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|Year : 2001 | Volume
| Issue : 11 | Page : 49--64
The influence of superoxide dismutase and glutathione peroxidase deficiencies on noise induced hearing loss in mice
Sandra L McFadden1, Kevin K Ohlemiller2, Dalian Ding1, Marlene Shero1, Richard J Salvi2,
1 Center for Hearing and Deafness, University at Buffalo, Buffalo, NY 14214, USA
2 Central Institute for the Deaf, St. Louis, MO 63110, USA
Sandra L McFadden
Center for Hearing and Deafness, University at Buffalo, Buffalo, NY 14214
One consequence of noise exposure is increased production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals, in the cochlea. ROS can cause oxidative damage to diverse cellular components, including membranes, proteins, and DNA, if they are not «DQ»neutralised«DQ» by antioxidant defences. Two important enzymes of the cochlear antioxidant defense system are cytosolic copper/zinc superoxide dismutase (SOD1) and selenium-dependent glutathione peroxidase (GPx1). These metalloenzymes work together to regulate ROS production in virtually every cell in the body, and they may be important for limiting cochlear damage associated with aging and acoustic overexposure. In this chapter, we describe a series of experiments using mice with targeted deletions of Sod1 or Gpx1, the mouse genes that code for SOD1 and GPx1, respectively, to study the cellular mechanisms underlying noise-induced hearing loss (NIHL). The results from Sod1 and Gpx1 knockout mice provide insights into the link between endogenous levels of antioxidant enzymes and susceptibility to NIHL.
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McFadden SL, Ohlemiller KK, Ding D, Shero M, Salvi RJ. The influence of superoxide dismutase and glutathione peroxidase deficiencies on noise induced hearing loss in mice.Noise Health 2001;3:49-64
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McFadden SL, Ohlemiller KK, Ding D, Shero M, Salvi RJ. The influence of superoxide dismutase and glutathione peroxidase deficiencies on noise induced hearing loss in mice. Noise Health [serial online] 2001 [cited 2022 May 16 ];3:49-64
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Acoustic overexposure can cause cochlear damage and noise-induced hearing loss (NIHL) in susceptible individuals. Many factors are likely to influence individual susceptibility to NIHL, including an imbalance between reactive oxygen species (ROS) produced during cellular metabolism and the cochlea's defences against them. Four lines of evidence support a link between ROS and NIHL. First, noise exposure leads to increased levels of ROS such as superoxide radicals (O 2 -), hydroxyl radicals (OH) and hydrogen peroxide (H 2 O 2 ) in the cochlea (Nicotera et al., 1999; Ohlemiller et al., 1999; Yamane et al., 1995a,b). Second, activity levels of cochlear antioxidants and related enzymes increase as a result of noise exposure (Bobbin et al., 1995; Jacono et al., 1998; Yamasoba et al., 1998a). Third, noise-induced temporary threshold shifts (TTS), permanent threshold shifts (PTS) and hair cell losses can be reduced with pharmacological treatments that target ROS or induce antioxidant activity (Hight et al., 1999; Hight et al., 2000; Liu et al., 1999; Hu et al., 1997; Seidman et al., 1993; Yamasoba et al., 1998b;Yamasoba et al., 1999). Fourth, noise-induced TTS, PTS and hair cell damage can be increased by pharmacological manipulations that deplete cochlear antioxidants (Henderson et al., 1999; Yamasoba et al., 1998b). In light of this evidence, it is reasonable to expect that individuals with impaired cochlear defences against ROS would be more susceptible to NIHL than individuals with normal antioxidant defences. Conversely, treatments that enhance antioxidant defences might protect susceptible individuals from NIHL.
We have been exploring the effects of specific antioxidant enzyme deficiencies on cochlear vulnerability to aging and noise using two types of mice with targeted gene mutations ("knockout" mice). One mouse model has a targeted deletion of Sod1, the gene that codes for cytosolic copper/zinc superoxide dismutase (Cu/Zn SOD or SOD1). SOD1 is the most prominent member of a family of SOD enzymes that also includes mitochondrial manganeseSOD (Mn-SOD, or SOD2) and extracellular Cu/Zn SOD (EC-SOD, or SOD3). The second mouse model has a targeted mutation of Gpx1, the gene that codes for the cytosolic seleniumdependent form of glutathione peroxidase (GPx1). GPx1 is the predominant form of glutathione peroxidase, and the isoform believed to participate most widely in protecting cells from oxidative stress (Brigelius-Flohe, 1999). SOD1 and GPx1 are found in the cytosol of virtually every cell in the body, and they work together to regulate levels of several ROS [Figure 1]. To understand how this is accomplished, and to lay the groundwork for interpreting the results of our experiments with Sod1 and Gpx1 knockout mice, it may be useful to provide a brief overview of ROS and how SOD1 and GPx1 regulate ROS levels in the cochlea and other tissues.
Reactive Oxygen Species and Cochlear Defences Against Them
What are ROS? ROS are ions, atoms or molecules that have the ability to oxidize (take electrons from) reduced molecules. ROS with one or more unpaired electrons are referred to as "free radicals." Free radicals in the cochlea and other tissues include •O 2 -, various peroxyl radicals (RO 2 ), and •OH (note that the dot in each formula represents an unpaired electron).
Nonradical ROS include H 2 O 2 and peroxynitrite (ONOO-). Many ROS perform useful functions in the body, such as regulating vascular tone, acting as intra- and intercellular signalling molecules, promoting cell proliferation, and acting as antimicrobial agents (Gutteridge and Halliwell, 1999; Babior, 1999). However, ROS can damage cells when they react indiscriminately or with inappropriate targets, as described below. To a large extent, cellular damage is prevented by endogenous antioxidant defences. These are not completely effective, however, and some oxidative damage occurs constantly in the human body (Gutteridge and Halliwell, 1999). Oxidative damage is exacerbated when cells are metabolically challenged or defence mechanisms are impaired.
How do ROS damage cells? ROS can damage cells by attacking a variety of cellular components and disrupting normal cellular processes (Adams et al., 1996; Halliwell, 1992; Halliwell et al., 1992, Orrenius and Nicotera, 1994). They can oxidize lipids and proteins (including membrane bound enzymes and receptors), destroy or destabilize membranes, disrupt ionic balance, interfere with cellular signalling and calcium homeostasis, attack DNA and disrupt protein synthesis, alter cytoskeletal components, and damage DNA repair and transcription processes. ROS can also trigger the generation of excitatory amino acids, leading to excitotoxic damage to neurons.
The toxicity of a particular ROS can be influenced by its biological milieu, including the presence of transition metals (metal ions with one or more unpaired electrons) and other biomolecules and ROS (Halliwell, 1992; Halliwell et al., 1992). The pH of the chemical environment is particularly important, because it affects both the formation and the stability of ROS. For example, the lifetime of •O 2 - increases by a factor of 10 for each pH unit above pH 6.0 - i.e., stability increases as alkalinity increases.
Irrespective of the biological milieu, some ROS are consistently more reactive and harmful than others. One of the least reactive ROS is •O 2 -. Nonradical ROS such as H 2 O 2 and ONOO- tend to be moderately reactive. -OH is at the extreme end of the reactivity continuum. Because it reacts almost instantaneously with any substance (DNA, proteins, lipids and carbohydrates) near its site of generation, it is the most dangerous ROS known. When •OH reacts with other biological molecules (i.e., by addition to unsaturated bonds, hydrogen abstraction, and electron transfer), it can initiate and propagate chain reactions that lead to extensive cell and membrane damage.
How are ROS formed? ROS are formed in a variety of ways in vivo, with all but a few arising from •O 2 - (Babior, 1997; Halliwell and Gutteridge, 1999). Metabolic processes that generate •O 2 - include: (a) aerobic cellular respiration and the activity of electron transport chains during oxidative phosphorylation; (b) the oxidation of thiols such as cysteine, methionine and glutathione (GSH); (c) the activity of phagocytes, which use ROS to kill foreign invaders; and (d) the auto-oxidation of biomolecules such as catecholamines and hemoglobin. It is estimated that the human body generates more than 2 kg of •O 2 - per year during normal cellular metabolism. •O 2 - production is increased under conditions of metabolic stress induced by heavy exercise, certain drugs and toxins, ischemia/reperfusion, infections, and various disease states (Evans and Halliwell, 1999).
O2- can damage cells directly, by initiating lipid peroxidation and by reducing the activity of many enzymes, including mammalian creatine kinase (McCord and Russell, 1988), the NADH dehydrogenase complex of the mitochondrial electron transport chain (Zhang et al., 1990), antioxidant enzymes such as catalase and GPx (Halliwell and Gutteridge, 1999), and enzymes that include or depend on sulfhydryls, including glutamic acid decarboxylase (GAD), acetylcholinesterase (AChE), and sodium-potassium ATP-ase (Na,K-ATPase) (Pierson and Gray, 1982). •O 2 - can also damage cells indirectly, through the formation of other ROS with greater toxicity.
Four important pathways by which •O 2 -participates in the formation of more toxic ROS are illustrated in [Figure 1]. The first pathway involves the protonation of •O 2 - to form the hydroperoxyl radical (HO 2 ). HO 2• exists in equilibrium with •O 2 - at physiological pH and is favoured at more acidic pH. Because it is uncharged, HO 2• can cross membranes more easily than •O 2 - to initiate lipid peroxidation (Halliwell and Gutteridge, 1999). Peroxyl radicals can also attack membrane proteins such as receptors and membrane-bound enzymes, oxidize cholesterol, and modify DNA (Evans and Halliwell, 1999). The second pathway involves the dismutation of •O 2 - to H 2 O 2 and molecular oxygen (O2), either spontaneously or through an enzymatic process catalyzed by SOD [Figure 1]. SOD-catalyzed dismutation occurs at a rate that is approximately four orders of magnitude faster than the nonenzymatic reaction at pH 7.4. The third pathway involves the combination of •O 2 - and nitric oxide (NO) to form ONOO-. This is a nonenzymatic, diffusioncontrolled reaction that occurs at an extremely fast rate at physiological pH (Halliwell and Gutteridge, 1999).
The fourth reaction shown in [Figure 1] is the formation of •OH from H 2 O 2 in Haber-Weiss reactions and Fenton reactions (i.e., reactions catalyzed by metal ions). •O 2 - contributes to OH formation by generating H 2 O 2 , participating in Haber-Weiss reactions, and facilitating Fenton reactions. •O 2 - facilitates Fenton reactions by liberating iron from iron-sulphur clusters (Keyer and Imlay, 1996), and by "recycling" metals. Metal recycling by •O 2 - is illustrated in [Figure 1] as the reduction of ferric iron (Fe 3 +) back to ferrous iron (Fe 2 +), making it available for another round of •OH production. As a result of metal recycling, trace levels of metals can catalyse the formation of very large quantities of OH. Consequently, intracellular levels of free metals (e.g., iron, copper and platinum) are extremely important in determining how much •OH is produced from •O 2 - and H 2 O 2 .
How are cells protected from ROS damage? To regulate ROS levels and protect itself from ROS damage, the body maintains a complex set of defences. Among these are agents that sequester or bind free metal ions that stimulate ROS formation or convert less reactive species to more reactive ones through Fenton reactions, and molecules that help repair DNA, proteins and membranes after they are damaged. Molecules such as vitamin C, vitamin E, and GSH are "chain-breaking" antioxidants; they inhibit lipid and protein peroxidation chain reactions by reducing cellular components after they have been oxidized by ROS. Cellular defences also include antioxidant enzymes that prevent ROS formation or remove ROS after they are formed. Prominent antioxidant enzymes in the cochlea and other tissues include the SODs, GPxs, and catalases (Jacono et al., 1998; Pierson and Gray, 1982; Rarey and Yao, 1996; Reaume et al., 1996; Yao and Rarey, 1996). SOD plays a role in regulating the levels of many ROS by controlling the availability of both •O2 - and H 2 O 2 . GPx and the catalases convert H 2 O 2 to water (H 2 O), thereby limiting the formation of OH• through Haber-Weiss and Fenton reactions. As shown in [Figure 1], GPx converts H 2 O 2 to H 2 O by using it convert GSH to oxidized GSH (GSSG). Another enzyme, glutathione reductase, then reduces GSSG back to GSH.
Pierson and Gray (1982) measured endogenous activity levels of SOD, GPx and catalase in brainstem, cochlea, cerebellum, retina, cortex and lung tissues from guinea pigs. Activity levels of each of these antioxidant enzymes tended to be higher in the cochlea than in other tissues, and higher in the lateral (stria vascularis) fraction of the cochlea than in the medial (organ of Corti) fraction. SOD and GPx activity levels measured in the cochlea (medial fraction), retina and cortex are compared in [Figure 2]. Other measurements of endogenous SOD in the cochlea (Yao and Rarey, 1996) confirm that SOD levels are higher in the stria vascularis than in the organ of Corti; intermediate levels of SOD activity are observed in the spiral ligament [Figure 3]. In each cochlear region, activity levels of cytosolic SOD1 exceed those of mitochondrial Mn-SOD [Figure 3]. In another report, Rarey and Yao (1996) described intense immunostaining for SOD1 in Reissner's membrane, IHCs, OHCs, and supporting cells of the organ of Corti.
Rationale for Experiments with Knockout Mice
Although we know that intense noise exposure is associated with increased levels of ROS in the cochlea (Nicotera et al., 1999; Ohlemiller et al., 1999; Yamane et al., 1995a,b), we do not yet know the mechanisms responsible for ROS generation, the actual ROS that initiate cochlear damage, or the pathways and mechanisms by which ROS damage occurs. With respect to sources of increased ROS during and after noise exposure, we can consider at least four possibilities: (1) increased activity of mitochondria to meet the increased energy demands of cells in the organ of Corti and stria vascularis; (2) ROS-generating phagocytes (e.g., monocytes, neutrophils, eosinophils or macrophages) recruited to areas of cellular injury; (3) events triggered by ischemia/reperfusion (Yamane et al., 1995a; Yamane et al., 1995b; Ohlemiller and Dugan, 1999), and (4) injury to tissues that causes the release of transition metals into the surrounding fluid and subsequent formation of ROS through Fenton reactions. The actual ROS that promote cochlear damage and the mechanisms by which damage occurs are even more poorly understood. Does •O 2 - cause cochlear damage directly (e.g., through lipid peroxidation or enzyme inactivation)? Is ONOO- a major contributor to noise-induced pathology? Does H 2 O 2 cause damage directly, or must it be converted to •OH? Is •OH the major source of ROS damage in the noise-exposed cochlea?
One way to approach questions about ROS and antioxidant contributions to NIHL is to observe the consequences of eliminating specific antioxidant enzymes. This has been our approach with Sod1 and Gpx1 knockout mice. Theoretically, experiments with Sod1 knockout mice should reveal the consequences of unopposed •O2 - generation and/or diminished levels of H 2 O 2 on susceptibility to NIHL. If •O2 -or its immediate products (e.g., HO 2 , ONOO-) are direct contributors to noise-induced cochlear pathology, then SOD1 deficiencies would be expected to promote damage from a noise exposure. If, however, noise-induced cochlear pathology depends more on H 2 O 2 pathways, then SOD1 deficiencies might actually protect the cochlea from damage. Experiments with Gpx1 knockout mice should show the effects of elevated levels of H 2 O 2 on susceptibility to NIHL. If H 2 O 2 and/or •OH are major contributors to NIHL, then GPx1 deficiencies might increase noise-induced cochlear pathology and hearing loss.
Subjects. Experiments utilized "SOD1 mice" (129/CD-1-Sod1 tm1 Cep; Cephalon, Inc., West Chester, PA) and "GPx1 mice" (obtained from Dr. Ye-Shih Ho). SOD1 mice were produced by replacing the Sod1 gene with the neomycin resistance gene in embryonic stem (ES) cells derived from an F1 cross of 129/Sv and 129/Re mice, and inserting the ES cells into donor embryos of CD-1 females. Germline positive chimeric males were mated with CD-1 females, and their heterozygous offspring were used to produce wild-type (WT) mice, heterozygous (HET) knockout mice, and homozygous knockout (KO) mice (Reaume et al., 1996). Although HET mice were used in most SOD1 experiments, our current presentation is limited to results from WT and KO mice.
GPx1 mice were produced by transplanting 129/SvJ-derived ES cells containing a targeted mutation of Gpx1 into donor embryos of C57BL/6 (B6) females. Germline positive chimeric males were mated with B6 females, and their heterozygous offspring were crossed. Mice used in the GPx1 experiment were the product of homozygous matings (KO x KO, WT x WT), hence no HET mice were available. Both WT and KO parentals were, however, the offspring of multiple HET X HET mating pairs; hence, the effects of background genetic variability were randomised.
The genotype of each mouse was determined through tissue typing (whole-blood or liver enzyme activity and protein concentrations) or polymerase chain reaction (PCR) of DNA [Figure 4]. SOD1 or GPx1 enzyme activity and protein were virtually eliminated in KO mice (Ohlemiller et al., 1999; Ohlemiller et al., 2000; Reaume et al., 1996).
Four experiments were performed using SOD1 mice: Exp. I: 15 WT and 15 KO mice aged 1-3 months (35-91 days); Exp. II: 5 WT and 5 KO mice aged 6 months (192-194 days); Exp. III: 6 WT and 3 KO mice aged 3 months (102 days), and 8 WT and 7 KO mice aged 7 months (208 days); and Exp. IV: 4 WT and 2 KO mice aged 7 months (206-234 days). Exp. V utilized GPx1 mice: 14 WT and 19 KO mice aged 2 months (52-60 days).
Auditory brainstem response (ABR) measurements and histology. Mice were anaesthetized with Avertin (=0.35 mg/g body weight, i.p.) or ketamine/xylazine (80 mg/kg/15 mg/kg, i.p.) and placed on a homeothermic blanket inside a sound chamber. Core temperature was maintained at 37.5 ± 1 °C. Auditory brainstem responses (ABRs) were recorded from subdermal electrodes inserted at the midline of the animal's head, posterior to the left ear, and at the back. Responses to pure-tone stimuli were amplified, filtered, digitised and averaged for 250-1000 stimulus presentations. Response threshold was defined as the lowest SPL at which any wave of the ABR could be recognized. ABRs were recorded before and after noise exposure to determine noise-induced TTS and PTS. At the end of the experiment, cochleas were removed and stained for hair cell counts, as described previously (Spongr et al., 1997; McFadden et al., 1999b).
Noise exposures. The effects of antioxidant enzyme deficiencies were tested under a variety of noise exposure conditions: filtered white noise (4.0-45.0 kHz) at 110 dB SPL for 1 h (Exps. I and V); 4 kHz octave band noise (OBN) at 110 dB SPL for 2 h (Exp. II); 4 kHz sine wave at 105 dB SPL for 168 h (Exp. III); and impulse noise at 150 dB peak SPL (Exp. IV). Details of noise generation and calibration can be found in previous publications (McFadden et al., 1999a; McFadden et al., 2000b; Ohlemiller et al., 1999).
Thresholds prior to noise exposure. SOD1 and GPx1 mice exhibited high-frequency hearing loss relative to CBA/CaJ mice, even at 1-3 months of age [Figure 5]. The high-frequency hearing losses in SOD1 and GPx1 mice arise from the parental strains (129, CD-1, and B6) used to create knockout mice. Within each strain, antioxidant enzyme deficiencies were associated with additional threshold elevations. Young adult Sod1 KO mice had significantly higher thresholds than Sod1 WT mice at low frequencies (5 and 10 kHz), and young adult Gpx1 KO mice had significantly higher thresholds than Gpx1 WT mice at 28.3 kHz. The magnitude of the effect was greater for GPx1 mice (=16 dB) than for SOD1 mice (=6-7 dB). The early appearance of genotypic differences suggests that antioxidant enzyme deficiencies either influence the development of the cochlea, or increase the vulnerability of the cochlea to environmental stimuli after the onset of hearing.
SOD1 mice of various ages were tested, allowing us to examine the influence of SOD1 deficiencies on age-related hearing loss (AHL) as well as NIHL. The results clearly showed that SOD1 deficiency exacerbated AHL, producing progressive elevations of ABR thresholds and dramatically greater losses of IHCs, OHCs, spiral ganglion cells, and primary auditory nerve fibers (McFadden et al., 1999b,c; McFadden et al., 2000a). Because of the progressive nature of hearing loss in SOD1 mice and the exacerbation of AHL by SOD1 deficiency, Sod1 KO mice were essentially deaf (mean thresholds >80 dB SPL at all frequencies) by 13 months of age [Figure 6]. It should be noted that the preexisting threshold differences between WT and KO mice complicates the interpretation of noiseinduced PTS, particularly for older mice.
Experiments I and II: Does SOD1 deficiency increase NIHL from acute exposure?
In our first experiment, young SOD1 mice were exposed to broadband noise at 110 dB SPL for 1 hr, and threshold shifts were measured 1, 14 and 28 days later (Ohlemiller et al., 1999). There were no significant changes in thresholds between 14 and 28 days after exposure; thus, TS at 14 days or later represents PTS [Figure 7]. As shown in [Figure 7], PTS of KO and WT mice differed significantly at 20 kHz (p Experiment III: Does SOD1 deficiency exacerbate NIHL caused by chronic overexposure?
Given the results from acute noise exposure experiments, we were interested in seeing if the effects of SOD1 deficiency would be magnified under conditions of chronic acoustic overstimulation and prolonged oxidative stress.
In Exp. III, young and middle-aged mice were exposed to a 4 kHz tone at 105 dB SPL for 168 hr, and PTS was measured 14 days later (McFadden et al., 2000a). In this experiment, the average PTS of KO mice was actually lower than that of WT mice, for both age groups [Figure 8]. There was a significant effect of age, with middle-aged mice developing more PTS than young mice, but there was no significant effect of genotype. Furthermore, there were no significant differences in hair cell losses between WT and KO mice in either age group.
Exp. IV: Does SOD1 deficiency influence NIHL caused by impulse noise?
Our final experiment with SOD1 mice utilized impulse noise at peak levels of 150 dB SPL. This type of noise has been shown to produce both mechanical and metabolic damage to the cochlea (Hamernik et al., 1984). One possible outcome of mechanical damage is the release of transition metals and the subsequent formation of •OH through Fenton reactions (Halliwell and Gutteridge, 1992). In addition, impulse noise may lead to higher levels of peroxides and ONOO- in the cochlea than continuous noise (Nicotera et al., 1999).
In Exp. IV, impulse noise produced mean PTS ranging from 0 to 25 dB SPL, with the greatest losses at 3 and 6 kHz [Figure 9]. Interestingly, KO mice developed approximately 20 dB less PTS at 3 kHz than WT mice. Although the number of mice exposed to the impulse noise was small, the difference between Sod1 KO and Sod1 WT mice in PTS at 3 kHz was statistically significant (p Does GPx1 deficiency influence NIHL in young adult mice?
The results from Exp. I supported a relationship between SOD1 deficiency and enhanced susceptibility to NIHL in young mice. Would GPx1 deficiency have a similar effect? In Exp. V, young GPx1 mice were exposed to the same broadband noise used in Exp. I (Ohlemiller et al., 2000). Two weeks after exposure, Gpx1 KO mice showed approximately 10-15 dB more PTS than Gpx1 WT mice at 10 and 20 kHz [Figure 10], even though thresholds of KO mice were already elevated at these frequencies prior to exposure (see [Figure 5]). Gpx1 KO mice also had significantly more IHC and OHC loss (p Sod1 WT mice, compared to 20 dB for Gpx1 WT mice. The differences between SOD1 and GPx1 WT mice reflect the influence of genetic background (parental strain differences). Mice with a 129,CD-1 parental background appear to have greater inherent susceptibility to NIHL than mice with a 129,B6 parental background. In young mice of both strains, however, antioxidant enzyme deficiencies significantly increased susceptibility to NIHL, enhancing losses by as much as 20 dB.
Summary and Discussion
The results clearly demonstrate that endogenous levels of SOD1 and GPx1 can influence susceptibility to NIHL. However, the nature of the ROS/antioxidant enzyme/NIHL relationship is complex. Young mice lacking SOD1 or GPx1 showed increased susceptibility to NIHL, by approximately 20 dB at 20 kHz [Figure 7],[Figure 10]. The magnitude of the effect may appear modest, but it is consistent with other studies in which TTS or PTS has been attenuated by pharmacological manipulations (e.g., Hight et al., 1999; Hu et al., 1997; Liu et al., 1999; Seidman et al., 1993). In middle-aged mice, homozygous deletion of Sod1 had little effect on NIHL associated with acute continuous noise exposure, and it may actually have protected the cochlea from NIHL associated with chronic exposure [Figure 8] and impulse noise [Figure 9].
One issue that must be addressed concerns the impact of genetic background. The parental strains that are commonly used to produce mutant (transgenic and knockout) mice (i.e., inbred B6 and 129; outbred CD-1) were originally selected for their advantages in breeding, fertility, viability and gene manipulation, not for any feature related to their hearing. Unfortunately for auditory research, each of these parental strains exhibits early onset, progressive hearing loss (Shone et al., 1991; Zheng et al., 1999), and this feature is consistently passed on to mutant models derived from them, including the SOD1 and GPx1 mice used in our noise exposure experiments. The hearing loss genes in our mice may have modified or dwarfed the effects of disabling antioxidant enzyme genes. That is, pre-existing cochlear pathology in our mice may have masked some of the effects of noise exposure, since a cochlea that is already damaged cannot be damaged again by acoustic trauma. Consequently, our data may underestimate the effect of SOD1 and GPx1 deficiency on susceptibility to NIHL. It will be important to test normal-hearing gene knockout models as they become available.
Despite complications introduced by genetic background, results from our experiments with SOD1 and GPx1 mice provide important insights into the relationship between ROS, antioxidant enzymes, and NIHL. The data from young and middle-aged SOD1 mice suggest that there may be developmental trends, whereby mechanisms of ROS generation, antioxidant protection, and/or cochlear vulnerability to noise change over an animal's lifetime. Developmental changes of this nature might be a factor contributing to changes in susceptibility to NIHL during development (Yanz and Abbas, 1982) and in adulthood (SOD1 mice, [Figure 8]; CBA mice, Miller et al., 1998).
Many factors complicate our understanding of ROS damage and the actions of antioxidants in the cochlea. Two factors relevant here are the effects of imbalances between different ROS, and imbalances between different antioxidant enzymes. A relevant example of how balances between different ROS can affect their toxicity is provided by Brune et al., (1999). They found that •NO alone could initiate apoptosis in rat mesangial cells, whereas the simultaneous generation of •NO and •O2 - actually protected the cells from apoptosis. If the generation of either NO or •O2- was offset relative to the other, protection from apoptosis was diminished. Extending this finding to our experiments, it is possible that the balance between •NO and •O2 -generated during noise exposure not only influenced the magnitude of NIHL, but also determined whether the effects of SOD1 deficiency were detrimental (Exp. I) or protective (Exps. III and IV).
Studies of other organ systems have shown that the balance between different antioxidants is important for normal cellular functioning; an excess of one antioxidant in relation to the other may have deleterious effects. In several studies, age-related increases in SOD activity that are not matched by increases in GPx activity have been implicated as a source of cell damage during aging (Cristiano et al., 1995; de Haan et al., 1995; Park et al., 1996). This type of antioxidant imbalance, favouring the generation of H2O2 and
OH, could account for the results seen in our KO mice under different noise exposure conditions. It will be interesting to see if susceptibility to NIHL changes in GPx1 mice as they age. It would also be interesting to examine the effects of eliminating both Sod1 and Gpx1 genes on AHL and NIHL.
A final point to consider is that there may be compensatory mechanisms in the KO mice to deal with increased levels of ROS generated during noise exposure. Indeed, Ghoshal et al., (1999) recently reported that levels of two other metalloenzymes, metallothionein-I and -II, are elevated in livers of homozygous Sod1 KO mice. If metallothioneins or other antioxidants are overexpressed in the cochleas of Sod1 KO mice, this might contribute to protection from ROS damage during noise exposure. However, if compensatory mechanisms protect KO mice from acoustic trauma but not from aging, this implies that different pathways mediate ROS damage in AHL versus NIHL.
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