Protective effects of apocynin on cisplatin-induced ototoxicity in an auditory cell line and in zebrafish June Choi, a Gi Jung Im, a Jiwon Chang, a Sung Won Chae, a Seung Hoon Lee, a Soon-Young Kwon, a Ah-Young Chung, b Hae-Chul Park b and Hak Hyun Jung a * ABSTRACT: Cisplatin is a very effective anticancer drug and generates reactive oxygen species (ROS) such as superoxide anions that can deplete antioxidant protective molecules in the cochlea. These processes result in the death of cochlear hair cells by induction of apoptosis. Apocynin, which is used as a specific nicotinamide adenine dinucleotide phosphate oxidase inhibitor, has a preventive effect for intracellular ROS generation. In this study, the effect of apocynin was investigated in a cochlear organ of Corti-derived cell line, HEI-OC1 cells, and in transgenic zebrafish (Brn3C: EGFP). To investigate the protec- tive effects of apocynin, HEI-OC1 cells were treated with various concentrations of apocynin and a 20 mM concentration of cisplatin, simultaneously. An in vivo study of transgenic zebrafish (Brn3C: EGFP) was used to investigate the protective effects of apocynin on cisplatin-induced hair cell death. In an in vitro study, apocynin appeared to protect against cisplatin-induced apoptotic features on Hoechst 33258 staining in the HEI-OC1 cells. Treatment of the HEI-OC1 cells with 100 mM of apocynin, significantly decreased caspase-3 activity. Treatment of the cells with a 100 mM concentration of apocynin and a 20 mM concentration of cisplatin significantly decreased the intracellular ROS production. In the in vivo study, apocynin significantly decreased the TUNEL reaction and prevented cisplatin-induced hair cell loss of the neuromasts in the transgenic zebrafish at low concentrations (125 and 250 mM). These findings suggest that apocynin has antioxidative effects and prevents cisplatin- induced apoptotic cell death in HEI-OC1 cells as well as in zebrafish. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: apocynin; cisplatin; ototoxicity; HEI-OC1 cells; zebrafish INTRODUCTION Cisplatin is one of the most effective chemotherapeutic agents used for the treatment of a wide variety of human solid tumors, including ovarian, testicular, cervical, head and neck, lung, and bladder cancer. However, it has significant side effects such as nephrotoxicity, neurotoxicity and ototoxicity (Rybak and Kelly, 2003; Rybak, 2007; Rybak et al., 2007). The ototoxic symptoms of cisplatin include tinnitus and high-frequency sensorineural hearing loss, which can be permanent and progressive, and also involve the lower frequencies. Cisplatin has been shown to cause the death of the outer hair cells in the organ of Corti (Takeno et al., 1994; Wake et al., 1994; Ou et al., 2007). Cisplatin-induced ototoxicity is closely associated with the pro- duction of reactive oxygen species (ROS), resulting in depletion of glutathione and antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) in cochlear tissues. These reactions can increase concentrations of malondialdehyde, toxic lipid peroxides, 4-hydroxynonenal and peroxynitrite (Lee et al., 2004; Rybak et al., 2007). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a potential source for the production of toxic free radicals follow- ing cisplatin exposure; it catalyzes the formation of superoxide radi- cals (Rybak and Whitworth, 2005). Many agents have been studied to evaluate their protective effects against cisplatin-induced ototoxicity, including L-methionine, L-N-acetylcysteine, fosfomycin, sodium salicylate, D-methionine, diethyldithiocarbamate, lipoic acid, glutathione ester and epicatechin (Schweitzer et al., 1986; Rybak et al., 1995, 1999; Campbell et al., 1996, 2003a; Feghali et al., 2001; Li et al., 2001, 2002; Kim et al., 2008). Apocynin, a speci fic NADPH oxi- dase inhibitor (Muijsers et al., 2000; Erdos et al., 2006; Chan et al., 2007; Gracia-Sancho et al., 2007; Chirino et al., 2008), has been shown to protect tissue damage in several experimental models (Rugale et al., 2005; Pech et al., 2006; Cui et al., 2007; Chirino et al., 2008). Zebrafish have hair cells on the surface of their body in a sensory system referred to as the lateral line. Hair cells of the neuro- masts in the zebrafish lateral line are emerging as a useful system for studying hair cell loss after exposure to ototoxic agents. The trans- genic zebrafish (Brn3C: EGFP) has naturally occurring green-colored neuromasts that are visible without any staining under a fluorescent microscope. The purpose of this study was to investigate the effects *Correspondence to: Hak Hyun Jung, Department of Otolaryngology–Head and Neck Surgery, Korea University College of Medicine, 126–1 Anam-dong 5-Ga, Seongbuk-Gu, Seoul, South Korea 136–705. E-mail: ranccoon@naver.com a Department of Otorhinolaryngology–Head and Neck Surgery, Korea University College of Medicine, Seoul, Korea b Laboratory of Neurodevelopmental Genetics, Graduate School of Medicine, Korea University, Seoul, Korea J. Appl. Toxicol. 2011 Copyright © 2011 John Wiley & Sons, Ltd. Research Article Received: 27 January 2011, Revised: 27 July 2011, Accepted: 27 July 2011 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jat.1729