Ebselen attenuates cochlear damage caused by acoustic trauma

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Abstract

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a seleno-organic compound, mimics glutathione peroxidase and reacts with peroxynitrite. It is reported to protect against gentamicin- and cisplatin-induced ototoxicity. We investigated whether it protects the cochlea from acoustic trauma. Male pigmented guinea pigs (250–300 g) with normal auditory brainstem response (ABR) thresholds were exposed for 5 h to 125 dB sound pressure level octave band noise centered at 4 kHz. One hour before and 18 h after exposure, they received orally 0.25 ml chloroform solution containing 0, 10, or 30 mg/kg ebselen (n=6, 5 and 5, respectively). The protective effect of ebselen was evaluated by ABR measurement and quantitative hair cell assessment. Treatment significantly (P<0.01) reduced the extent of permanent threshold shifts and outer hair cell loss. Interestingly, the protective effect of a 30 mg/kg dose was less than that of a 10 mg/kg dose. There were no adverse systemic or auditory function effects in three unexposed control subjects given 30 mg/kg ebselen. These findings indicate that ebselen attenuates noise-induced cochlear damage. The concentration that provides optimal protection against such damage has now to be determined.

Introduction

Noise-induced hearing loss (NIHL) is the primary cause of acquired hearing loss in industrialized countries. It is particularly important because it may affect the individual’s safety and work performance. A variety of mechanisms have been proposed to account for hearing loss after high intensity sound exposure. There are two main ones: (1) direct mechanical trauma to the organ of Corti and (2) overuse of metabolically dependent processes in the inner ear. The generation of reactive oxygen species (ROS) in the cochlea constitutes a mechanism for metabolic damage to the organ of Corti. The importance of ROS in NIHL is supported by previous findings: (1) ROS are significantly increased in the cochlea after noise exposure (Ohlemiller et al., 1999, Yamane et al., 1995); (2) noise trauma is also associated with significant increases in the activity level of endogenous antioxidant enzymes such as glutathione (GSH) (Yamasoba et al., 1998b); and (3) enhancement of ROS scavengers by various antioxidant interventions attenuates NIHL (Seidman et al., 1993, Quirk et al., 1994, Hu et al., 1997, Yamasoba et al., 1999).

Another suggested mechanism in NIHL is NO-induced tissue damage. NO is present in several cochlear mechanisms, including neurotransmission and blood flow regulation, and may be cytotoxic under pathological conditions (Takumida et al., 1997, Takumida and Anniko, 1988, Amaee et al., 1997, Franz et al., 1996). The NO generated under pathological conditions reacts with superoxide anion (O2), forming peroxynitrite (ONOO) which may mediate oxidation, nitration, or nitrosation reactions leading to impaired function, toxicity and alteration of signaling pathways (Arteel et al., 1999). Oxidative stress after anoxia and noise trauma increases the release of excitatory amino acid (EAA) (Coyle and Puttfarcken, 1993) and produces excess release of EAA, causing excitotoxic damage to dendrites beneath the inner hair cells (IHCs) (Pujol and Puel, 1999). Excessive release of EAA contributes to ROS formation and the excessive synthesis of NO (Dawson et al., 1991, Yamauchi et al., 1998).

Neuronal-type NO synthase (NOS) I and endothelial-type NOS III are present in guinea pig cochlea (Fessenden and Schacht, 1998). Popa et al. (2001) reported NOS I staining in the spiral ganglion, cochlear nerve fibers, outer hair cells (OHCs), supporting cells and stria vascularis. Compared to NOS I, there was less intense staining by NOS III in the OHCs, supporting cells and spiral ganglion cells. Weak NOS II staining was present only in a few cochlear nerve fibers. The fluorescence intensity of NO in guinea pig cochlea is enhanced by L-arginine or glutamate stimulation and increases significantly after inoculation with lipopolysaccharide (LPS) (Takumida and Anniko, 2001). Carboplatin increases NO levels significantly, as well as the activities of xanthine oxidase and manganese-superoxide dismutase, in rat cochlea (Husain et al., 2001). Shi and associates (Shi et al., 2002) showed that broadband noise exposure (3 h/day at 120 dB sound pressure level (SPL) on three consecutive days) increases the NO concentration in the perilymph of guinea pig cochlea and that specific dyes for NO and ROS show greater fluorescence in the IHCs and OHCs of noise-exposed animals than in unexposed controls. Moreover, a neuronal NOS inhibitor reduces the threshold shift of the compound action potential in guinea pigs subjected to 30 min of ischemia (Tabuchi et al., 2001). These findings indicate that NO has importance in noise-induced cochlear damage.

Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, mimics GSH peroxidase (GSH Px), reacts with ONOO and inhibits the enzymes lipoxygenase, NO synthase, NADPH (reduced nicotinamide adenine dinucleotide phosphate) oxidase, protein kinase C and H+/K+-ATPase (Schewe et al., 1994). Although not a strong scavenger of ROS, it effectively scavenges organic hydroperoxides. Because organic hydroperoxides promote lipid peroxidation, their removal is analogous to scavenging ROS. Ebselen is reported to protect against the cochlear damage induced by gentamicin (Takumida et al., 1999), cisplatin (Kopke et al., 1997, Rybak et al., 1999, Rybak et al., 1999, Rybak et al., 2000) and LPS-induced labyrinthitis (Takumida et al., 1999), probably by scavenging NO and inhibiting lipid peroxidation. Because NO generation is also thought to be involved in noise-induced cochlear damage, we investigated whether ebselen prevents or attenuates NIHL in the guinea pig.

Section snippets

Animals

Nineteen healthy, otomicroscopically normal, pigmented guinea pigs weighing 250–300 g were used. Three days after arrival, their hearing was confirmed to be within the normal range (within one standard deviation of the normative lab baseline) by auditory brainstem response (ABR) measurements.

Animals were randomly allocated to the treatment and control groups. Treatment guinea pigs were randomly assigned to one of three groups, then exposed to noise for 5 h. Treatment and control solutions were

Baseline ABR thresholds

ABR thresholds 2 days before noise exposure were essentially equivalent in all the ears. No significant differences in the baseline ABR thresholds among the groups were found at any frequency.

Protective effects on permanent threshold shifts

Fig. 1 shows the ABR threshold shifts (average change in the threshold level, relative to the pre-noise ABR threshold) across the frequencies tested 14 days after noise exposure. Means of threshold shifts are plotted for the left ears as a function of the treatment group. The vehicle-only animals (group I)

Discussion

We found that ebselen (10 and 30 mg/kg) administered orally by gavage to guinea pigs attenuated noise-induced permanent threshold shifts and cochlear damage. Its protective effects on hearing function and hair cell survival were statistically significant for both concentrations. The higher dose, however, produced less attenuation of ABR threshold shifts and greater hair cell damage in the noise-exposed animals; however, it was not harmful to cochleas of control animals unexposed to noise. These

Acknowledgments

We thank Professor Josef M. Miller, Kresge Hearing Research Institute, The University of Michigan, and Professor Kimitaka Kaga, Department of Otolaryngology, The University of Tokyo, for their valuable advice.

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