Use of Isotopes and LC-MS-ESI-TOF for Mechanistic Studies of Tienilic Acid Metabolic Activation

Belghazi, Maya; Jean, P.; Poli, Sonia; Schmitter, Jean‐Marie; Mansuy, Daniel; Dansette, M. Patrick

doi:10.1007/978-1-4615-0667-6_17

Cited by 19 publications

(24 citation statements)

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“…These observations implied that M10 and M8 were derived from a common reactive intermediate. The evidence for oxidative metabolic activation of MP at the thiophene ring, derived from the characterization of M8, immediately suggested two possibilities for the identity of M10: 1) that it was by analogy with a major metabolite of tienilic acid (Belghazi et al, 2001) 5-hydroxythiophene MP formed by rearrangement of either MP S-oxide or the 4,5-epoxide, or 2) that it was by analogy with a metabolite of 2-phenylthiophene ) the cycloadditive dimer of MP S-oxide. However, neither of these possibilities was confirmed by LC-MS analyses of hepatic microsomal incubations of MP.…”

Section: Downloaded Frommentioning

confidence: 99%

“…The results of these deuterium exchange experiments can be tested against the three proposed mechanisms of oxidative bioactivation of monocyclic 2-thiophenes (Kalgutkar et al, 2005): S-oxidation (Belghazi et al, 2001;Dansette et al, 2005), 4,5-epoxidation without opening of the thiophene ring (O'Donnell et al, 2003;Dansette et al, 2005), and 4,5-epoxidation with concerted opening of the epoxide and thiophene rings to produce a ␥-thioketo-␣,␤-unsaturated aldehyde (O'Donnell et al, 2003) (Fig. 12).…”

Section: Downloaded Frommentioning

confidence: 99%

“…Tienilic acid (Belghazi et al, 2001) and its 3-thiophene isomer (Valadon et al, 1996) are reported to be metabolized to S-oxides by hepatic microsomes. Tienilic acid forms numerous protein adducts in rat hepatocytes, although no GSH adduct has been found (López-García et al, 2005).…”

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confidence: 99%

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Identification of the Thiophene Ring of Methapyrilene as a Novel Bioactivation-Dependent Hepatic Toxicophore

Graham¹,

Walsh²,

Hirst³

et al. 2008

J Pharmacol Exp Ther

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Methapyrilene (MP), a 2-thiophene H 1 -receptor antagonist, is a model toxicant in the genomic and proteomic analyses of hepatotoxicity. In rats, it causes an unusual periportal necrosis that is hypothetically attributed to chemically reactive and cytotoxic metabolites. We have characterized the bioactivation of MP by hepatic microsomes and primary rat hepatocytes, and we established a possible causal linkage with cytotoxicity. Methapyrilene tritiated at C-2 of the diaminoethane moiety ([ 3 H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat Ͼ mouse Ϸ human). This binding was attenuated by the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole and thiols but not by trapping agents for iminium ions and aldehydes. Reactive intermediates were trapped as thioether adducts of monooxygenated MP. Mass spectrometric and hydrogen/deuterium exchange analysis of the glutathione adduct produced by rat liver microsomes indicated that the metabolite was most probably a thioether of MP S-oxide substituted in the thiophene ring. The glutathione adduct was formed by rat hepatocytes and eliminated in bile by rats administered [ 3 H]MP intravenously. MP produced concentration-and time-dependent cytotoxicity, depleted glutathione, and underwent irreversible binding to the hepatocytes before a significant increase in cell damage was observed. P450 inhibitors reduced turnover of the drug, production of the glutathione adduct, irreversible binding, and cytotoxicity but inhibited glutathione depletion selectively. MP underwent lesser turnover and bioactivation in mouse hepatocytes and was not cytotoxic. Analogs with phenyl and p-methoxyphenyl rings were much less hepatocytotoxic than MP. Hepatotoxicity in rats was diminished by predosing with 1-aminobenzotriazole. For the first time, a thiophene ring substituent is identified as a bioactivation-dependent toxicophore in hepatocytes. Fig. 1], a 2-thiophene H 1 -receptor antagonist, was not associated with hepatotoxicity in humans, but it causes unusual dose-dependent periportal damage in adult male rats (Graichen et al., 1985;Ratra et al., 1998a;Ratra et al., 2000). Typical hepatotoxic regimens (150 -300 mg/kg/day ϫ 3) produce a mild to moderate injury characterized by hepatocellular necrosis, bile duct proliferation, and inflammatory cell infiltration. MP is also toxic to isolated rat hepatocytes (McQueen and Williams, 1982;Ratra et al., 1998b), which are reported to be more susceptible than mouse hepatocytes (Kelly et al., 1992).MP has come to be regarded as a model investigatory compound among drug hepatotoxicants, and it is used fre- Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.135483.ABBREVIATIONS: MP, methapyrilene; P450, cytochrome P450; tGSH, total glutathione (GSH plus glutathione disulfide); MeOD, monodeuteromethanol; HPLC, high-performance liquid chromatography; [ 3 H]MP, methapyrilene tritiated at C-2 of the diaminoethane moie...

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Section: Downloaded Frommentioning

confidence: 99%

Section: Downloaded Frommentioning

confidence: 99%

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Identification of the Thiophene Ring of Methapyrilene as a Novel Bioactivation-Dependent Hepatic Toxicophore

Graham¹,

Walsh²,

Hirst³

et al. 2008

J Pharmacol Exp Ther

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“…Analysis of sera obtained from patients suffering from liver damage following TA exposure revealed the presence of highly specific anti-liver and kidney microsomal (anti-LKM2) autoantibodies [6] known to specifically recognize CYP450 2C9 in humans [7,8] and the corresponding isoform 2C11 in rats [9]. Subsequent studies have revealed that tienilic acid undergoes a selective CYP450 2C9-mediated bioactivation on its thiophene ring, resulting in the formation of a reactive intermediate that covalently modifies the enzyme [10] despite that a stable peptide conjugate has never been characterized [11,12]. A relatively limited number of proteins appear to be modified by TA metabolites in both human and rat liver microsomes [9,10,13,14].…”

Section: Introductionmentioning

confidence: 95%

Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

Methogo

Dansette

Klarskov

2007

International Journal of Mass Spectrometry

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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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“…It should be noted that, to maintain consistency, tienilic acid concentrations need to be adjusted for each new HLM pool (data not shown). Tienilic acid-treated microsomes should be diluted and/or washed to minimize unbound molecules, because tienilic acid has been reported to react with protein nucleophiles, to deplete glutathione levels, and to up-regulate genes involved in oxidative stress responses and phase II drug metabolism (Belghazi et al, 2001;López-García et al, 2005;Nishiya et al, 2008); this is most applicable to hepatocyte preparations.…”

Section: Kinetic Parameters Of the Effect Of Tienilic Acid On Non-cypmentioning

confidence: 99%

Development of an In Vitro System with Human Liver Microsomes for Phenotyping of CYP2C9 Genetic Polymorphisms with a Mechanism-Based Inactivator

Flora

Tracy

2011

Drug Metab Dispos

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ABSTRACT:Polymorphisms in cytochrome P450 enzymes can significantly alter the rate of drug metabolism, as well as the extent of drug-drug interactions. Individuals who homozygotically express the CYP2C9*3 allele (I359L) of CYP2C9 exhibit ϳ70 to 80% reductions in the oral clearance of drugs metabolized through this pathway; the reduction in clearance is ϳ40 to 50% for heterozygotic individuals. Although these polymorphisms result in a decrease in the activity of individual enzyme molecules, we hypothesized that decreasing the total number of active enzyme molecules in an in vitro system (CYP2C9*1/*1 human liver microsomes) by an equivalent percentage could produce the same net change in overall metabolic capacity. To this end, the selective CYP2C9 mechanismbased inactivator tienilic acid was used to reduce irreversibly the total CYP2C9 activity in human liver microsomes. Tienilic acid concentrations were effectively titrated to produce microsomal preparations with 43 and 73% less activity, mimicking the CYP2C9*1/*3 and CYP2C9*3/*3 genotypes, respectively. With probe substrates specific for other major cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), no apparent changes in the rate of metabolism were noted for these enzymes after the addition of tienilic acid, which suggests that this model is selective for CYP2C9. In lieu of using rare human liver microsomes from CYP2C9*1/*3 and CYP2C9*3/*3 individuals, a tienilic acid-created knockdown in human liver microsomes may be an appropriate in vitro model to determine CYP2C9-mediated metabolism of a given substrate, to determine whether other drug-metabolizing enzymes may compensate for reduced CYP2C9 activity, and to predict the extent of genotypedependent drug-drug interactions.

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Use of Isotopes and LC-MS-ESI-TOF for Mechanistic Studies of Tienilic Acid Metabolic Activation

Cited by 19 publications

References 4 publications

Identification of the Thiophene Ring of Methapyrilene as a Novel Bioactivation-Dependent Hepatic Toxicophore

Identification of the Thiophene Ring of Methapyrilene as a Novel Bioactivation-Dependent Hepatic Toxicophore

Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

Development of an In Vitro System with Human Liver Microsomes for Phenotyping of CYP2C9 Genetic Polymorphisms with a Mechanism-Based Inactivator

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