activation of its isomer with both systems. Rates of oxidation of Tienilic Acid (and its isomer) by yeast microsomes are 6–8-fold higher than those found in human liver microsomes, which would be in agreement with the previously reported amount of P450 2C9 in human liver. These results not only suggest the important role of P450 2C9 in the oxidative metabolism of Tienilic Acid in human liver, but also indicate that the 5-hydroxylation reaction could be a useful marker for P450 2C9 activity and underline the interest of human liver P450s expressed in yeast as tools for studying the formation of reactive metabolites.
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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
Biochemical Pharmacology, 1991Co-Authors: Patrick M Dansette, Claudine Amar, P Valadon, C Pons, Ph Beaune, D MansuyAbstract:Abstract 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 ( K i ≅ 25 μ M) 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.
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oxidative activation of the thiophene ring by hepatic enzymes hydroxylation and formation of electrophilic metabolites during metabolism of Tienilic Acid and its isomer by rat liver microsomes
Biochemical Pharmacology, 1990Co-Authors: Patrick M Dansette, C Pons, C Amar, C Smith, D MansuyAbstract:Tienilic Acid (TA) is metabolized by liver microsomes from phenobarbital-treated rats in the presence of NADPH with the major formation of 5-hydroxyTienilic Acid (5-OHTA) which is derived from the regioselective hydroxylation of the thiophene ring of TA. During this in vitro metabolism of TA, reactive electrophilic intermediates which bind irreversibly to microsomal proteins are formed. 5-Hydroxylation of TA and activation of TA to reactive metabolites which covalently bind to proteins both required intact microsomes, NADPH and O2 and are inhibited by metyrapone and SKF 525A, indicating that they are dependent on monooxygenases using cytochromes P-450. Microsomal oxidation of an isomer of Tienilic Acid (TAI) bearing the aroyl substituent on position 3 (instead of 2) of the thiophene ring also leads to reactive intermediates able to bind covalently to microsomal proteins. Covalent binding of TAI, as that of TA, depends on cytochrome P-450-dependent monooxygenases and is almost completely inhibited in the presence of sulfur containing nucleophiles such as glutathione, cysteine or cyteamine. These results show that 5-OHTA, which has been reported as the major metabolite of TA in vivo in humans, is formed by liver microsomes by a cytochrome P-450-dependent reaction. They also show that two thiophene derivatives, TA and TAI, bind to microsomal proteins after in vitro metabolic activation, TAI giving a much higher level of covalent binding than TA (about 5-fold higher) and a much higher covalent binding: stable metabolites ratio (4 instead of 0.5).
Sunao Manabe - One of the best experts on this subject based on the ideXlab platform.
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involvement of cytochrome p450 mediated metabolism in Tienilic Acid hepatotoxicity in rats
Toxicology Letters, 2008Co-Authors: Takayoshi Nishiya, Michiyuki Kato, Takami Suzuki, Chikako Maru, Hiroko Kataoka, Chiharu Hattori, Kazuhiko Mori, Toshimasa Jindo, Yorihisa Tanaka, Sunao ManabeAbstract:Abstract Tienilic Acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that Tienilic Acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l -buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-Tienilic Acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of Tienilic Acid (300 mg/kg). Furthermore, a Tienilic Acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66 mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and Tienilic Acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of Tienilic Acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of Tienilic Acid under impaired GSH biosynthesis.
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the crucial protective role of glutathione against Tienilic Acid hepatotoxicity in rats
Toxicology and Applied Pharmacology, 2008Co-Authors: Takayoshi Nishiya, Hiroko Kataoka, Chiharu Hattori, Kazuhiko Mori, Toshimasa Jindo, Noriko Masubuchi, Sunao ManabeAbstract:Abstract To investigate the hepatotoxic potential of Tienilic Acid in vivo , we administered a single oral dose of Tienilic Acid to Sprague–Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile Acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S -transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3–6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by Tienilic Acid. In a subsequent experiment, Tienilic Acid in combination with l -buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of Tienilic Acid as the upregulation of the Nrf2-regulated genes by Tienilic Acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of Tienilic Acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.
