Abstract
Methapyrilene (MP), a 2-thiophene H1-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 ([3H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat > mouse ≈ 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 [3H]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.
Footnotes
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This work was supported by studentships funded by AstraZeneca (to E.E.G. and R.J.W.) and AstraZeneca/Biotechnology and Biological Sciences Research Council (to C.M.H.).
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The data presented here have appeared in abstract form as follows: Dalton-Brown E, Hirst C, Maggs J, Williams D, Wild MJ, Wilson I, Harding J, Kenna G, and Park K (2006) The metabolic basis of methapyrilene-induced hepatotoxicity. Drug Metab Rev 38 (Suppl 1):175–176; and Hirst C, Dalton-Brown E, Williams D, Maggs J, Powell H, Harding J, Wilson I, Wild MJ, Kenna G, and Park K (2006) Chemical, biological, and toxicological mechanisms of hepatotoxins. Drug Metab Rev 38 (Suppl 1):169–170.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.135483.
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ABBREVIATIONS: MP, methapyrilene; P450, cytochrome P450; tGSH, total glutathione (GSH plus glutathione disulfide); MeOD, monodeuteromethanol; HPLC, high-performance liquid chromatography; [3H]MP, methapyrilene tritiated at C-2 of the diaminoethane moiety; GSH, glutathione; GSH-EE, glutathione O-ethyl ester; NAC, N-acetylcysteine; ABT, 1-aminobenzotriazole; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; LC-MS, liquid chromatography-mass spectrometry; RLM, rat liver microsomes; MLM, mouse liver microsomes; HLM, human liver microsomes; LC-MS/MS, liquid chromatography-tandem mass spectrometry; βME, β-mercaptoethanol; ALT, alanine amino transferase; GLDH, glutamate dehydrogenase; H/D, hydrogen/deuterium exchange; equiv, equivalent; amu, atomic mass unit; MD, molecular weight of fully deuterated analyte; SKF-525A, 2-diethylaminoethyl 2,2-diphenyl pentanoate.
- Received December 17, 2007.
- Accepted April 29, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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