Functional and toxicological consequences of metabolic bioactivation of methapyrilene via thiophene S-oxidation: Induction of cell defence, apoptosis and hepatic necrosis Amy E. Mercer a, 1 , Sophie L. Regan a, 1 , Charlotte M. Hirst a, 1 , Emma E. Graham a , Daniel J. Antoine a , Craig A. Benson a , Dominic P. Williams a, ⁎, John Foster b , J. Gerry Kenna b , B. Kevin Park a a MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, The University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool L69 3GE, UK b AstraZeneca plc, Alderley Park, Cheshire, UK abstract article info Article history: Received 11 February 2009 Revised 22 May 2009 Accepted 31 May 2009 Available online 10 June 2009 Keywords: Methapyrilene Hepatotoxicity Glutathione Thiophene Apoptosis Necrosis Methapyrilene, [N,N-dimethyl-N′-pyridyl-N′(2-thienylmethyl)-1,2-ethanediamine] (MP) was withdrawn from, clinical use due to reported periportal hepatic necrosis and hepatocarcinogenicity in the rat, via S- oxidation of the thiophene group.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. © 2009 Elsevier Inc. All rights reserved. Introduction Drug induced liver injury (DILI) is one of the leading causes of serious ill health in man, and affects many hundreds of marketed and withdrawn drugs (Park et al., 2006). It is clear that, for at least some hepatotoxic drugs, metabolic bioactivation of structural toxicophores within hepatocytes plays a key role in initiating toxicity (Park et al., 2006). However, in relatively few instances have the molecular events, by which reactive intermediates derived from pharmaceuticals cause cellular injury, been defined. The elucidation of the mechanisms associated with each toxicophore is an important objective, since this will be required to underpin the safe clinical use of currently licensed drugs and to improve safety evaluation and risk assessment of new candidate drugs. Recently we demonstrated that the withdrawn antihistamine methapyrilene [N,N-dimethyl-N′-pyridyl-N′(2-thienylmethyl)-1,2- ethanediamine] (MP) is hepatotoxic in the rat due to cytochrome P450 mediated bioactivation of the thiophene ring, which has been identified as a structural alert for hepatotoxicity (Graham et al., 2008). We also demonstrated the detoxication of the S-oxide via its conjugation with glutathione (GSH) in vivo (Fig. 1). The hepatotoxi- city of MP led to its withdrawal from the market due to reports of hepatocarcinogenicity in the rat, despite having previously been widely used in cold remedies and sleep aids for 25 years (Lijinsky et al., 1980). MP toxicity is of particular interest due to the unusual and distinctive pattern of liver injury observed (i.e. periportal hepatocel- lular hepatic injury accompanied by bile duct hyperplasia) and its progression to hepatic tumours (Graichen et al., 1985; Ratra et al., 1998), and also because of its definite dependence on the activation of the thiophene toxicophore to defined reactive metabolites (Graham et al., 2008). MP is one of several drugs containing thiophene functional groups which have been withdrawn due to toxicity, for example tienilic acid (TA). MP and TA share similar mechanisms of thiophene Toxicology and Applied Pharmacology 239 (2009) 297–305 Abbreviations: MP, methapyrilene; HO-1, heme-oxygenase 1; GCLC, glutamate cysteine ligase, catalytic subunit; GSH, glutathione; Nrf2, nuclear factor-erythroid 2- related factor 2; DEM, diethyl maleate; TBS-T, Tris-buffered saline containing 0.1% Tween 20; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul- fophenyl)-2H-tetrazolium; SEM, standard error of the mean; ALT, alanine aminotrans- ferase; AST, aspartate aminotransferase; GLDH, glutamate dehydrogenase; ALP, alkaline phosphatase; Apaf-1, apoptosis activating factor-1; TA, tienilic acid. ⁎ Corresponding author. Fax: +44 151 794 5540. E-mail address: dom@liv.ac.uk (D.P. Williams). 1 These authors contributed equally to this work. 0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.05.027 Contents lists available at ScienceDirect Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap