N-acetylcysteine, coenzyme Q10 and superoxide dismutase mimetic prevent mitochondrial cell dysfunction and cell death induced by d-galactosamine in primary culture of human hepatocytes

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Abstract

d-Galactosamine (d-GalN) induces reactive oxygen species (ROS) generation and cell death in cultured hepatocytes. The aim of the study was to evaluate the cytoprotective properties of N-acetylcysteine (NAC), coenzyme Q10 (Q10) and the superoxide dismutase (SOD) mimetic against the mitochondrial dysfunction and cell death in d-GalN-treated hepatocytes. Hepatocytes were isolated from liver resections. NAC (0.5 mM), Q10 (30 μM) or MnTBAP (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (1 mg/mL) were co-administered with d-GalN (40 mM) in hepatocytes. Cell death, oxidative stress, mitochondrial transmembrane potential (MTP), ATP, mitochondrial oxidized/reduced glutathione (GSH) and Q10 ratios, electronic transport chain (ETC) activity, and nuclear- and mitochondria-encoded expression of complex I subunits were determined in hepatocytes. d-GalN induced a transient increase of mitochondrial hyperpolarization and oxidative stress, followed by an increase of oxidized/reduced GSH and Q10 ratios, mitochondrial dysfunction and cell death in hepatocytes. The cytoprotective properties of NAC supplementation were related to a reduction of ROS generation and oxidized/reduced GSH and Q10 ratios, and a recovery of mitochondrial complexes I + III and II + III activities and cellular ATP content. The co-administration of Q10 or MnTBAP recovered oxidized/reduced GSH ratio, and reduced ROS generation, ETC dysfunction and cell death induced by d-GalN. The cytoprotective properties of studied antioxidants were related to an increase of the protein expression of nuclear- and mitochondrial-encoded subunits of complex I. In conclusion, the co-administration of NAC, Q10 and MnTBAP enhanced the expression of complex I subunits, and reduced ROS production, oxidized/reduced GSH ratio, mitochondrial dysfunction and cell death induced by d-GalN in cultured hepatocytes.

Introduction

Apoptosis is considered to be involved in the normal regulation of the organ size, as well as in the underlying mechanism of cell death in liver diseases. The induction of oxidative stress is a key event in the intracellular pathways leading to cellular apoptosis [1]. Several free radical generation sites, such as mitochondria or cytochrome P450-dependent metabolism, are involved in cell death [2], [3]. The depletion of cellular reduced glutathione (GSH) content, as a consequence of intense intracellular oxidative stress, has been observed during cell death induced by different agents [4], [5]. d-Galactosamine (d-GalN) is a suitable experimental model of human liver failure [6]. d-GalN induces oxidative stress and cell death in human and rat cultured hepatocytes [7], [8]. The rapid generation of reactive oxygen species (ROS) was related to hyperpolarization of the mitochondrial membrane potential and apoptosis in d-GalN-treated human hepatocytes [9]. In addition, d-GalN-dependent cell necrosis was related to mitochondrial membrane depolarization [9].

Mitochondria provide most of the cell energy through the fatty acid β-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation. Endogenous compounds (such as cytokines or female sex hormones) or xenobiotics (including toxins, such as ethanol and drugs, such as aspirin, valproic acid, ibuprofen, or zidovudine) can inhibit β-oxidation directly or through a primary effect on the mitochondrial genome or the respiratory chain itself [10]. The mitochondrial ROS generation can be significantly enhanced by a rise of NADH supplementation or with the functional impairment of complexes I and III of electron transport chain (ETC) [11]. Although energy production is an important function of mitochondria, these organelles also participate in the initiation and execution of cell death, and maintaining calcium and iron homeostasis. The regulation of intracellular oxidative stress by antioxidants may determine cell fate and the mode of cell death [12]. Different antioxidant strategies have been shown to be useful to reduce oxidative stress and cell death in hepatocytes. GSH is the most abundant non-protein thiol present in mammalian cells, and acts as an essential component of the cellular antioxidant defense system. Due to its very reactive cysteine sulfhydryl moiety, GSH is enzymatically conjugated to toxic electrophiles by cellular GSH transferases. Additionally, GSH participates as a co-substrate in various GSH-dependent peroxidase reactions, protecting cells or tissues against oxidative damage by metabolizing toxic organic molecules and hydrogen peroxides. The maintenance of mitochondrial GSH content prevents cell damage in different in vivo[13] and in vitro[14] experimental models of hepatotoxicity. The administration of N-acetylcysteine (NAC), an excellent source of intracellular cysteine and free radical scavenger, has been shown to have clinical applications in HIV infection, cancer, heart disease, as well as in smoking, kidney and liver diseases [15]. The regulation of mitochondrial oxidative stress may be a useful strategy to prevent the depletion of GSH in the organelle. The aim of our study was the identification of the beneficial properties of NAC, coenzyme Q10 (Q10) and superoxide dismutase (SOD) mimetic (MnTBAP) on oxidative stress, expression and/or activity of the mitochondrial electron transport chain (ETC) components and cell survival in d-GalN-treated hepatocytes. The study showed that the co-administration of NAC, Q10 and MnTBAP enhanced the expression of complex I subunits, and reduced ROS production, oxidized/reduced GSH ratio, mitochondrial dysfunction and cell death in d-GalN-treated hepatocytes.

Section snippets

Materials

All reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated. DME:Ham-F12 and William's E culture mediums were obtained from Sigma Chemical Co. and Applichem (Applichem GmbH, Darmstadt, Germany), respectively. Antibiotics–antimycotic solution and fetal bovine serum were obtained from Life Technologies Inc. (Paisley, UK). NAC and Q10 were obtained from Sigma Chemical Co. MnTBAP (Mn (III)tetrakis(4-benzoic acid) porphyrin chloride was obtained from Calbiochem

NAC reduced cell death in d-GalN-induced cytotoxicity

d-GalN induces oxidative stress and cell death in primary culture of rat and human hepatocytes [7], [9]. d-GalN induced a rapid and transient induction of mitochondrial ROS generation measured by DCFDA (Fig. 2A) (p  0.05). The contribution of hydrogen peroxide (H2O2) to the oxidation of the DCFDA was determined with the pre-incubation (30 min) of the cells with catalase (500 U/mL). Catalase showed that 50% of DCFDA oxidation was due to H2O2 (Fig. 2B). The profile of ROS generation mimicked the

Discussion

Oxidative stress plays a relevant role in the induction of cell death in hepatocytes. NAC has been shown to exert cytoprotection in different thiol-depleting experimental models of cell death in hepatocytes [25], [27]. d-GalN-induced ROS production, mitochondrial dysfunction and cell death in hepatocytes. The study showed that the reduction of oxidative stress, recovery of mitochondrial oxidized/reduced GSH and reduced Q10 content, and the restoration of ETC by antioxidants are key factors for

Conflict of interest

None declared.

Acknowledgments

This study has been supported by the Instituto de Salud Carlos III (FIS 02/0181, FIS 05/0703) and the Consejería de Salud (SAS 50/03). CIBERehd is funded by the Instituto de Salud Carlos III.

Funding source: The research has been carried out with public national funding.

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