Mitochondrial involvement in cocaine-treated rat hepatocytes: effect of N-acetylcysteine and deferoxamine.

The cytotoxicity of cocaine (0–1000 μM), was studied on parameters related to the mitochondrial role and the cascade of events that lead to apoptosis in hepatocyte cultures from phenobarbitone (PB) pretreated rats. Cytotoxicity was dose-dependent and LDH leakage was significantly enhanced above 100 μM cocaine. Apoptosis was visualized by DNA fragmentation on agarose gel, and appeared at 50 and 100 μM cocaine. Cocaine induced biphasic changes in mitochondrial transmembrane potential and significantly increased the mitochondrial release of cytochrome c, the caspase-3 like DEVDase activity and the level of 20 kDa subunit, a product of pro-caspase-3 cleavage. The protective effect of N-acetylcysteine (NAC) and deferoxamine (DFO) on all these parameters confirmed the involvement of oxygen radicals in cocaine-induced necrosis/apoptosis. We conclude: first, that the biphasic changes recorded in mitochondrial inner membrane potential by the effect of cocaine, were parallel to apoptosis; second, that caspase-3 activity and cleavage to it p20 subunit increased sharply in parallel to the translocation of cytochrome c from mitochondria to cytosol; and third, that the antioxidants, NAC or DFO exerted a noticeable protective role in counteracting the cytotoxicity of cocaine, these effects being more pronounced in the case of DFO than NAC. These findings demonstrate that cocaine cytotoxicity involves mitochondrial damage. Keywords: Cocaine cytotoxicity, oxidative stress, mitochondria, cytochrome c, caspase-3, apoptosis, antioxidants Introduction Experimental and clinical evidence has demonstrated that cocaine is an hepatotoxic drug that significantly alters liver function and induces liver injury characterized by an unevenly distributed intraacinar necrosis (Marks & Chappel, 1967; Kanel et al., 1990; Cascales et al., 1994). Cocaine hepatotoxicity is mediated by a series of sequential oxidations catalyzed by cytochrome P450 and flavin-mono-oxygenases (Kloss et al., 1984; Boelsterli & Godlin, 1991). It is well known that phenobarbitone (PB) by inducing the expression of P-450 microsomal mono-oxygenases responsible for cocaine oxidative metabolism (Cascales et al., 1994; Powell et al., 1994) enhances the rate of cocaine oxidation and consequently the hepatotoxicity of this drug. Two major pathways have been postulated to explain cocaine-induced hepatotoxicity, as the result of oxidative metabolism: (a) either through depletion of cellular reducing equivalents (NADPH) and generation of reactive oxygen species (ROS), superoxide radical (O2.−) and hydrogen peroxide (H2O2), during the redox cycling between N-hydroxynorcocaine and norcocaine nitroxide: or (b) through the production of the nitrosonium ion or other as yet unidentified reactive metabolites that bind covalently to cellular macromolecules (Kloss et al., 1984; Bouis & Boelsterli, 1990; Boelsterli & Godlin, 1991; Jover et al., 1993; Ponsoda et al., 1999). In a previous study we demonstrated that apoptosis occurs in liver following the in vivo administration of cocaine to mice either pre-treated or non-pretreated with PB (Cascales et al., 1994). Apoptosis was also detected in cultures of hepatocytes from PB-pretreated rats (Zaragoza et al., 2000). Several experiments have demonstrated that cocaine can induce apoptosis in cultured foetal neurons (Nassogne et al., 1997), mouse thymocyte populations (Wu et al., 1997) and foetal myocardial cells (Xiao et al., 2000). Pathological studies have implicated oxidative damage in the mechanisms of cocaine-induced liver injury (Godlin & Boelsterli, 1991; Boelsterli et al., 1993) Recent data of our group (Diez-Fernandez et al., 1999) have also shown that reactive oxygen species contribute directly or indirectly to cocaine-induced apoptosis in cultured hepatocytes. Actually, although many factors can be involved in apoptosis, ROS generated as subproducts derived from the metabolism of toxic agents are important mediators in this form of cell death (Gardner et al., 1997; Hildeman et al., 1999). Several studies have also shown that mitochondrial alterations are involved in these processes (Masini et al., 1997), and that structural and functional changes in mitochondria are impaired by cocaine and its metabolites (Devi & Chan, 1996; Yuan & Acosta, 1996; Xiao et al., 2000). Recent studies suggest that mitochondria could be the main sensor in apoptosis, since the release of mitochondrial factors in the cytosol, such as cytochrome c, can initiate the activation cascade of caspases (Liu et al., 1996; Green & Reed, 1998), a crucial event in the apoptotic death program. The activity of these proteases, related to interleukin-1β-converting enzyme (ICE) is involved in the multiple steps, such as cell shrinkage, membrane blebbing and chromatin degradation, that constitute apoptosis. It is well known that exogenous antioxidants play an important role against oxidant injury induced by excess of ROS generation. The hepatoprotective action of N-acetylcysteine (NAC), a thiol containing a compound that acts as a nucleophile and as a precursor of reduced glutathione, is well defined (Cotgreave, 1997); and the effect of deferoxamine (DFO), an iron chelating agent that consequently prevents ROS generation by inhibiting Fenton reaction, has also been well studied (Susa et al., 1997; Zaragoza et al., 2000). The purpose of the present investigation was to find, in a model of cocaine cytotoxicity, using rat hepatocyte cultures, a potential relationship between apoptosis and a series of related parameters: the presence of cytochrome c in the cytosol, caspase activity and the levels of 20 kDa caspase subunit, and mitochondrial transmembrane potential (ΔΨm). Afterwards, the effect of NAC or DFO as antioxidants coadministrated with cocaine, was evaluated in the same experimental conditions to determine the way by which these substances protect against the toxic effects of cocaine.