RESUMEN
UNLABELLED: Methapyrilene, [N,N-dimethyl-N'-pyridyl-N'(2-thienylmethyl)-1,2-ethanediamine] (MP) was withdrawn from, clinical use due to reported periportal hepatic necrosis and hepatocarcinogenicity in the rat, via S-oxidation of the thiophene group. In this study MP is used as a model hepatotoxin to further characterise the functional consequences of S-oxidation of the thiophene group in vivo, in rat models and in vitro, in freshly isolated rat hepatocyte suspensions. In vivo histological studies revealed the early depletion of glutathione (GSH), which was confined to the damaged periportal area, in contrast to an increase in GSH levels in the centrilobular region. Additionally, the induction of cell defence was demonstrated by an increase in the protein levels of heme-oxygenase 1 (HO-1) and glutamate cysteine ligase, catalytic subunit (GCLC) in vivo. Histological examination demonstrated that cytotoxicity progresses initially via apoptosis before an increase in necrosis over the 3-day administration. An apoptotic-like mechanism was observed in vitro via the measurement of cytochrome c release and caspase activation. CONCLUSION: This study provides evidence for a complex pathway of MP-induced hepatotoxicity which progresses through early adaptation, apoptosis, necrosis and inflammation, all underpinned by the zonal induction and depletion of GSH within the liver.
Asunto(s)
Apoptosis/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hígado/efectos de los fármacos , Metapirileno/toxicidad , Tiofenos/metabolismo , Animales , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Glutamato-Cisteína Ligasa/biosíntesis , Glutatión/metabolismo , Hemo-Oxigenasa 1/biosíntesis , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Metapirileno/farmacocinética , Necrosis , Oxidación-Reducción , Ratas , Ratas WistarRESUMEN
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 ([3H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat > mouse approximately 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.