Glutathione limits noise-induced hearing loss

Abstract

The generation of reactive oxygen species (ROS) is thought to be part of the mechanism underlying noise-induced hearing loss (NIHL). Glutathione (GSH) is an important cellular antioxidant that limits cell damage by ROS. In this study, we investigated the effectiveness of a GSH supplement to protect GSH-deficient animals from NIHL. Pigmented guinea pigs were exposed to a 4 kHz octave band noise, 115 dB SPL, for 5 h. Group 1 had a normal diet, while groups 2, 3 and 4 were fed a 7% low protein diet (leading to lowered tissue levels of GSH) for 10 days prior to noise exposure. One hour before, immediately after and 5 h after noise exposure, subjects received either an intraperitoneal injection of 5 ml/kg body weight of 0.9% NaCl (groups 1 and 2), 0.4 M glutathione monoethyl ester (GSHE; group 3) or 0.8 M GSHE (group 4). Auditory thresholds were measured by evoked brain stem response at 2, 4, 8, 12, 16 and 20 kHz before and after noise exposure. Ten days post exposure, group 1 showed noise-induced threshold shifts of approximately 20 dB at 2, 16 and 20 kHz and 35 to 40 dB at other frequencies. Threshold shifts in group 2 were significantly greater than baseline at 2, 4, 16 and 20 kHz. GSHE supplementation in a dose-dependent fashion attenuated the threshold shifts in the low protein diet animals. Hair cell loss, as evaluated with cytocochleograms, was consistent with the auditory-evoked brainstem response results. Group 2 exhibited significantly more hair cell loss than any of the other groups; hair cell loss in group 3 was similar to that seen in group 1; group 4 showed less loss than group 1. These results indicate that GSH is a significant factor in limiting noise-induced cochlear damage. This is compatible with the notion that ROS generation plays a role in NIHL and that antioxidant treatment may be an effective prophylactic intervention.

Introduction

A variety of mechanisms have been proposed to account for hearing loss following high intensity sound exposure. They are classified into two main categories: (1) direct mechanical trauma to the organ of Corti and (2) ‘metabolic stress’ through increased oxidative metabolism in the inner ear. One important factor associated with tissue destruction through metabolic stress is the generation of reactive oxygen species (ROS). ROS formation results from increased mitochondrial activity and may also follow changes in cochlear blood flow associated with intense sound exposure (Lamm and Arnold, 1996, Scheibe et al., 1990, Thorne and Nuttall, 1987). The importance of ROS in noise-induced hearing loss (NIHL) and tissue damage is supported by findings that: (1) superoxide anion radicals emerge in the stria vascularis after intense sound exposure (Yamane et al., 1995); (2) hydroxyl radical levels significantly increase in the cochlea following intense sound exposure (Ohlemiller and Dugan, 1998); (3) conditioning noise exposure which reduces NIHL increases the activity of some antioxidant enzymes (Jacono et al., 1997), (4) reduction in the endogeneous antioxidant, glutathione (GSH, L-glutamyl-L-cysteinylglycine), increases NIHL (Yamasoba et al., 1998b), (5) GSH is upregulated in the lateral wall following noise exposure (Yamasoba et al., 1998a) and (6) NIHL is attenuated by a variety of antioxidant interventions. The latter include: superoxide dismutase-polyethylene glycol, a scavenger of ROS, and allopurinol, a blocker of ROS production and potential scavenger of ROS (Seidman et al., 1993); lazaroids, lipid peroxidation inhibitors and ROS scavengers (Quirk et al., 1994); R-phenylisopropyl adenosine, an adenosine analogue effective in upregulating antioxidant enzyme activity levels (Hu et al., 1996); and the antioxidant mannitol and the iron chelator deferoxamine mesylate (Yamasoba et al., 1999).

GSH is the most abundant non-protein thiol in mammalian cells. It serves as an antioxidant by reacting with ROS either directly or in reactions catalyzed by GSH peroxidases or GSH transhydrogenases (Meister and Anderson, 1983, Meister, 1991, Orrenius and Moldéus, 1984). GSH may be especially important for organs that are exposed to exogenous toxins or oxidative stress.

In the inner ear, substantial amounts of GSH are normally present (Lautermann et al., 1997). GSH immunoreactivity is preferentially distributed in the stria vascularis, spiral ligament and vestibular end organs (Usami et al., 1996). This GSH distribution overlaps with that of GSH S-transferase, whose activity is highest in the lateral wall tissues, intermediate in the neurosensory epithelium, and lowest in the modiolus (El Barbary et al., 1993). Compared to brain tissues GSH S-transferase activity is much higher in cochlear lateral wall and neurosensory epithelium.

Increasing cochlear GSH may protect against gentamicin ototoxicity (Garetz et al., 1994a, Garetz et al., 1994b), whereas reduction of GSH enhances hearing loss induced by gentamicin (Lautermann et al., 1995a), cisplatin (Lautermann and Schacht, 1996) or the combination of aminoglycosides and ethacrynic acid (Hoffman et al., 1987, Hoffman et al., 1988, Lautermann and Schacht, 1996). Cisplatin-induced tissue damage in the inner ear correlates with a decrease in GSH levels following cisplatin administration (Ravi et al., 1995).

Glutathione monoethyl ester (GSHE) is an effective delivery agent for GSH. It is transported into cells and hydrolyzed, presumably by intracellular esterases, to GSH and ethyl alcohol (Puri and Meister, 1983, Anderson and Meister, 1989). In contrast, GSH itself does not readily enter cells. After intraperitoneal injections into mice, GSHE effectively increased the GSH levels in liver and kidney (Puri and Meister, 1983). Likewise, GSH levels in the cochlea that were decreased by a low protein diet could be restored by supplementary administration of GSHE (Lautermann et al., 1995b).

We examine here the influence of a low protein diet on auditory threshold shifts following noise exposure and the effect of dietary GSHE supplementation.