Establishment of knockdown of superoxide dismutase 2 and expression of CYP3A4 cell system to evaluate drug-induced cytotoxicity
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.
Section snippets
Materials
Cell Counting Kit-8s (CCK-8) for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, testosterone, corticosterone, carbamazepine, dantrolene, flutamide, nifedipine, sulfamethoxazole, trazodone, and zidovudine were obtained from Wako Pure Chemical Industries (Osaka, Japan). Troglitazone and rosiglitazone were kindly provided by Daiichi-Sankyo (Tokyo, Japan). Albendazole, dapsone, isoniazid, nimesulide, and 2,7-dihydro dichlorofluorescein (DCF) were obtained from
MOI-dependent knockdown effect of AdSOD2-shRNA in BRL3A cells
To investigate the most efficient multiplicity of infection (MOI), BRL3A cells were infected with AdSOD2-shRNA at MOI 0, 5, 10, 20, 50, 100 or 200 for 3 days (Fig. 1). SOD2 mRNA was decreased MOI-dependently, but some cells began floating at MOI 200. Thus, infection at MOI 100 (70% decrease of SOD2 mRNA) could be an appropriate condition of AdSOD2-shRNA infection.
Changes of SOD2 mRNA expression in various hepatic cell lines
To investigate the knockdown effect on the cells, various hepatic cell lines were infected with AdSOD2-shRNA or AdLuc-shRNA (negative
Discussion
In this study, we constructed a recombinant adenovirus vector expressing shRNA-directed rat superoxide dismutase 2 (AdSOD2-shRNA). In BRL3A cells, the SOD2 mRNA was decreased MOI dependently (Fig. 1). At MOI 200, some cells floated and SOD2 mRNA was decreased, but glyceraldehydes-3-phosphate (GAPDH) mRNA was not changed, suggesting slight toxicity by the adenovirus itself was appeared. Thus, infection of AdSOD2-shRNA at MOI 100 was an appropriate condition to suppress the SOD2 mRNA without
Conflict of Interest Statement
None of the authors has any conflicts of interest related to this manuscript.
Acknowledgments
This work was supported by Health and Labor Sciences Research Grants from the Ministry of Health, Labor, and Welfare of Japan. We thank Mr. Brent Bell for reviewing the manuscript.
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