Role of reactive oxygen species, glutathione and NF-kB in apoptosis induced by 3,4-methylenedioxymethamphetamine (‘‘Ecstasy’’) on hepatic stellate cells Cristina Montiel-Duarte a , Eduardo Ansorena a , Maria Jesu ´s Lo ´pez-Zabalza a , Edurne Cenarruzabeitia b , Marı ´a J. Iraburu a,* a Department of Biochemistry, Universidad de Navarra, C/Irunlarrea 1, 31008 Pamplona, Navarra, Spain b Department of Pharmacology, Universidad de Navarra, C/Irunlarrea 1, 31008 Pamplona, Navarra, Spain Received 30 July 2003; accepted 20 October 2003 Abstract ‘‘Ecstasy’’ (3,4-methylenedioxymethamphetamine, MDMA), is a derivative of amphetamine with hepatotoxic effects that has been shown to induce apoptosis of cultured liver cells. In the present work, we studied the role played by oxidative stress in the apoptotic response caused by MDMA on a cell line of hepatic stellate cells (HSC). MDMA-treatment provoked oxidative stress determined as reactive oxygen species (ROS) accumulation and decrease of intracellular reduced glutathione levels. Pre-treatment with the antioxidant pyrrolidine dithiocarbamate blocked ROS production but did not prevent MDMA-induced apoptosis of HSC. The pro-oxidant menadione induced in HSC ROS production and apoptosis that were prevented by pyrrolidine dithiocarbamate, showing HSC to be susceptible to oxidative stress-induced apoptosis. Addition of exogenous GSH or its precursor NAC potentiated the apoptotic action of MDMA but blocked apoptosis induced by menadione. Pre-treatment of HSC with the cytochrome P450 inhibitor quinine diminished the extent of apoptosis caused by MDMA, suggesting the involvement of a metabolic derivative of MDMA on its apoptotic effect. Nuclear factor NF- kB was activated by MDMA in a oxidative stress independent fashion and played a protective role in the apoptotic response, since inhibition of NF-kB by treatment with parthenolide or by viral infection with a dominant-negative form of NIK (Ad5dnNIK) resulted in an increase of MDMA-induced cell death. In summary, MDMA-induced apoptosis of HSC is accompanied, but not caused by oxidative stress; a metabolic derivative of the drug is responsible for the apoptotic effect of MDMA, which is partially blocked by NF-kB activation. # 2003 Elsevier Inc. All rights reserved. Keywords: MDMA; Apoptosis; Hepatic stellate cells; Glutathione; Reactive oxygen species; NF-kB 1. Introduction Apoptosis is an endogenous cell death program that can be triggered by different stimuli and is characterized by morphological features such as reduction in cell volume, membrane blebbing, chromatin condensation and nuclear DNA fragmentation [1,2]. A family of specific cysteine proteases, caspases, plays a key role in the apoptotic response [3]. There are reports suggesting that oxidative stress is involved in the induction of programmed cell death in some systems. Addition of oxidants like hydrogen per- oxide or menadione can lead to cell death by apoptosis [4,5], and an increased production of reactive oxygen species (ROS) seems to be a critical step for apoptosis associated with ionizing radiation or chemotherapeutic drugs [6–8]. Moreover, antioxidants such as N-acetylcysteine (NAC) and reduced glutathione (GSH) can block apoptosis induced by agents different from oxidants [9,10]. Mitochondria could be involved in some of these events, since they are both a source of ROS and a target for ROS-induced toxicity, and mitochondria disfunction has been proposed as one of the pathways leading to caspase activation and cell death [11]. Biochemical Pharmacology 67 (2004) 1025–1033 0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2003.10.020 * Corresponding author. Tel.: þ34-48-425600x6481; fax: þ34-48-425649. E-mail address: miraburu@unav.es (M.J. Iraburu). Abbreviations: MDMA, 3,4-methylenedioxymethamphetamine; GSH, glutathione; HSC, hepatic stellate cells; PDTC, pyrrolidine dithiocarba- mate; NAC, N-acetylcysteine; MEM, minimum essential medium; FBS, fetal bovine serum; BSO, buthionine sulfoximine; CM-H 2 DCFDA, 5-6- chloromethyl-2 0 ,7 0 -dichlorohydrofluorescein diacetate; ROS, reactive oxy- gen species; NIK, NF-kB-inducing kinase; DHMA, 3,4-dihydroxy- methamphetamine.