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|Year : 2010 | Volume
| Issue : 48 | Page : 159--165
Dosing study on the effectiveness of salicylate/N-acetylcysteine for prevention of noise-induced hearing loss
John Coleman1, Xinyan Huang2, Jianzhong Liu1, Richard Kopke3, Ronald Jackson1,
1 Spatial Orientation Centre, Department of Otolaryngology, Naval Medical Centre San Diego, 34800 Bob Wilson Drive, San Diego, CA 92134, USA
2 Springfield Clinic, 1025 S 7th St Springfield, IL 62703, USA
3 Hough Ear Institute, 3400 N.W. 56th St, Oklahoma City, OK 73112-4463, USA
Spatial Orientation Center, Department of Otolaryngology, Naval Medical Center San Diego, San Diego, CA 92134
The efficacy of three different doses of sodium salicylate (SAL) in combination with one dose of N-acetylcysteine (NAC) to prevent noise-induced hearing loss was studied in chinchillas. After obtaining baseline-hearing thresholds, the chinchillas were randomly assigned to one of four treatment groups: three sets were injected intraperitoneally with 325 mg/kg NAC combined with 25, 50, or 75 mg/kg SAL, and a separate control group was injected with an equal volume of saline. Animals were injected twice daily for 2 days prior to and 1 hour before the noise exposure (6 hours to a 105-dB Standard Pressure Level octave band noise centered at 4 kHz). Immediate post-noise hearing thresholds were obtained followed by post-noise treatments at 1 hour then twice-daily for 2 days. Hearing tests continued at 1, 2, and 3 weeks post-noise, and immediately after the last hearing test, animals' cochleae were stained for hair cell counts. All the groups showed hearing improvement until week 2. However, at week 3, saline treated animals demonstrated a 17-33 dB SPL permanent threshold shift (PTS) across the test frequencies. Hearing loss was lowest in the 50 SAL/325 NAC mg/kg group (all frequencies, P < 0.001), and although PTS was reduced in the 25 and 75 mg/kg SAL dosage groups compared to the saline group, only the 75 mg/kg SAL group was significantly different at all but 2 kHz frequency. Coupled with the hearing loss, outer hair cell (OHC) loss was maximal in the 4-8 kHz cochlear region of saline treated animals. However, there was a substantial reduction in the mean OHC loss of the NAC plus 50 or 75 mg/kg (but not the 25 mg/kg) SAL groups. These findings suggest that SAL in combination with NAC is effective in reducing noise damage to the cochlea, but SAL has a relatively narrow therapeutic dosing window.
|How to cite this article:|
Coleman J, Huang X, Liu J, Kopke R, Jackson R. Dosing study on the effectiveness of salicylate/N-acetylcysteine for prevention of noise-induced hearing loss.Noise Health 2010;12:159-165
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Coleman J, Huang X, Liu J, Kopke R, Jackson R. Dosing study on the effectiveness of salicylate/N-acetylcysteine for prevention of noise-induced hearing loss. Noise Health [serial online] 2010 [cited 2023 Mar 30 ];12:159-165
Available from: https://www.noiseandhealth.org/text.asp?2010/12/48/159/64972
Noise is ubiquitous in today's increasingly industrialized world. Noise induced-hearing loss (NIHL) results not only from recreational sound exceeding safe limits but also can occur from damaging occupational exposures experienced in industry and military service. With the recent Middle East conflicts, nearly 68% of all injuries in action evacuations of US service personnel was due to exposures to extreme impulse noise or blasts from rocket propelled grenades, artillery, and improvised explosive devices (IEDs). It was reported that  60% of US personnel exposed to blasts suffer from permanent hearing loss and 49% also suffer from tinnitus. Hearing protection devices are only partially effective and their theoretical maximal potential for preventing hearing loss still does not afford complete protection due to extremely high noise levels of the newer weapons platforms and ototoxic effects of non-noise factors, such as industrial solvents and fuels, as well as non-compliance issues. Recent success of a number of compounds in preventing hearing loss suggests other strategies for otoprotection, namely, making the cochlea biologically more resistant to acoustic injury or treating the acutely injured cochlea through pharmacologic intervention. Free radicals, especially reactive oxygen species (ROS), have been linked to acoustic trauma-induced hair cell loss. ,, Several earlier studies revealed the specific damaging effects of ROS on the cochlea morphology and cochlear function. ,, In recent years, inflammation is also considered one of the mechanisms of acoustic trauma. Hirose et al,  found a large increase in CD45 (+) cells in the cochlea after noise exposure. Shi and Nuttall  reported loud sound activated expression of adhesion molecular protein and leukocyte migration. Tornabene et al,  studied the time course of immune cell recruitment after exposing mice to octave-band noise (8-16 kHz) at 118 dB and found a chemokine-induced increase of leukocytes, especially CD45 within days and F4/80-positive cells over the course of a week. Miyao et al,  also reported stronger immune response to antigen after acoustic trauma. It is possible that the inflammatory response plays an important role in propagating cellular damage in the cochlea; therefore, an anti-inflammatory drug such as salicylate may prevent cochlear damage.
Many studies have focused on the use of pharmacologic agents to help prevent or reduce NIHL. Antioxidants are among the most studied in recent years, and our laboratory, among others, has validated the use of N-acetylcysteine (NAC) in preventing noise-induced permanent threshold shift (NIPTS). NAC, a thiol compound, is the active ingredient in a Mucomyst; , an oral agent given to counteract liver oxidative damage in cases of acetaminophen overdose. Acetaminophen depletes hepatic intracellular-reduced glutathione (GSH), the major cellular antioxidant, while increasing the oxidized form (GSSG). Increased GSSG not only deactivates many enzymes but also strongly inhibits intracellular antioxidases such as superoxide dismutase.  By acting as a free radical scavenger through replenishment of GSH and by preventing c-Jun N-terminal kinase (JNK) activation (a cell death initiator) in a neuronal model of cell death, NAC can protect against this acetaminophen-induced damage. , NAC is deacetylated in the gut and well-absorbed, taken up as cysteine by membrane transporters into cells and converted to GSH. At therapeutic doses it has few side effects. , NAC has shown otoprotection when used in combination with other antioxidants such as acetyl-l-carnitine (ALCAR) and 4-hydroxy phenyl N-tert-butylnitrone (4-OHPBN). ,
Salicylate is an aspirin-related compound, distinct from aspirin. It was selected for this study due to its dual effects as an antioxidant as well as an anti-inflammatory agent. The hydroxyl free radical (OH-) was reported to be the prominent type of ROS formed in the cochlea secondary to acoustic overexposure.  Salicylate is chemically converted to dihydroxybenzoate (DHB) which is a specific scavenger of the (OH-) and DHB itself is important in preventing other free radical generating reactions.  As an iron chelator, DHB can prevent ROS formation by inhibiting the Fenton reaction.  Salicylate may also inhibit NF-gamma B, a potential activator of inflammatory or cell death pathways, , or induce heat shock proteins which are known to have a protective effect on the cochlea.  Salicylate also has been broadly used in the treatment of noise-induced , and ototoxic drug-induced ,, cochlear damage.
Unlike NAC which has relatively broad safety dosing window, salicylate has been reported to cause reversible hearing threshold shift and tinnitus. In fact, Jastreboff et al,  developed the first animal model of tinnitus using salicylate administration. Controversial results for salicylate have been reported as to whether it may enhance NIHL. ,,
The combination of NAC and salicylate was selected because each compound is Food and Drug Administration (FDA) approved; both the agents can be administrated orally, a clinically desirable delivery strategy; and each may prevent oxidative stress through different yet synergistic mechanisms. Our previous studies have demonstrated that NAC combined with salicylate was effective in reducing NIHL and hair cell death.  This study is a continuation of our previous research on the effectiveness of applying combinational antioxidant therapy by investigating the relative dose-response curve using one dose of NAC with varying doses of the second antioxidant, salicylate.  Utilization of combinatorial otoprotection is an active area of investigation in our group. There may be a greater level of otoprotection when applying different combinations of proven otoprotective compounds that have synergistic and additive effects against NIPTS than when these agents are administered individually.
Materials and Methods
The Institutional Review Board and the Institutional Animal Care and Use Committee of Naval Medical Center San Diego (NMCSD) approved the experimental protocol. The animals involved in this study were procured, maintained, and used in accordance with the Animal Welfare Act of 1996, as amended, and the Guide for the Care and Use of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, National Academy of Sciences - National Research Council. The animals were housed in the NMCSD animal facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.
Twenty-four female, adult Chinchilla laniger were equally divided into four study groups. All the animals underwent baseline-hearing thresholds using skin auditory brainstem response (ABR) testing within 2 days of the initial noise exposure. All subsequent measurements were represented as shifts from each animal's individual baseline. The four groups consisted of a saline control group and three salicylate-dosing groups of 25, 50, and 75 mg/kg. All the animals, with the exception of the saline group, also received 325 mg/kg NAC in the same injection. Animals were injected twice daily for 2 days prior to and 1 hour before the noise exposure then starting at 1 hour after the immediate post-noise hearing measurement and twice-daily for 2 days giving a total of 10 injections over 5 days. Control animals were injected with a similar volume of saline over the same schedule as the treatment groups. All four groups had audiologic tests performed pre-noise, 1-hour post-noise, and once a week for 3 weeks after noise exposure. Shortly after the last audiometric determination, animals were humanely euthanized, and the temporal bones were harvested and then stained with a vital dye to indicate living hair cells.
Auditory brainstem response measurement
Hearing thresholds were determined by ABR via subcutaneous needle electrodes placed in the skin of the head and posterior to the ear. Digitally generated stimuli consisted of alternating tone pips (2, 1, 0.5, 0.25 millisecond Blackman rise/fall ramp, 1.0, 0.5, 0.25, 0.125 millisecond duration at 2, 4, 6, and 8 kHz, respectively). The stimulus was routed through a computer-controlled attenuator to an insert earphone (Etymotic Research ER-2), which was positioned approximately 5 mm from the tympanic membrane. The output of the insert earphone was calibrated by measuring the sound pressure level at a position 4-5 mm away from the tympanic membrane. Animals were placed in a plastic restraint tube during the 30-minute recording procedure. The electrical response from the recording electrode was amplified (100,000Χ), filtered (100-3000 Hz), and fed to an A/D converter on a signal processing board in the computer. One thousand samples were averaged at each level. Stimuli were presented at the rate of 21/second and the stimulus level was varied in 10 dB descending steps until threshold was reached, then 5 dB ascending step to confirm. Threshold was defined as the mid-point between the lowest level at which a clear response was seen and the next lower level where no response was seen. The ABR thresholds were determined before injections of drugs and again before noise exposure, 1 hour following the first dose of post-noise exposure rescue therapy, and at various time points after the noise exposure (7, 14, and 21 days, respectively).
Before noise exposure, the animals were both handled (gentling) and acclimated to the wire cage, breeding collar, and booth for several hours on at least two consecutive days. The noise exposure, consisting of an octave band noise centered at 4 kHz, was generated by a standard audiometer set to white noise. This signal was routed through an attenuator (HP 350 D), a filter (Krohn-Hite 3550R), and a power amplifier (NAD S200) to an acoustic compression driver and horn (JBL 2446H and 2360A). The loudspeaker was suspended directly above the cages (112 ± 1 cm from driver opening to cage tops). During the noise exposure, the animals were restrained by a breeding collar and tether system secured in small wire animal cages with free access to water. This system secured each chinchilla in a comfortable position while preventing the animal from assuming a posture that would shield either of its ears. Each animal was exposed to noise at a level of 105 dB SPL (±1 dB SPL) for 6 hours. Whenever the animals were not exposed to noise, they were housed in a quiet animal colony with food and water available ad libitum.
Noise levels were measured using a sound pressure level meter (Larson and Davis 800B), a pre-amplifier (Larson and Davis model 825), and a condenser microphone (Larson and Davis, LDL 2559). The microphone was positioned within the cage at the level of the animal's head. The sound pressure level meter was used immediately before and immediately after each animal's noise exposure. In addition, a dosimeter was used to monitor each noise exposure to assure that sound levels were maintained throughout the 6-hour period.
After final physiological assessments (21 days after noise exposure), the animals were deeply anesthetized with ketamine/xylazine and decapitated with a guillotine. Each bulla was quickly removed from the skull. Cochleae were slowly perfused from oval window to the round window with 0.2 M sodium succinate and 1% nitrotetrazolium blue (NTB) in 0.1 M phosphate buffer (pH 7.4), then immersed in the same solution for 1 hour at 37°C. Cochleae were then fixed with 4% paraformaldehyde for 24 hours. Subsequently, the dissected cochleae and sections of the organ of corti were mounted on a slide. All specimens were examined for loss of hair cells under a light microscope (Olympus BH-2; Olympus Optical Co. Ltd., Tokyo, Japan). Data from each section were put into a worksheet (Origin, version 6.0, Micro Software Inc., Northampton, MA, USA) to construct a continuous range of data from the hook area to the apex of the cochlea. Absolute hair cell counts were converted to percentages of missing outer hair cells (OHCs) by dividing the cell count for the experimental animals by control value cell counts for normative values developed for each cochlear region. Each cochlea's length was normalized to 100%.  The number of OHCs as a function of percent distance from the apex was established using a linear regression model, and the percent distance from the apex was converted to frequency corresponding to the relative location. The percentage of missing hair cells was then plotted as a function of frequency. Histograms were developed for OHCs for each cochlea, and histogram means were computed and graphed for the four frequencies corresponding to the ABR test frequencies.
All noise-induced threshold shifts were calculated relative to the baseline thresholds measured before the administration of pharmacologic therapy or saline, and mean threshold shifts were analyzed using a repeated measures multiple analysis of variance (ANOVA) (Treatments Χ Time Χ Frequency). A two-way ANOVA (Treatments Χ Frequency) was applied to analyze group differences for OHC counts. Finally, a post hoc test (Newman-Keuls testing) was applied to determine the most significant comparisons for threshold shifts and hair cell counts. All testing was performed at a level of significance of PAudibility threshold measurements
The baseline audibility thresholds of all the animals used in this study are presented in [Figure 1]. As there were no statistically significant differences between the control, 25, 50, and 75 mg groups before noise exposure, all data are presented as means for the ABR frequencies of 2, 4, 6, and 8 kHz.
Immediately post-noise, the audibility thresholds as measured by ABR were not statistically significant for the control vs. 25, 50, and 75 mg/kg groups at any of the thresholds measured, except for control vs. 25 mg/kg group at 2 kHz [Table 1]. By week 2, there was still little difference between control and 25 mg/kg group (except week 1 at 2 kHz and week 3 at 6 kHz). However, there were significant differences in tested ABR thresholds between the control and the 50 and 75 mg/kg groups [Table 1]. [Table 2] provides the statistical comparisons between the experimental groups. There were significant differences between the lowest sodium salicylate (SAL) dose of 25 mg compared to 50 and 75 mg groups during recovery, and at week 3 in some of the tested frequencies. However, by week 3 post-noise, there were no significant differences between the two highest SAL doses of 50 and 75 mg as illustrated in [Figure 2] (a-d show the responses at 2, 4, 6, and 8 kHz, respectively). The significant differences observed between the 25 mg group vs. 50 and 75 mg groups are most likely due to the fact that the 25 mg group was not remarkably different from the control group. [Figure 2] indicates that there was a steady threshold improvement across all groups except the control group ([Figure 2]a-d show the responses at 2, 4, 6, and 8 kHz, respectively). At each frequency, the NAC/SAL 50 mg/kg is significantly improved over control at the 3-week time period (P Hair cell counts
Cytocochleograms quantified the percent of OHC loss as a function of distance in millimeters from the apex.  Mean OHC cytocochleogram data for the NAC/salicylate dose-response groups is shown in [Figure 3] and correspond to the threshold shift data shown in [Figure 2]. There was little difference in OHC loss between treatment groups in the 2 kHz region. There was maximal OHC loss of about 75-85% in the 4-8 kHz region of the cochlea in saline treated noise-exposed controls. There was a substantial reduction in mean OHC loss to around 40-50%, in the NAC plus 50 or 75 mg/kg salicylate groups corresponding to the reduced permanent threshold shift (PTS) seen in [Figure 2]A-D. When mean OHC loss was compared at selected frequencies, there was a statistically significant reduction in hair cell loss for the 50 mg/kg group at 4, 6, and 8 kHz. The 75-mg/kg group was significantly different at 6 and 8 kHz from the hair cell loss observed for the control group. There was no statistical difference between the 25 mg/kg group and the saline control OHC counts at any of the measured frequencies.
High-intensity noise can damage the cochlea by causing mechanical damage to sensory and supporting structures. However, if the noise level is at a certain threshold or at a lower level for a longer duration, it can permanently injure the cochlea by inducing metabolic "exhaustion". , Acoustic over-stimulation of the cochlea generates the production or formation of free radicals and ROS, which may induce OHC death. ,, Such high levels of ROS activate the up-regulation of cochlear antioxidant enzyme activity  and modulate the key antioxidant compound, GSH. , A variety of agents with antioxidant properties have been shown to attenuate threshold shifts and/or hair cell loss when given prior to a damaging noise exposure. These include 2-oxothiazolidine-4-carboxylate (OTC); , U74389F;  allopurinol, and superoxide dismutase;  R-phenylisopropyladenosine; ,, Ebselen, ,, d-methionine, NAC and ALCAR, ,,, vitamins A, C, E, and magnesium. 
Despite the intensity of noise exposure in this study, the treatment combination, when given shortly before noise exposure, reduced threshold shifts by up to 20 dB and OHC loss by up to 40% in the 50 mg/kg group. Further, a dose-response type interaction was noted for all the three treatment groups. The results of the 25 mg group showed little effect on hearing recovery, with one notable exception at week 3 at 6 kHz; the frequency demonstrating the greatest NIPTS and OHC loss showed significant attenuation of both when compared to controls, indicating that this dose did have some effect in this region of greatest damage. In comparison to 25 mg/kg group, the 50 mg/kg group showed significant treatment effects as early as week 1 for the ABR data at 6 kHz and significant treatment effects at week 2 for OHC loss at 4, 6, 8 kHz. These data also suggest that the nature of the dose-response curve for the therapeutic effect (otoprotection) of salicylate closely approximates the curve for the toxic effect (decreasing protection), with a very small therapeutic window under these conditions. This is consistent with previous research which demonstrated that high dose salicylate can decrease hearing thresholds. ,
In contrast, NAC protects sensory cells by various molecular mechanisms including: 1) acting as an ROS scavenger, 2) replenishing intracellular GSH, 3) reducing apoptosis by inhibiting activation of caspase-3 and JNK, 4) reducing lipid peroxidation, and 5) improving icrocirculation. ,,,,, The elevation of GSH in the cochlea plays a significant role in protecting the cochlea from oxidative stress resulting from loud noise. , Therapeutically, NAC has been used to treat patients with AIDS at doses of up to 8 g/day prescribed for extended periods without significant side effects, , and as mentioned earlier, NAC is used very aggressively (up to 102 g for 4 days) for acetaminophen overdose where it counters the liver damage (which can be fatal) induced by oxidative stress and GSH depletion.  These data are consistent with previous studies using other antioxidant compounds or compounds with antioxidant actions. When given either systemically ,, or applied to the round window membrane ,,,, prior to noise exposure, antioxidant agents used in this study resulted in a reduction in threshold shift and/or hair cell loss.
This article delineates conditions whereby a combination of antioxidant compounds given before noise exposure may help ameliorate the damage caused by excessive sound. Our results further support the hypothesis that antioxidants play a significant role in preventing or reversing noise-induced PTS in a chinchilla model. The use of combinations of agents may represent strategy for ameliorating NIHL in the clinical setting. In the laboratory, many investigators have shown positive results from this combinational approach. Lamm  et al, used a variety of blood flow enhancing agents and anti-inflammatory agent combinations. Hight et al, who used the combination of glutathione monoethylester and R(−)-N6 -(2-phenylisopropyl) adenosine (R-PIA) to effectively treat NIHL in a chinchilla model.  Yamashita  and colleagues investigated treatment with ROS and reactive nitrogen species (RNS) scavengers, salicylate and trolox in a guinea pig model of NIHL and Minami et al,  used creatine and tempol and Le Prell et al,  focused on a combination of several vitamins and magnesium as an effective treatment for PTS in an animal model.
The potential that combinations of compounds with antioxidant and cell protective properties of different but reinforcing mechanisms may act synergistically to provide greater otoprotection from acoustic trauma is also strengthened by these data.
The authors gratefully acknowledge the generous support of the National Organization of Hearing Research. They also recognize the help of Office of Naval Research, Department of the Navy, Department of Clinical Investigation at Naval Medical Center San Diego. Thanks are especially to Ms. Waine Macalister for her excellent review and editing, and Department of the Army for its support of this project.
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