Establishment of knockdown of superoxide dismutase 2 and expression of CYP3A4 cell system to evaluate drug-induced cytotoxicity

Abstract

Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. In this study, we established SOD2-knockdown cells. An adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA) was constructed, and infection of AdSOD2-shRNA to rat hepatic BRL3A cells resulted in significant decreases of SOD2 mRNA and protein by 60%, and SOD2 activity by 50% after 3 days infection. We previously constructed an adenovirus expressing cytochrome P450 3A4 (AdCYP3A4). Co-infection of AdSOD2-shRNA and AdCYP3A4 to BRL3A cells was carried out to evaluate the superoxide- and CYP3A4-mediated formation of active metabolites, and mitochondrial toxicity, ROS and superoxide radical production and lipid peroxidation were selected to assess the cell viability. Albendazole, carbamazepine, dapsone, flutamide, isoniazid, nifedipine, sulfamethoxazole, trazodone, troglitazone, and zidovudine demonstrated significant increases of SOD2- and CYP3A4-mediated cytotoxicity. In conclusion, we constructed a highly sensitive cell system to evaluate oxidative stress and CYP3A4 mediated cytotoxicity that could be useful in preclinical drug development.

Introduction

Oxidative stress is one of the causes of drug-induced hepatotoxicity (Kaplowitz, 2005) and is induced by superoxide. Superoxide is mainly generated in mitochondria. Superoxide can react with NO to form peroxynitrite or, after conversion to hydrogenperoxide, undergo metal-catalysed Fenton reactions to form highly reactive hydroxyl radicals (Boelsterli et al., 2006). Superoxide dismutases (SODs) are the first and most important line of antioxidant enzyme defense systems against reactive oxygen species (ROS) and particularly superoxide anion radicals (Zelko et al., 2002). At present, three distinct SOD isoforms, SOD1, SOD2, and SOD3, have been identified in mammals. SOD1, SOD2 and SOD3 are mainly localized to the cytoplasm, mitochondria and plasma, respectively (Zelko et al., 2002). In addition, SOD2 is known to be induced by a wide variety of compounds, including lipopolysaccharide and anticancer drugs (Visner et al., 1990, Das et al., 1998). Meanwhile, the importance of SOD2 function in mammalian organisms was confirmed by disruption of the SOD2 gene, which turns out to be lethal for mice due to neurodegeneration and damage to the heart (Lebovitz et al., 1996). These suggest that SOD2 plays an especially important role in detoxification.

Recently, recombinant adenovirus methods are being developed and used for the purpose of gene delivery (Akai et al., 2007, Pérez and Cederbaum, 2003, Hosomi et al., submitted). Furthermore, a small interfering RNA strategy, which has been proven to be more specific and efficient than the full-length antisense cDNA strategy, has been established (Meister and Tuschl, 2004). In the present study, we constructed a recombinant adenovirus expressing SOD2-short hairpin RNA (AdSOD2-shRNA) that could knockdown rat SOD2 mRNA efficiently. In general, since cytochrome P450 (CYPs) are hardly expressed in cell lines, we infected adenovirus expressing CYP3A4 (AdCYP3A4). CYP3A4 is the dominant CYP3A enzyme in the liver and intestine (Rendic and Di Carlo, 1997, Shimada et al., 1994), and contributes to the metabolism of more than 50% of drugs clinically in use today (Rendic and Di Carlo, 1997, Nelson, 1999, Guengerich, 2001). In addition, metabolic activations of drugs by CYP3A4 are thought to induce hepatotoxicity (Vignati et al., 2005). Based on these considerations, in the present study we established an SOD2-knockdown and CYP3A4-expressing cell system to evaluate the oxidative stress and the CYP3A4-mediated cytotoxicity with high sensitivity for preclinical drug development studies.