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Year : 2011  |  Volume : 13  |  Issue : 55  |  Page : 452--458

Effect of vitamin E supplementation on carbogen-induced amelioration of noise induced hearing loss in man

Neeru Kapoor, Kumar Vyonkesh Mani, Radhey Shyam, Raj Kumar Sharma, Ambika Prasad Singh, William Selvamurthy 
 Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organization, Ministry of Defence, Timarpur, Delhi, India

Correspondence Address:
Neeru Kapoor
Occupational Health Division, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi - 110 054


The study explores the effect of occupational noise on oxidative stress status and prophylactic effect of Vitamin E and carbogen (5% CO 2 +95%O 2 ) breathing in alleviating the oxidative damage and conserving the hearing in human volunteers exposed to intense occupational noise. Plasma total antioxidant status, blood glutathione (GSH), malondialdehyde (MDA), antioxidant enzyme activities of GSH peroxidase (EC, GPx), superoxide dismutase (EC; SOD) in erythrocytes, nitric oxide and nitric oxide synthase in plasma were assessed before and after 6 days of administration of Vitamin E and Carbogen. Results of the study indicate that the exposure to noise for 6 days increased blood concentration of MDA, decreased concentrations of reduced GSH, antioxidant enzyme activity of SOD and plasma total antioxidant status in control (noise) group. Vitamin E- supplemented group showed decline in oxidative stress reflected by significant decrease in blood concentration of MDA and increase in antioxidant enzyme activity of erythrocyte SOD. Results of audiometric studies revealed that breathing of carbogen prevented the development of temporary threshold shift; thereby reducing the risk of noise induced hearing loss.

How to cite this article:
Kapoor N, Mani KV, Shyam R, Sharma RK, Singh AP, Selvamurthy W. Effect of vitamin E supplementation on carbogen-induced amelioration of noise induced hearing loss in man.Noise Health 2011;13:452-458

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Kapoor N, Mani KV, Shyam R, Sharma RK, Singh AP, Selvamurthy W. Effect of vitamin E supplementation on carbogen-induced amelioration of noise induced hearing loss in man. Noise Health [serial online] 2011 [cited 2020 Mar 29 ];13:452-458
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Noise has been recognized as an important occupational hazard affecting general well being and quality of human life. High noise levels are known to cause hearing impairment and disturb the functioning of other non- auditory systems. Several studies have reported these adverse effects of noise in men and animals. [1],[2] Industrial noise is one of the major contributory factors to various noise induced health effects of workers, due to continuous and repeated exposures to intense noise. Studies on industrial workers has brought out that people in noisy industries suffer from circulatory, digestive, metabolic and neurobiological difficulties. [3]

Surge in noise levels due to spreading industrialization have contributed to make Noise Induced Hearing Loss (NIHL) the forerunner of environmental hazards globally. In a military environment, exposure of the soldier to offensive noise becomes inescapable due to the operation of heavy armored vehicles, weapon and equipment systems, turbine engines, and high-performance aircraft and helicopters. Controlling noise and its harmful effects on human beings has been a challenging task for the environmental management and safety professionals around the world and the success achieved is debatable.

The mechanism of NIHL is not yet clearly understood. As a "mechanical stressor", noise may lead to NIHL by incapacitating or partially destroying the sensory hair cells in the cochlea due to reduced blood supply as a result of cerebral vasoconstriction. This reduces the oxygen supply and impairs the normal functioning of the hearing organ and may give rise to Noise Induced Temporary Threshold Shift (NITTS). [4],[5] It was also conjectured that if the constrictive influence of noise could be countered and oxygen supply to the organ restored by any technique, NIHL might be controlled. [6] Recently, it has been proposed that both auditory as well as non-auditory effects are regulated by a series of enzyme activities. [7],[8] These chemical changes are mediated by free radical reactions. [9],[10] The free radicals are removed both by enzymatic and non- enzymatic reactions comprising antioxidant defense system of the body. [11] The antioxidants in general contribute to the non-enzymatic cellular defense against lipid peroxidation by donating hydrogen atoms to free radicals resulting in their inactivation. Vitamin E functions as a free radical scavenger that quenches free radicals responsible for lipid peroxidation. [12]

Curtailing the transmission of sound energy to the inner ear by using personal hearing protective devices is a vital countermeasure against the debilitating effects of noise. However, though ear defenders offer adequate protection, they suffer from several limitations and hence have restricted utility and useability. This has led to a search for effective, simple, and easy-to-use modalities. Carbogen breathing as a prophylactic treatment was found to be useful in laboratory investigations on human beings. [13],[14] Since Carbon dioxide (CO 2 ) is a cerebral vasodilator, its supplementation along with high concentration of oxygen in carbogen proved to be very effective in maintaining the cerebral supply and conserving the hearing of workers at industrial workplace by the laboratory earlier. Vitamin E (a-tocopherol) is a well known antioxidant and free radical scavenger. [15] These properties, as adjunct to carbogen inhalation, may have benefits in hearing conservation by preventing the free radical mediated damage due to intense noise exposures.

The present study was undertaken to assess the effect of occupational noise on oxidative stress status of man and explore the role of supplementation of Vitamin E with or without administration of Carbogen as prophylactic measures in alleviating the effects of free radical - mediated injuries and prevent hearing impairment in industrial workers.

 Materials and Methods

A group of 40 male industrial Army Base Workshop workers with mean age 36.4 ± 7.3 years and having exposure to intense occupational noise were selected to participate in the study. They were screened on the basis of audiometry. Individuals with normal hearing or those with average hearing threshold of 25.4 ± 7.1 dB across 0.5, 1, 2, and 4 kHz were included in the study. All of them were explained about the aims, objectives, methodology, and the anticipated benefits of the protocol, which was duly approved by the Institute's Ethics Committee. The subjects were divided into four equal groups, namely (i) Control group (ii) Carbogen group (iii) Vitamin E Group and (iv) Carbogen and Vitamin E Group. The experiments were conducted during six consecutive working days. The subjects were exposed to noise for 5 hours during their work schedule at Engine Test House (A and B), Air Compression Section and tank testing room. The sound pressure levels (SPL) at these places were measured for exposure assessment. The initial audiometry of the subjects was recorded in a sound attenuated chamber after overnight rest and post-exposure audiometry was carried out on the last day of the study and completed in quick succession after the cessation of the exposure for each subject and before breathing of carbogen gas. The schedule of 5 minutes breathing of Carbogen, twice daily before going to the work place and soon after completion of daily routine, was followed for every one of the carbogen and carbogen + Vitamin E group of subjects. The interval between the two-carbogen inhalations was 5 hours. Vitamin E (dose 400 mg/day) following breakfast was given to the Vitamin E and combined Carbogen and Vitamin E groups from day one of the study.

The A-weighted SPL (dBA), A-weighted equivalent sound level (LAeq), and noise dose were measured at different work stations using B&K Type 2260 Type 1 Modular Precision Sound Analyzer. The audiometry of the subjects was carried out with duly calibrated GSI 61 Grason-Stadler Audiometer having 1 dB resolution. The audiometric test was carried out in air conduction mode at frequency range of 0.125 to 8 kHz for both the ears.

Exposure duration

Subjects of each group were exposed to noise at their workstations for 5-hour duration. During testing of the engines and tanks, the personnel remained at their respective workstations without wearing any hearing protective device.

Biochemical estimations

Blood was collected before and after 6 days exposure to occupational noise, along with supplementation with either Carbogen and/or Vitamin E alone. Blood was collected in heparinized tubes and centrifuged at 1000 g for 10 minutes for separating plasma. RBCs were washed with 150 mmol chilled KCl and both plasma and RBC samples were stored in aliquots at -80 o C until assayed. Blood concentrations of malondialdehyde (MDA) and reduced glutathione (GSH) were estimated immediately after the collection of blood samples. Oxidative stress parameters were evaluated before and after the exposures and/or supplementation. Erythrocytes activity of antioxidant enzymes, GSH peroxidase (EC, GPx), Superoxide dismutase (EC; SOD), and total antioxidant status were estimated using commercially available kits of Randox Laboratories, UK. Blood concentrations of MDA and reduced GSH were determined immediately after the collection of blood samples by the method. [16],[17] Nitric Oxide (NO) and Nitric Oxide Synthase (NOS) were estimated by commercially available diagnostic kits (Oxis International, USA).

Statistical analysis

The data were analyzed using paired t-test. Significance level at P<0.05 was taken as the criteria of significance. For inter group comparisons, one way ANOVA followed by SNK test was carried out using SPSS 10 Software for windows.

 Results and Discussions

Physical characteristics of the subjects

[Table 1] presents the mean age, height, weight, and Body Mass Index (BMI) of the subjects. All four groups were homogenous with respect to age, height, weight and BMI. There was no significant difference with respect to any of these parameters between the groups.{Table 1}

Measurement of noise levels

The SPL (dBA) at the workplaces of the subjects are depicted in [Table 2].{Table 2}

The SPL measured at Engine Test House (A and B), Air Compression Section and testing site of Tanks ranged from 90.0 to 110.2 dBA, 100.9 to 106.8 dBA, and 100.7 to 113.5 dBA, respectively.

[Table 3] presents the noise dose received by the subjects and LAeq levels at the different locations of the workshop. During the period of maximum exposure of 10 minutes to approximately 90 minutes, the dose received ranged from 122.2% to 141.9%, with the prevailing LAeq values at the measurement sites of engine test house "A", Test house "B", air compression section and during testing of tanks respectively being 98.3, 101.7 101.8, and 108.1. At all other times during their normal duty hours, the subjects were exposed to SPL ranging between 76.5 to 81.8 dBA. In actual working condition, the noise levels cannot be the same at all the workstations.{Table 3}

Intensity of noise is the factor mainly affecting the hearing of persons working in noisy environment, while the duration of exposure alters the degree of impairment. Damage Risk Criteria (DRC) has been formulated on the basis of noise levels and the duration of the exposure for the assessment of auditory risk and determination of the allowable permissible duration in noisy environment when the noise level is known. Several research investigations conducted during the past few decades have brought out the permissible allowable level of 90 dBA for an 8-hour per day work schedule in continuous noise environment. [18] This is also termed as 100% noise dose for continuous noise. Comparison of the noise levels given in [Table 2] with the recommendations of the DRC formulated for the conservation of hearing of industrial work force [19],[20],[21] clearly brings about the magnitude of auditory hazard. In this study, the subjects were exposed to 90 to 113.5 dBA noise for 5 hours a day for 6 days a week without wearing any hearing protective device. Preventive measures are therefore essential to safeguard health and efficiency of the individuals working in such environments.


The mean hearing threshold levels (dB) with respect to combined ear of all the four groups of subjects before and after six consecutive days of exposure to noise are presented in [Figure 1]a to [Figure 1]d and [Table 4] representing the TTS of combined left and right ear in each group of subjects.{Table 4}{Figure 1}

The results indicated the development of significant temporary threshold shift among the noise group of subjects [Figure 1]a at audiometric frequencies above 1 kHz. The shift in hearing threshold ranged from 1.3 to 6.2 dB [Table 4].

The Carbogen group of subjects developed comparatively much lower temporary threshold shift. For frequencies above 3 kHz, the difference between pre and post mean hearing level was significant [Figure 1]b. Practically, no TTS developed at lower frequencies, which may be due to the hearing system being less sensitive to these frequencies and also due to carbogen breathing. Comparison between the audiometric observations presented in [Figure 1]a and b shows that carbogen definitely helps in controlling the impact of intense noise on hearing and corroborates with studies undertaken in controlled conditions. [22],[23] Similar trend was obtained with Vitamin E-supplemented group [Figure 1]c. It shows protection at frequencies 0.25, 0.5, and 1.0 kHz. It is possible that the protection in Vitamin E-supplemented group may have been obtained if Vitamin E supplementation was started weeks before the study. However, in our study, we have given the Vitamin E supplementation from the day one.

The mean pre- and post-exposure hearing threshold levels of the combined carbogen and Vitamin E-supplemented group is presented in [Figure 1]d. The hearing threshold was almost similar at all the frequencies. The shift in the hearing threshold levels at all audiometric frequencies ranged from -1 to 3 dB [Table 4].

The supplemented dose of Carbogen and Vitamin E did not cause any side effect in the volunteers.

In Group-1 volunteers, a significant increase in mean hearing threshold level has been observed at all the audiometric frequencies as a result of noise exposure. It is expected that similar level of shift would have set in had the group-4 subjects not been administered carbogen and Vitamin E. Carbogen inhalation is known to cause vasodilation [24],[25] and maintain the adequate supply of oxygen to cerebral blood vessels. In the present study, inhaling of carbogen probably reverses the vasoconstrictive effect of noise and helps in oxygenation of cochlear blood vessels that result in the hair cells retaining their sensitivity on exposure to noise. The study has brought out the synergistic action of carbogen and Vitamin E combination in controlling the development of hearing loss. It appears that this treatment did not allow the setting in of temporary shift in hearing that might not recover during rest and eventually lead to permanent loss of hearing.

The importance of this study lies in the fact that the carbogen breathing is for a very brief duration and that CO 2 and O 2 are intrinsic to the human system. Moreover, protection from TTS development in a noise field of 90 to 113.5 dBA is adequate. These findings also suggest and corroborate the result of similar work carried out against continuous and impulsive noise. [22],[23] The adaptation of this treatment would do away with the use of cumbersome and partially effective ear defenders which often hamper communication because of their attenuation to wanted sound. [26],[27] In the working conditions of the armed forces, right communication is of paramount importance and the lack of comprehension in combat due to use of ear defenders might result in blunders.

Biochemical parameters

It is observed that noise results in an increase in lipid peroxidation byproduct MDA concentration in blood, coupled with a decrease in the erythrocytes activity of SOD, reduced GSH in blood, and plasma total antioxidant status. These results were statistically significant at P<0.05. Inhalation of Carbogen alone or supplemented with 400 mg/day dose of Vitamin E for 6 days maintained the levels of all the above variables, while Vitamin E alone increased the erythrocytes activity of SOD (P<0.01) and decreased the activity of erythrocytes GPx (P<0.05) while maintaining the plasma total antioxidant status. Vitamin E treatment also resulted in lower MDA levels due to its quenching effect. The levels of the nitrosative stress variables, namely NO and NOS, remained almost similar to their baseline values in all the groups. The summarized results of the biochemical parameters are presented in [Table 5].{Table 5}

One way ANOVA for inter group comparison of biochemical variables following exposure and/or supplementation were found to be statistically significant at P<0.01 for MDA (between control and vitamin E group, carbogen and Vitamin E group, and, carbogen with Vitamin E group and Vitamin E group) and for SOD (between control and Vitamin E group and carbogen and Vitamin E group). For all other variables, results were not significant.

Lipid peroxidation is a chain reaction initiated by the attack on the membrane lipids by free radicals that has sufficient reactivity to abstract a hydrogen atom from the methylene group. Our results showed an increase in MDA level in the control group exposed to noise (Group I) indicating enhanced peroxidation of membrane lipids. Supplementation with Carbogen alone and Carbogen with Vitamin E helped maintain the levels of MDA, while supplementation with Vitamin E alone reduced MDA levels significantly. This indicates that Vitamin E helps in preventing lipid peroxidation.

The first line of cellular defense against radicals consists of antioxidative enzymes such as SOD and peroxidases. These enzymes react directly with the oxidizing radicals to yield non-radical product. Selenium-dependent GPx removes both H 2 O 2 and lipid peroxides by catalyzing the conversion of lipid hydroperoxides to hydroxy acids in the presence of reduced GSH. A reduction in the erythrocytic GPx in our investigations was observed in all the groups, but the decrease was statistically significant in the group supplemented with Vitamin E alone [Table 5]. This decline may help in maintaining the reducing environment of the cells in the Vitamin E-supplemented group, thus maintaining the viability of the cells indicative of beneficial effects of the supplementation. SOD enzyme is present in cells and tissues such as erythrocytes, liver, and brain. It is a free radical-metabolizing enzyme, catalyzing dismutation of superoxide anion to hydrogen peroxide. This protects the cell membrane from damage by highly reactive oxygen species. The low activity of total antioxidant status and SOD and in the noise-exposed group [Table 5] could be due to inactivation of the enzyme by cross linking or damage to DNA [28] by lipid peroxidation, hence causing a decrease in the expression of the enzyme. Increase in erythrocytes activity of SOD and maintained plasma total antioxidant status in the Vitamin E supplemented group indicates beneficial effects of the supplementation.

GSH plays a major role in cellular protection against oxidative damage. Conditions that perturb intracellular levels of GSH have been shown to result in significant alteration in cellular metabolism. The oxidation of GSH in various tissues has proven to be a consistent index of exercise-induced oxidative stress. When challenged with oxidative stress, intracellular GSH rapidly oxidizes to glutathione disulphide (GSSG). Previous studies have shown depletion of endogenous GSH potentiates NIHL, whereas replenishment of GSH attenuates NIHL. [29] Our results showed a significant decrease in GSH level in blood on exposure to noise [Table 5]. The decrease in GSH was in direct relation to the increase in MDA level. This shows that noise induced oxidative stress might have enhanced substrate utilization by GPx resulting in increase in GSSG level, so that the rate of GSSG formation exceeded the capacity of the cell to reduce the disulphide to GSH. GSH deficiency has been reported to cause a profound increase in tissue lipid peroxidation in rats. [30] Supplementation with Vitamin E helped in maintaining the reduced GSH levels, showing the beneficial effects of the supplementation.

It is being increasingly recognized that NO is involved in many physiological processes viz. neurotransmission in the nervous system, control of blood pressure and immune response. [31] Increased NO production is observed under the action of short-term or moderate stressor corresponding to the stage of mobilization in response to appropriate stress reaction; while decreased NO production is observed on application of long-term or detrimental impact of stressors corresponding to the stage of exhaustion in excessive stress reaction. [32] NOS is responsible for basal generation of NO that is critical for basal blood flow and pressure regulation across many organs and species. In the present study, no significant change in either NO or NOS were observed following exposure to noise or on supplementation with carbogen and Vitamin E [Table 5]. Hence, there are no adverse interactions of the different treatments. These results are in conformation with the observations of Vesela and Wilhelm [33] on the effects of CO 2 in scavenging of peroxynitrite and prevention of nitration and oxidative damage.

The free radical scavenger data are all supporting the evidence that the free radical mechanism might be of importance in NIHL. Persons exposed to occupational noise have increased lipid peroxidation reflected by increased concentration in blood MDA and decreased total antioxidant status reflected by decreased concentrations of antioxidants like GSH and SOD, revealing that occupation of these persons has a definite impact on these parameters.

The study brings out the utility and efficacy of the pre- and post-exposure supplementation of Carbogen against NIHL as it helps in attenuation of NITTS. Results also showed that Vitamin E supplementation decreased the oxidative stress by reducing blood MDA levels and increasing the antioxidative enzyme activity of SOD while maintaining the plasma total antioxidant status. Furthermore, Vitamin E has synergistic effect in attenuating NITTS. It is evident from the study that Carbogen and Vitamin E are providing protection against NIHL, possibly from two mechanisms; Carbogen through its vasodilatory and Vitamin E through its antioxidant effects.


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