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Noise and hearing conservation
Korean Journal of Audiology 2011;15(3):107-113.
Proteomic Analysis of the Protein Expression in the Cochlea of Noise-Exposed Mice.
Nam Kyung Yeo, Yun Suk Ahn, Ji Won Kim, Seung Hyo Choi, Gil Chai Lim, Jong Woo Chung
1Department of Otolaryngology, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Korea.
2Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. jwchung@amc.seoul.kr
3Department of Otolaryngology, Jeju University College of Medicine, Jeju, Korea.
When noise-induced hearing loss occurs, destruction of the hair cells is accompanied by mechanical injury, chemical injury, and hypoxia. Proteomics is a powerful tool for protein analysis, as it provides valuable information regarding the biochemical processes involved in diseases, monitors cellular processes, and characterizes protein expression levels. We attempted to identify the proteins associated with the pathophysiology of noise-induced hearing loss, as well as the mechanisms of this disease, using a proteomics approach.
We used BALB/C male mice. The control mice were placed in a booth without noise, while the experimental mice were exposed to noise for three hours daily for three consecutive days. Cochleae from each group were obtained for total protein extraction. The proteins were separated into numerous spots using two-dimensional electrophoresis. Seven protein spots that were strongly detected only in the noise-exposed cochleae were selected and subsequently analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry.
Approximately 286 protein spots were detected in the noise group. Seven selected spots were analyzed and various proteins identified, including tyrosine protein kinase MEG2, angiopoietin-like 1, heat shock 70 kDa protein, sodium dicarboxylate cotransporter 1, myeloid Elf-1-like factor, disintegrin, metalloproteinase domain 7, and activated leukocyte-cell adhesion molecule.
We identified several proteins expressed in noise-induced hearing loss using a proteomics approach. These proteins may help us to understand the pathogenic mechanisms of noise-induced hearing loss.
Keywords: Noise-induced hearing loss;Cochlea;Proteomics

Address for correspondence : Jong Woo Chung, MD, PhD, Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Korea
Tel : +82-2-3010-3718, Fax ; +82-2-489-2773, E-mail : jwchung@amc.seoul.kr


Noise is a common cause of sensorineural hearing impairment in industrialized countries. Millions of people currently have disabilities caused by noise-induced hearing loss and experience problems in communication with families, colleagues, and friends.1) In particular, tinnitus is one of the symptoms of noise-induced hearing loss that brings severe social isolation and leads to degraded quality of life. Noise exposure can physically destroy the tympanic membrane, middle ear, and inner ear and can alter the intracellular pathways that lead to cell necrosis or apoptosis, and induced hearing loss worsens. In addition, noise stimulation was recently found to induce metabolic changes inside the inner ear. Possible mechanisms underlying this noise-induced tissue damage are oxidative stress and the reduction of cochlear blood flow.2) Studies have been conducted in the last few decades on mechanical trauma and the metabolic damage of mechanical changes. Vascular endothelial growth factor, nuclear factor κB, glucose transporter-1, and hypoxia-inducible factor-1α are thought to be causes of noise-induced hearing loss. Most of them were histologically confirmed by immunohistochemical and fluorescence staining.4,5,6) 
Proteomics is a powerful tool for protein analysis, as it provides valuable information regarding the biochemical processes involved in diseases, monitors cellular processes, and characterizes protein expression levels. We can understand the pathophysiology of disease through analysis of the proteins involved using proteomics technology, which includes two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). These techniques can be used to produce a high-resolution two-dimensional map, in which individual (stained) proteins appear as spots of various sizes and intensities that depend on the amount of each protein in the sample.7,8)
To understand the proteins associated with the pathophysiology of noise-induced hearing loss, as well as the mechanisms of the disease, we compared expressed proteins in noise-exposed mice with those in unexposed mice using this proteomics approach. For this, we investigated expressed protein spots only in noise-exposed mice.

Materials and Methods

As experimental groups, 8-week-old BALB/c mice with normal Preyer's reflexes and normal hearing thresholds in auditory brainstem response were studied. 


Experimental setting
All mice were placed in separate soundproof booths with blocks to outside noise. We set an amplifier (R-399, INTER M, Seoul, Korea) in the left side of the room and placed an 8 Ω resistance speaker (290-8L, ALTEC LANSING, Oklahoma City, OK, USA) on the amplifier with a 45 degree horn.

Anesthesia of experimental groups
All mice were anesthetized (Ketamine hydrochloride 59 mg/kg and xylazine 1.3 mg/kg body weight) via intraperitoneal injection. If necessary, we injected an additional half dose of anesthestics.

Threshold measurement
The auditory brainstem response (ABR) to click stimuli was recorded, and thresholds were obtained for each ear. Hearing thresholds are measured at wave I lowering by 10 dB from an intensity of 90 dB hearing level (HL). When the wave was not definite, we checked threshold lowering by 5 dB. Click stimuli were filtered from 100 to 3,000 Hz, and the frequency was 1,024/min. 

Noise exposure procedure
Ten mice were exposed continuously for 3 h/day to a 120 dB sound pressure level (SPL) broad band click sound for 1-5 consecutive days. In the noise booth, mice were randomly divided into two groups, and their location inside the booth was changed daily so that each animal was exposed to the same level of noise. Ten normal BALB/c mice kept for 3 h/day for 5 consecutive days in the same noise booth without noise were used as the control group. Acoustic trauma was induced by a continuous pure tone of 6 kHz generated by a waveform generator (Cool Edit 1.52 software), and its level was checked to ensure that it was over 120 dB SPL at each corner of the booth with a level meter (B&K, Nærum, Denmark). The hearing level of each mouse was analyzed by measuring the ABR before and after noise exposure.

Tissue preparation and protein extraction
Following ABR measurements, the mice were sacrificed, and both cochleae were removed. Specimens were immediately frozen in liquid nitrogen and stored at -70℃ until used. Specimens were kept in ice-cold RIPA buffer for lysis and crushed and homogenized with a tissue grinder. Bradford protein assay (Bio-Rad, Hercules, CA, USA) was used to measure the protein samples. 

Two-dimensional electrophoresis
For first dimensional electrophoresis, immobilized 7 cm-sized, pH 3-10 linear gradient strips were used. The immobilized pH gradient strips were rehydrated with a solution of rehydration buffer (4 M urea, 2% 3-CHAPS, 20 mM DTT, and 2% v/v carrier ampholytes) for 12 hrs at 10 V followed by a voltage gradient of 500 to 8,000 V over 1 hr.
For the second dimension, the strips were equilibrated with a 12% agarose separating gel, and the agarose gel was covered with a mixture of bromophenol blue. Electrophoresis was carried out under the following conditions: 3W (for 6-7 hr). After completion of 2-DE, the gels were stained with silver and scanned into a computer. 

Image analysis
The stained gels were stored in 2% acetic acid solution after scanning. The digital image was processed with Adobe Photoshop and Illustrator software (Adobe Systems, San Jose, CA, USA). The total number of spots was recorded, and strong spots were selected by a software program (PDQuest Software, Bio-Rad, V7.1).

Protein spot extraction and trypsinization
The protein spots were cut out of the gel by sterilized pipette tip and were placed in 1.5 mL Eppendorf tubes. Silver staining was removed in 50 μL 30 mM potassium ferricyanide/100 mM sodium thiosulfate (1 : 1) solution for 10 mins followed by washing the gel pieces three times with saline. The gel pieces were allowed to react in 1 mL of Milli-Q water at room temperature for 5 minutes. After washing three times with saline, the gel pieces were dried for 30 min in a vacuum centrifuge. For protein digestion, the gel pieces were re-swollen in 20-30 mL of trypsin digestion buffer (3 µL trypsin at a concentration of 0.1 ng/µL in 50 mM ammonium hydrogencarbonate, pH 8), and each mixture was incubated at 37℃ for 16 hrs. After adding 100 µL 50 mM ammonium bicarbonate, samples were incubated at 37℃ in a shaking incubator for 1 hr. The samples were centrifuged briefly, and the supernatant that contained the tryptic peptides was collected. To remove residual peptides, the gel pieces were sonicated three times for 10 min in 100 µL acetonitrile. The extracts were combined and dried in a vacuum centrifuge over a period of 6 hrs. 

Protein identification and bioinformatics
Tryptic peptide digests were obtained with MALDI-TOF MS, and data were used to query the mammalian proteins in the SWISS-PROT protein sequence database (http://kr.expasy.org). The input parameters included the isoelectric point and molecular weight of the intact protein.


The change of hearing threshold after noise exposure
Mice exposed to noise for 3 consecutive days (n=10) experienced an increase in hearing threshold from 16.5±1.8 dB HL to 74.5±5.0 dB HL (Fig. 1).

Two-dimensional electrophoresis
By comparison of expressed proteins in noise-exposed mice cochleae with those in unexposed mice cochleae, 1,199 protein spots were identified in unexposed mice, while 1,221 protein spots were identified in normal exposed mice. 
Seven cochlear proteins were identified which showed increased expression only in noise-exposed mice. These proteins were the following: angiopoietin-like 1, heat shock 70 kDa protein, tyrosine protein kinase MEG2, sodium dicarboxylate cotransporter 1 (NaDC-1), myeloid Elf-1-like factor, activated leukocyte-cell adhesion molecule (ALCAM), disintegrin, and metalloproteinase domain 7. They have subsequently been characterized (Fig. 2).

Protein mass spectrometry
Seven proteins, which had strong densities compared with the other expressed proteins, were identified using the PDQuest software program. 
Peptides from protein spots were identified on the basis of MALDI-TOF MS-MS spectra and identified using peptide mass fingerprinting (PMF), which are shown in Fig. 3. Protein identification data are summarized in Table 1. The confidence of the identification is indicated by the number of matching peptides and the coverage of the protein's sequence by the matching peptides. For searches with PMF data, five or more matching peptides and a minimum of 15% sequence coverage are considered necessary for unambiguous protein identification. All proteins listed in Table 2 were the top candidates retrieved by the searches. Hypothetical proteins and unnamed protein products, which are unkown and innominated proteins, were not identified in this study.


The models of noise-induced hearing loss are transient threshold shift and permanent threshold shift. The present study focused on permanent threshold shift. Although we did not identify the changes of ABR thresholds in different noise-exposures over a 1 month period because of immediate specimen extraction, similar hearing loss has been reported by Chung, et al.,10) who used the same experimental setting.
Proteomics is a powerful tool for protein analysis which can analyze the mass (high-throughput screening) without destruction of cell structures.11) 
Proteomic efforts are an important component of functional genomics and will be critical to understanding and synthesizing the information generated by the Human Genome Project. The genome is individual and static; however, determination of protein profiles active in functional pathways in normal cells, as well as determining alterations associated with disease processes, can be performed.12)
Two-dimensional polyacrylamide gel electrophoresis and mass spectrometry are the main tools for biosample analysis and protein identification. Integration of these methods with tissue microdissection permits analysis of proteomes of defined cell populations derived from tissue samples.7,8,9) In addition, the development of staining technology and other technologies will improve proteomics.13) 
To understand the proteins associated with the pathophysiology of noise-induced hearing loss, as well as the mechanisms of the disease, we compared expressed proteins in noise-exposed mice with those in unexposed mice using the proteomics approach.
As the result of analysis using MALDI-TOF MS of 7 cochlear proteins which demonstrated increased expression only in noise-exposed mice, angiopoietin-like 1, heat shock 70 kDa protein, tyrosine protein kinase MEG2, NaDC-1, myeloid Elf-1-like factor, ALCAM, disintegrin, and metalloproteinase domain 7 have been characterized.
Angiopoietin-like 1 stimulates proangiogenic inflammatory cytokines and influences vascular permeability and migration, differentiation, and survival of vascular cells, similar to the activities of vascular endothelial growth factor (VEGF).5) 
It is known that VEGF expression is upregulated to compensate for ischemic damages in the stria vascularis at the onset of noise-induced damage.5) Similarly, angiopoietin-like 1 is thought to play a role in increasing blood supply like VEGF. To identify the exact site of action of this protein, additional immunohistochemical examinations are needed.
Heat shock 70 kDa protein (HSP 70) expression can determine the fate of the cell in response to a death stimulus and can be an apoptosis-inhibitory HSP.15)
Growth factor stimulation induces hypoxia-inducible factor (HIF)-1α protein synthesis via a signal transduction pathway leading from receptor tyrosine kinases in acute inflammation and especially hypoxic damage.16) Under conditions of tissue hypoxia, activation of HIF-1α can activate the adaptive response in hypoxic cells; the promotion of vulnerable hypoxic tissue survival may be involved in the damage to the inner ear caused by noise.10,17)
NaDC-1 compensates for acidic conditions at proximal loops in the kidney. As the environment increases in acidity, the expression of NADC-1 increases.18) Noise-induced ischemic changes of inner ear cells results in an acidic reaction, causing an increase in NaDC-1.
In epithelial cells, myeloid Elf-1-like factor (MEF) upregulated not only the activity of a transiently transfected lys5A promoter but also the transcription of the endogenous lysozyme gene and protein to increase immunity and anti-inflammatory reaction.19) In this study, MEF was shown to be increased, but the correlation was weak.
ALCAM is an adhesion molecule involved in leukocyte migration across the blood-brain barrier, lung, and liver. ALCAM is involved in angiogenesis, hematopoiesis, immunity, and oncogenesis.20) However, the purpose of ALCAM expression in the inner ear is currently unknown.
It is difficult to conclude the pathogenic mechanisms of noise-induced hearing loss based on the current understanding of these proteins' functions. However, it can be speculated that proteins, such as angiopoietin-like 1, heat shock 70 kDa protein, and tyrosine protein kinase MEG2, play a role to protect the structure of the inner ear. To understand the function of these proteins, further evaluation is necessary; additionally, increasing the sensitivity and specificity using MALDI-TOF/TOF and rechecking these proteins' expression levels through western blotting is needed. In addition, other proteins that are up- and downregulated during noise exposure should be analyzed to help elucidate the pathogenesis of noise-induced hearing loss.


To understand the proteins associated with the pathophysiology of noise-induced hearing loss, as well as the mechanisms of the disease, we compared expressed proteins in noise-exposed mice with those in unexposed mice using a proteomics approach.
In this study, angiopoietin-like 1, heat shock 70 kDa protein, tyrosine protein kinase MEG2, NaDC-1, myeloid Elf-1-like factor, ALCAM, disintegrin, and metalloproteinase domain 7 have been characterized. It can be assumed that proteins, such as angiopoietin-like 1, heat shock 70 kDa protein, and tyrosine protein kinase MEG2, play a role in the protection of inner ear structures.
These proteins may help us understand the pathogenic mechanisms of noise-induced hearing loss.

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