Su Hua Sha

Differential vulnerability of basal and apical hair cells is based on intrinsic susceptibility to free radicals.

The base of the cochlea is more vulnerable to trauma than the apex as seen in the pattern of hair cell damage by cisplatin or aminoglycosides. The differential vulnerability is maintained in organotypic cultures exposed directly to these drugs, suggesting there may be an intrinsic difference in sensitivity to damage along the cochlear spiral. We therefore investigated the survival capacity of isolated outer hair cells and strips dissected from different turns of the guinea pig organ of Corti in short-term culture. Cells were stained with fluorescent indicators of viable or dead cells, calcein-AM and ethidium homodimer. After 5 h at room temperature, up to 90% of outer hair cells from the apex survived, but less than 30% from the base. In contrast, basal inner hair cells remained viable, and supporting cells survived for at least 20 h. The difference in survival capacity between basal and apical outer hair cells coincided with a significantly lower level of the antioxidant glutathione in basal outer hair cells compared with apical outer hair cells. This suggested that basal outer hair cells may be more vulnerable to free-radical damage than apical outer hair cells. The survival of basal outer hair cells was significantly improved by addition of the radical scavengers n-acetyl cysteine, p-phenylenediamine, glutathione, mannitol or salicylate. The protection by antioxidants implies that the accelerated death of basal outer hair cells is due to free-radical damage. The results support an intrinsic susceptibility to free radicals that differs among cochlear cell populations. This differential provides a rational explanation for base-to-apex gradients observed in various forms of cochlear pathology.

Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague–Dawley rat

The availability of genetic information, transgenic and knock-out animals make the mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for kanamycin ototoxicity in adult CBA/J, C57BL/6 and BALB/c mouse strains and a comparison to Sprague-Dawley rats. Five-week-old mice were injected subcutaneously twice daily with 400-900 mg kanamycin base/kg body weight for 15 days. Kanamycin induced dose-dependent auditory threshold shifts of up to 70 dB at 24 kHz as measured by auditory brain stem-evoked responses. Vestibular function was also affected in all strains. The functional deficits were accompanied by hair cell loss in both cochlear and vestibular neurosensory epithelia. Concomitant administration of the antioxidant 2,3-dihydroxybenzoate significantly attenuated the kanamycin-induced threshold shifts. In adult male Sprague-Dawley rats, doses of 1 x 500 mg or 2 x 300 mg kanamycin base/kg body weight/day x 14 days induced threshold shifts of approximately 50 dB at 20 kHz. These were accompanied by loss of outer hair cells. The order of susceptibility, BALB>CBA>C57, was not due to differences in the pharmacokinetics of kanamycin. It also did not correlate with the presence of Ahl/Ahl2 genes which predispose C57 and BALB strains, respectively, to accelerated age-related hearing loss. Pigmentation, however, paralleled this rank order suggesting an influence of melanin on cochlear antioxidant status.

Oxidative imbalance in the aging inner ear

The mammalian inner ear loses its sensory cells with advancing age, accompanied by a functional decrease in balance and hearing. This study investigates oxidant stress in the cochlea of aging male CBA/J mice. Glutathione-conjugated proteins, markers of H2O2-mediated oxidation, began to increase at 12 months of age; 4-hydroxynonenal and 3-nitrotyrosine, products of hydroxyl radical and peroxynitrite action, respectively, were elevated by 18 months. Immunoreactivity to these markers was stronger in the supporting cells (Deiters and pillar cells) than the sensory cells and appeared later (23 months) in spiral ganglion cells and in the stria vascularis and spiral ligament. Conversely, antioxidant proteins (AIF) and enzymes (SOD2) decreased by 18 months in the organ of Corti (including the sensory cells) and spiral ganglion cells but not in the stria vascularis. These results suggest the presence of different reactive oxygen species and differential time courses of oxidative changes in individual tissues of the aging cochlea. An imbalance of redox status may be a component of age-related hearing loss.

Caspase-independent pathways of hair cell death induced by kanamycin in vivo.

Cochlear and vestibular sensory cells undergo apoptosis when exposed to aminoglycoside antibiotics in organ culture, but mechanisms of chronic drug-induced hair cell loss in vivo are unclear. We investigated cell death pathways in a mouse model of progressive kanamycin-induced hair cell loss. Hair cell nuclei showed both apoptotic- and necrotic-like appearances but markers for classic apoptotic pathways (cytochrome c, caspase-9, caspase-3, JNK, TUNEL) were absent. In contrast, drug treatment caused EndoG translocation, activation of μ-calpain, and both the synthesis and activation of cathepsin D. Poly (ADP-ribose) polymerase 1 (PARP1) was decreased, but a caspase-derived 89 kDa PARP1 fragment was not present. The mRNA level of PARP1 remained unchanged. Thus, chronic administration of aminoglycosides causes multiple forms of cell death, without a major contribution by classic apoptosis. These results provide a better understanding of the toxic effects of aminoglycosides and are relevant to design protection from aminoglycoside-induced hearing loss.

Stimulation of free radical formation by aminoglycoside antibiotics

We have previously shown gentamicin to form a redox-active iron chelate. This study investigates whether other aminoglycosides can likewise stimulate the generation of reactive oxygen species (free radicals). Kanamycin, neomycin and streptomycin were compared to gentamicin in intact cells and in cell-free in vitro assays using luminescence detection with lucigenin or luminol. Neutrophils and Epstein-Barr virus-transformed lymphoblastoid cells served as cell models in which a respiratory burst of superoxide was induced by phorbol ester. The addition of millimolar amounts of any of the aminoglycosides increased the luminescence significantly. The drugs also increased the formation of free radicals in an enzymatic (hypoxanthine-xanthine oxidase) and a non-enzymatic (phenazine methosulfate-NADH) superoxide-generating system. Half-maximal stimulation was reached with (0.4 mM gentamicin, and there was an absolute requirement for an electron donor, arachidonic acid. In both intact cells and cell-free systems, gentamicin-enhanced luminosity was suppressed by iron chelators. These results demonstrate that different aminoglycoside antibiotics can stimulate the formation of free radicals in biological and in cell-free systems. Luminescence detection is a convenient assay method to investigate the redox properties of these drugs.

NF-κB pathway protects cochlear hair cells from aminoglycoside-induced ototoxicity

Cell death in outer hair cells of the mammalian inner ear induced by aminoglycoside antibiotics is mediated by reactive oxygen species (ROS) and can be prevented by antioxidants. The current study investigates the role of the nuclear factor (NF)‐κB pathway in cell death or survival in adult CBA mice. Kanamycin (700 mg/kg subcutaneously, twice per day) progressively destroys hair cells but after 7 days of treatment auditory function and morphology are not yet affected significantly, permitting investigations of early events in drug‐induced cell death. Immunostaining for 4‐hydroxynonenal, indicative of lipid peroxidation, was elevated in the cochlea, but there was no effect on nitrotyrosine, a marker for peroxynitrite. NF‐κB was increased at 3 hr, 3 days, and 7 days of treatment, with p50 and p65 proteins as its most abundant subunits. Immunoreactivity for p50 was present in nuclei of inner hair cells and supporting cells that survive the drug treatment. In contrast, nuclei of outer hair cells were devoid of label. Concomitant injections of antioxidants, however, such as 2,3‐dihydroxybenzoic acid or salicylate (which prevent cell death induced by kanamycin), promoted the translocation of NF‐κB into the nuclei of outer hair cells. In addition, kanamycin treatment decreased tyrosine phosphorylation of the inhibitory IκBα protein, leading to increased IκBα levels in the cochlea; the effect was reversed by cotreatment with antioxidants. These results suggest that changes in the redox state of the cochlea stimulate the activation of NF‐κB and that this activation is cell protective.

Tanshinone (Salviae miltiorrhizae Extract) Preparations Attenuate Aminoglycoside-Induced Free Radical Formation In Vitro and Ototoxicity In Vivo

ABSTRACT Antioxidant therapy protects against aminoglycoside-induced ototoxicity in animal models. A clinically suitable antioxidant must not affect the therapeutic efficacy of aminoglycosides or exhibit any side effects of its own. In addition, the treatment should be inexpensive and convenient in order to be implemented in developing countries where the use of aminoglycosides is most common. Standardized Salviae miltiorrhizae extracts (Danshen) are used clinically in China and contain diterpene quinones and phenolic acids with antioxidant properties. We combined in vitro and in vivo approaches to investigate the effect of a clinically approved injectable Danshen solution on aminoglycoside-induced free radical generation and ototoxicity. In vitro, Danshen inhibited gentamicin-dependent lipid peroxidation (formation of conjugated dienes from arachidonic acid), as well as the gentamicin-catalyzed formation of superoxide (in a lucigenin-based chemiluminescence assay) and hydroxyl radicals (oxidation of N,N-dimethyl-p-nitrosoaniline). Danshen extracts were then administered to adult CBA mice receiving concurrent treatment with kanamycin (700 mg/kg of body weight twice daily for 15 days). Auditory threshold shifts induced by kanamycin (approximately 50 dB) were significantly attenuated. Danshen did not reduce the levels in serum or antibacterial efficacy of kanamycin. These results suggest that herbal medications may be a significantly underexplored source of antidotes for aminoglycoside ototoxicity. Such traditional medicines are widely used in many developing countries and could become an easily accepted and inexpensive protective therapy.

Formation of reactive oxygen species following bioactivation of gentamicin

The present study investigated the ability of gentamicin to catalyze free radical reactions and probed the underlying mechanisms by hydroethidine imaging, oxygen consumption, and reduction of cytochrome c. In Epstein-Barr virus-transformed lymphoblastoid cells, a respiratory burst was induced by phorbol ester and detected by hydroethidine, a fluorescent indicator of superoxide radical. The addition of gentamicin increased the fluorescence two-fold while gentamicin did not produce fluorescence in the absence of phorbol ester. In membrane preparations, gentamicin did not enhance NADPH consumption ruling out a direct activation of NADPH oxidase. The formation of reactive oxygen species by gentamicin was additionally supported by experiments that showed gentamicin increased oxygen consumption two-fold in intact cells and a cell-free system. In addition, generation of superoxide was indicated by the gentamicin-stimulated reduction of cytochrome c. The stimulation by gentamicin depended upon the presence of iron (FeII/FeIII) and of arachidonic acid as an electron donor. These results support the hypothesis that an iron-gentamicin complex can increase reactive oxygen species in nonenzymatic and in biological systems. The requirement for a reductive activation in intact cells (e.g., by a respiratory burst) is interpreted as the conversion of an inactive FeIII-gentamicin to a redox-active FeII-gentamicin complex.

Aspirin attenuates gentamicin ototoxicity : From the laboratory to the clinic

This article reviews recent advances in the protection from the adverse auditory or vestibular side effects associated with antibacterial treatment with aminoglycoside antibiotics. Compelling evidence from animal models suggests that reactive oxygen species are part of the initial mechanisms that trigger apoptotic and necrotic cell death in the inner ear. Consequently, antioxidants protect against aminoglycoside-induced hearing loss in animals and, importantly, they do so without compromising drug serum levels or antibacterial efficacy. While clinical studies have long confirmed the ototoxicity of aminoglycosides in human, a trial on protection was only recently reported (Sha, S.-H., Qiu, J.-H., Schacht, J., 2006. Aspirin attenuates gentamicin-induced hearing loss. New Engl. J. Med. 354, 1856-1857). Based on the finding that salicylate afforded protection in animals, the efficacy of aspirin (acetyl salicylate) was tested in a randomized double-blind placebo-controlled study in patients receiving gentamicin for acute infections. Fourteen of 106 patients (13%) met the criterion of hearing loss in the placebo group while only 3/89 (3%) were affected in the aspirin group (p=0.013). Aspirin did not influence gentamicin serum levels or the course of therapy. These results indicate that therapeutic protection from aminoglycoside ototoxicity may be extrapolated from animal models to the clinic. Furthermore, medications as common as aspirin can significantly attenuate the risk of gentamicin-induced hearing loss.

Recent Advances in Understanding Aminoglycoside Ototoxicity and Its Prevention

Studies over the last decade have left little doubt that reactive oxygen species (ROS) participate in the cellular events leading to aminoglycoside-induced hearing loss. The evidence ranges from the demonstration of aminoglycoside-mediated ROS formation in vitro to the prevention of ototoxicity by antioxidants in guinea pig in vivo. Here we review a hypothesis of the mechanism of toxicity, discuss possible causes underlying the gradient in base-to-apex sensitivity of outer hair cells, and present recent results on the adult mouse as a new animal model of aminoglycoside ototoxicity and its prevention.