Qualitative and quantitative assessment of noise at moderate intensities on extra-auditory system in adult rats Noura Gannouni1,2, Abada Mhamdi2, Olfa Tebourbi1, Michèle El May3, Mohsen Sakly1, Khémais Ben Rhouma1 Laboratory of Integrated Physiology, Faculty of Sciences of Bizerte, Carthage University, Jarzouna, ²Laboratory of Toxicology, Ergonomics and Occupational Environment, Faculty of Medicine of Tunis, El-Manar University, ³Research Unit 01/UR/08-07, Faculty of Medicine of Tunis, El-Manar University, Tunisia ¹
Abstract Noise has long been realized as an environmental stress causing physiological, psychological and behavioral changes in humans. The aim of the present study was to determinate the effect of chronic noise at moderate intensities on both glandular and cardiac function and oxidative status. Our problem comes from working conditions in call centers where operators are responsible for making simple and repetitive tasks. One wishes to ascertain the effects of moderate sound levels on rats exposed to the same noise levels during similar periods to those experienced by call center operators. Male Wistar rats were exposed to 70 and 85 dB(A) to an octave-band noise (8-16 kHz) 6 h/day for 3 month. Corticosterone levels, oxidative status and functional exploration of adrenal and thyroid glands and cardiac tissue were determined. Exposure to long-term noise for different intensities (70 and 85 dB(A)) resulted in increased corticosterone levels, affected various parameters of the endocrine glands and cardiac function. Markers of oxidative stress (catalase, superoxide dismutase and lipid peroxidation) were increased. These results imply that long-term exposure to noise even at moderate levels may enhance physiological function related to neuroendocrine modulation and oxidative imbalance. In these data, the physiological changes occur during the different sounds suggests the concept of allostatic load or homeostatic response of the body. Keywords: Corticosterone, dilated veins, glandular dysfunction, moderate intensities, noise stress, oxidative stress
Introduction During daily life, people are exposed to potentially hazardous noise levels related to work environment, urban traffic, household appliances, discos and the like.[1,2] Extra-auditory effects of noise have been related to psychophysiologic stress and the involvement of the pituitaryadrenocortical axis.[3,4] According to World Health Organization (WHO), environmental noises cause stress and have direct consequences on the psychological and physiological health: Hearing loss, sleep disorders, cardiovascular problems, increased levels of stress hormones with effects on metabolism and the immune Access this article online Quick Response Code:
Website: www.noiseandhealth.org DOI: 10.4103/1463-1741.121236 PubMed ID: ***
Noise & Health, November-December 2013, Volume 15:67, 406-11
system. Romuald Abadie and Jean-Paul Bernadat consultantstrainers by CS3 have considered that when we living in houses exposed to more than 60 dB could be responsible for hormonal stimulation (adrenaline and cortisone) while our body did not need it. This release of hormones grows 38% all 5 dB between 45 and 65 dB.[5] The biological effects of noise does not only reduce auditory effects due narrow interconnection of nerve pathways and the acoustic nerve messages attain to other nerve centers, which can cause specific reactions in biological functions or other physiological systems as those related to hearing.
Methods Animals Male Wistar rats weighing 200-250 g (Institut of Pasteur, Tunisia) were used for the experiments. Animals were housed in the animal facility, fed ad libitum and kept under closely controlled environmental conditions (12 h light: Dark cycle, lights on between 07:00 and 19:00 h; room temperature 21°C). Animals were treated in accordance with the European Convention (1986) for the protection and use of 406
Gannouni, et al.: Effect of noise on extra-auditory system in rats
vertebrate animals. All possible efforts were made to reduce animal suffering and minimize the number of animals used. Noise exposure Two groups were exposed in this study for 3 months (9 rats of each group), the acoustic exposure was done using the audio software Audacity 1.3.12 (Unicode). The noise level was set at 70 dB(A) and 85 dB(A) to an octave-band noise (8-16 kHz) by the use of two loud speakers installed at a distance of 10 cm to each cage. The noise level was monitored using an Integrating Sound Level Meter with Class 1 accuracy-Type 2238 Bruel and Kjaer. The signals duration was 6 h/day and repeated automatically after selecting start and end of noise. Before starting exposure sessions, the sound was passed through a band-pass filter. During noise exposure, noise levels were controlled using a sound level meter, a preamplifier and a condenser microphone positioned at the level of the animal’s head. Control rats (N = 9) were placed in the same kind of cage without being exposed to noise when the background sound level was 32-35 dB(A). In order to avoid circadian variation, all animals were sacrificed by decapitation and the blood samples were obtained always between 7:00 and 8:00 A.M. Determination of corticosterone levels The assay[6] is to put in a buffer medium of labeled hormone and cold hormone in the presence of corticosteroid binding globulin. Briefly, 500 µl of ethanol was added to 100 µl of plasma assay. After agitation and centrifugation at 3000 rpm for 10 min, the supernatant was aspirated and evaporated under a stream of air. The same protocol is used for the standard range (0, 1, 2, 4, 8, 16, 32 and 64 ng/100 µl), using 100 µl of plasma adrenalectomized rats. After evaporation, 1 ml of 3H-corticosterone (specific radioactivity 82 Ci/mmol) was added and incubation 7 mn at 45°C. For determination of non-specific binding, 0.5 ml of charcoal-dextran solution was added followed by 10 mn incubation in ice and centrifugation at 3000 tr/mn. Aliquot of supernatant fraction was taken for scintillation counting (µβ2Perkin Elmer 2450 Microplate counter).
The superoxide dismutase (SOD) activity was assessed by the addition of bovine catalase and epinephrine. For catalase estimation, standard hydrogen peroxide (0.3 M) was provided as substrate and the catalase activity was terminated at intervals of 0, 15, 30, 60, 90, 120, 150, 180 s by the addition of sodium-potassium phosphate buffer, pH 7.[7] Lipid peroxidation was measured by estimating malonyldialdehyde, an intermediary product of lipid peroxidation, using thiobarbituric acid.[8] Bovine serum albumin (20 µl/ml) obtained from Sigma chemical, was used as standard for protein estimation.[9] Statistical analysis The results are expressed as mean ± standard error of mean the data collected were statistically analyzed using Student’s t-test. A value of P ˂ 0.05 was considered to be significant.
Results Noise induced corticosterone levels and altered adrenal gland During noise exposure, marked differences in endocrine hormone activation were observed. The corticosterone level was higher significantly (P < 0.05) in both exposed animals to 70 dB(A) and 85 dB(A) compared with the control [Figure 1]. However, no significant increase in the animals exposed to 85 dB(A) compared with animals exposed to 70 dB(A). The structural disorganization in the adrenal gland was shown in Figures 2 and 3 at both sound levels. Noise protocol 70 dB(A) showed morphological alterations at the various adrenocortical zonae [Figure 2]. The first layers of the zona fasciculate are eosinophils, not containing lipids and showed the absence of arranged cords [Figure 2b]. Figure 2c shows that the abnormal segregation between the glomerulosa and the fasciculata, the fibroblasts which develop and form a capsule. However, Figure 2d shows that the cortex was
Exploration methods of heart, adrenal and thyroid function The tissue was rapidly removed, washed for several times with 0.1 M phosphate buffer saline (pH 7.4), fixed in 10% formalin for 24 h at real time, dehydrated by graded ethanol and embedded in paraffin. During experiment, sections (5-6 µm thick) were deparaffinized with xylene, stained with hematoxylin and eosin, after that observed by light microscopy. Measurement of various markers of oxidative stress A total of 400 mg of heart were homogenized in phosphate buffer, followed by centrifugation at 10,000tr/10 mn. 1 ml of aliquot is reserved for the determination of protein. 407
Figure 1: Plasma corticosterone concentration in noise exposure. Significant differences compared with control are indicated by asterisks: *P < 0.05 Noise & Health, November-December 2013, Volume 15
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completely disorganized and zona reticularis was located between two layers of zona fasciculata. Animals exposed to 85 dB(A) showed same changes [Figure 3], cell damage at both areas glomerulosa and fasciculate were showed in Figure 3a and glomerulosa was disarranged and thin. The adrenocortical zonae showed in Figure 3b was small and specially the zona reticularis. Architecture change of the cells of the zona fasciculate was demonstrated in Figure 3c. In contrast, Figure 3d shows that zona fasciculate contains a lot of lipids. Noise induced oxidative stress and injury in cardiac cell The noise induced oxidative stress is estimated by analyzing the levels of free radical scavenging enzymes and lipid peroxidation. The noise exposure showed a significant
a
c
(P < 0.05) increase in lipid peroxidation levels in the exposed group to 70 dB(A) when compared with the control group [Figure 4]. However, higher significantly lipid peroxidation in group exposed to 85 dB(A). The catalase levels [Figure 5] showed significantly (P < 0.05) elevated in both noise exposed groups when compared with controls. The SOD levels were significantly (P < 0.05) increased in the entire exposed group after noise exposure [Figure 6]. Increased lipid peroxidation and SOD levels depend of noise intensity. This finding was not demonstrated to the catalase level. Histological sections of heart tissue of animals exposed to noise, showed the presence of highly inflamed areas containing lymphocytes, macrophages and polynucleaire in the pericardium groups exposed to 70 dB(A) [Figure 7a]. Lots of dilated veins in the periphery [Figure 7b and c] were observed for both noise exposure to 70 dB(A) and 85 dB(A).
b
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d
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Figure 2: Morphological evaluation of adrenal gland in rats exposed to noise. (a) (×40): Control group, (b-d) (×40): Group exposed to noise at 70 dB(A). Normal adrenal gland characterized by two concentric layers: Adrenal cortex and adrenal medulla, the cells of both layers are grouped in cords along capillaries. After noise exposure to 70 dB(A), adrenal cortex characterized by disorganization of each zona. ZG: Zona glomerulosa, ZF: Zona fasciculata, ZR: Zona reticularis, M: Medulla, Eo: Eosinophils, blue arrows: Abnormal segregation between zona glomerulosa and zona fasciculata
Figure 4: Lipid peroxidation after noise exposure. The results are expressed as mean ± standard error of mean. *Significance compared with control, **Higher significance compared with control Noise & Health, November-December 2013, Volume 15
Figure 3: Morphological evaluation of adrenal gland in rats exposed to noise. (a-d) (×100): Animals exposed to 85 dB(A). (a) The cells of zona glomerulosa and zona fasciculata lose their arrangement in cords. (b) Decrease in thickness of adrenal cortex. (c) Structural alteration in cells of zona fasciculata. (d) Zona fasciculata rich in lipid vesicle. ZF: Zona fasciculata, C: Capsule, M: Medulla, AC: Adrenal cortex, Eo: Eosinophils, LV: Lipid vesicle
Figure 5: Catalase after noise exposure. The results are expressed as mean ± standard error of mean. **Higher significance compared with control 408
Gannouni, et al.: Effect of noise on extra-auditory system in rats
Figure 6: Superoxide dismutase after noise exposure. The results are expressed as mean ± standard error of mean. **Higher significance compared with control
Noise induced morphological changes in thyroid gland After noise exposure, the histological sections of thyroid glands showed animals exposed to 70 dB(A) had colloids filled with inflammatory cells compared with the controls [Figure 8a and b]. Complete disorganization of the thyroid gland [Figure 8c and d] showed that colloids of animals exposed to 85 dB(A) are damaged and replaced by connective tissue.
Discussion Noise is one of the most widespread sources of environmental stress in living environment.[10] WHO has declared noise to be an international health problem. The present study showed that moderate intensity 70 dB(A) and 85 dB(A) induced in long-term an increased corticosterone level followed by alteration on the adrenal gland. Exposure to noise stress is known to activate the hypothalamicpituitary-adrenal (HPA) axis in different species.[11-13] The concentration of corticosterone was elevated in stressed animals between 3 and 6 h post-noise stress, confirming the activation of the HPA axis.[14] The few experiments on chronic noise exposure (>1 week) also showed a significant activation of the HPA axis although a habituation was observed to repeated noise sessions.[15] Increased levels of cortisol were reported in persons, who were experimentally exposed to aircraft noise with maximal levels of 55-65 dB(A)[16] during sleep at home. Our results show that long-term exposure to noise may lead to chronic increase of above the normal range. The effects of chronic exposure of rats to intensities noise of the call center were evaluated on both adrenal cortex structure and on plasma corticosterone levels. Cellular examination showed a marked involvement of each zona of the adrenal cortex. 409
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Figure 7: Morphological evaluation in the heart of rats exposed to noise. (a) (×100): Control group. (b) (×100): Animal exposed to 70 dB(A) showed inflamed area of pericardium and dilated veins. (c) (×100) and (d) (×40): Animals exposed to 85 dB(A) showed a lot of dilated veins in the periphery of pericardium
Our findings indicate that each zona of the adrenal cortex show a differential reaction to noise stress. In particular, we observed various alterations, the most frequent consisting of disorganization of zona fasciculata. This zona, producing cortisol (controlled by adrenocorticotropic hormone) showed clearly after noise exposure affected cells, they are not arranged in cords as in control, but become anarchically arranged due to the effect of noise. Similarly, corticosterone plasma levels significantly increased over the time of application of noise stimulus. These morphological changes seem to be in accord with the increased corticosterone levels. These results indicate that prolonged exposure to noise, whatever the sound intensity, induced structural and functional modifications in the adrenal gland. This finding has also been described in rats exposed to loud noise stimulation of 100 dB.[17,18] Moderate noise stress induced structural alterations of the adrenal cortex and medulla in association with noise density, indicating that noise dose-over a period of time and frequency of noise events may have an impact on adrenal morphology with possible consequences to endocrine dysfunction.[19] The association between call center noise and cardiovascular disorders were showed in this study. Oxidative stress, dilated veins and inflammatory responses in the heart of rats treated with different intensities of noise was investigated. Marked oxidative stress was induced as evaluated by antioxidants activity and increasing of lipid peroxidation. One consequence of noise exposure is increased production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide and hydroxyl radicals. In this study, the lipid peroxidation level increased markedly after chronic Noise & Health, November-December 2013, Volume 15
Gannouni, et al.: Effect of noise on extra-auditory system in rats
The increased activity of all these enzymes in chronic noise exposure may be a protective mechanism.
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Figure 8: Morphological evaluation of thyroid gland in rats exposed to noise. (a) (×40): Control group showed normal thyroid with colloids of different size (c: Colloid). (b) (×40): Animal exposed to 70 dB(A) showed damage cells of thyroid gland with inflammatory reaction. (c and d) (×100): Animals exposed to 85 dB(A). Thyroid gland is completely disorganized when the colloids are filled with inflammatory cells (c) and pathological effect demonstrated in (d), few intact colloids and the other replaced by connective tissue
noise exposure following by the significant increase in SOD and catalase levels, which is in agreement with the findings of Samson.[20] The significant increase observed in SOD and catalase levels in chronic noise exposure is an indicator of a relative increase in the superoxide radical production. The increased SOD activity is therefore an indication that the heart’s antioxidant machinery is activated in response to excessive generation of free radicals. The damage mechanisms were possibly associated with oxidative stress,[21] which is a common denominator in many aspects of heart-related diseases.[22] Histological detection of heart tissue showed damage cellular related to the creation of oxidative stress and increased levels of corticosterone. Some newer publications report significantly increased cortisol levels and changes in cardiovascular parameters in subjects exposed to noise.[23-30] The results of epidemiological studies on traffic noise and the cardiovascular risk can be explained theoretically by noise-induced chronic increase of cortisol.[31] The negative effects of noise on cell structure and function were supposed to be, at least in part, mediated by the increase of reactive oxygen species (ROS). The involvement of ROS might play a causal role in the induction and persistence of genetic damage related to loud noise exposure also in extra-auditory organs.[32] Elevation of the corticosterone level accelerates the generation of free radicals.[33] However, in the present study corticosterone, lipid peroxidation, SOD and catalase levels were significantly increased in animals exposed to chronic noise at moderate intensities. Noise & Health, November-December 2013, Volume 15
The relationship of noise stress at different intensities identified in call center and thyroid gland was investigated in this study however, the thyroid gland is one of the largest endocrine glands. The thyroid gland controls how quickly the body uses energy, makes proteins and controls how sensitive the body is to other hormones. The responses to various stress stimuli report that certain forms of stress such as noise exposure altered the thyroid activity in the guinea-pig and it has been observed in other species.[34] The thyroid allows the perception of sound and especially those of the human voice, which it allows understanding of the symbolism and fixing our language. It proceeds to the establishment of our verbal images and all the feelings resulting from human speech. Our study showed structural disorders in the thyroid gland after noise exposure for both noise levels. These facts indicate that endocrine noise effects may play a role in widespread functional disorders or even diseases.[35]
Conclusions The results of the present study indicate that exposure of rats to occupational call center noise causes morphological alterations at endocrine gland. Furthermore, it has been shown that noise exposure induces increase of corticosterone concentration and also affects the morphology of the adrenal gland and heart tissue. Oxidative stress and glandular changes occur during the different sound levels. All the results, implicate that chronic noise at moderate intensity sound exerted tissue injuries and damage mechanisms. The noise does not necessarily have to be strong to be annoying; it also depends on the duration of exposure. Finally, the data from this study suggests two concepts; the concept of allostasis and ischemia-reperfusion phenomena. Allostasis concept results in resilience of the organism to stress and their repetition in time. Address for correspondence: Dr. Noura Gannouni, Imm Elezz Villa, J11 Radès Méliane, Radès 2040, Tunis, Tunisia. E-mail: [email protected]
References 1. Kawecka-Jaszcz K. Effect of professional work and environmental factors on arterial blood pressure. Med Pr 1991;42:291-6. 2. Lang T, Fouriaud C, Jacquinet-Salord MC. Length of occupational noise exposure and blood pressure. Int Arch Occup Environ Health 1992;63:369-72. 3. Axelrod J, Reisine TD. Stress hormones: Their interaction and regulation. Science 1984;224:452-9. 4. Ising H, Braun C. Acute and chronic endocrine effects of noise: Review of the research conducted at the Institute for Water, Soil and Air Hygiene. Noise Health 2000;2:7-24.
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Gannouni, et al.: Effect of noise on extra-auditory system in rats 5. Abadie R, Bernadat JP. Le Bruit, Source de stress. Actualité 445. Available from: http://www.gereso.com/service-public/le-bruit-sourcede-stress.html. [Last cited on 2008 Jan 01]. 6. Murphy BE. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J ClinEndocrinolMetab 1967;27:973-90. 7. Aebi H. Catalase. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis, Vol. 2. NY: Academic Press; 1974. p. 673-84. 8. Devasagayam TP, Tarachand U. Decreased lipid peroxidation in the rat kidney during gestation. Biochem Biophys Res Commun 1987;145:134-8. 9. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75. 10. Wallenius MA. The interaction of noise stress and personal project stress on subjective health. J Environ Psychol 2004;24:167-77. 11. Engeland WC, Miller P, Gann DS. Pituitary-adrenal and adrenomedullary responses to noise in awake dogs. Am J Physiol 1990;258:R672-7. 12. van Raaij MT, Dobbe CJ, Elvers B, Timmerman A, Schenk E, Oortigiesen M, et al. Hormonal status and the neuroendocrine response to a novel heterotypic stressor involving subchronic noise exposure. Neuroendocrinology 1997;65:200-9. 13. Evans GW, Lercher P, Meis M, Ising H, Kofler WW. Community noise exposure and stress in children. J Acoust Soc Am 2001;109:1023-7. 14. Mazurek B, Haupt H, Joachim R, Klapp BF, Stöver T, Szczepek AJ. Stress induces transient auditory hypersensitivity in rats. Hear Res 2010;259:55-63. 15. Armario A, Castellanos JM, Balasch J. Adaptation of anterior pituitary hormones to chronic noise stress in male rats. Behav Neural Biol 1984;41:71-6. 16. Maschke C, Ising H, Arndt D. Nocturnal traffic noise and health: Results from laboratory and field studies. Federal Health Gazette 1995;4:130-7. 17. Pellegrini A, Soldani P, Gesi M, Lenzi P, Natale G, Paparelli A. Effect of varying noise stress duration on rat adrenal gland: An ultrastructural study. Tissue Cell 1997;29:597-602. 18. Soldani P, Gesi M, Lenzi P, Natale G, Fornai F, Pellegrini A, et al. Longterm exposure to noise modifies rat adrenal cortex ultrastructure and corticosterone plasma levels. J Submicrosc Cytol Pathol 1999;31:441-8. 19. Kanitz E, Otten W, Tuchscherer M. Central and peripheral effects of repeated noise stress on hypothalamic-pituitary-adrenocortical axis in pigs. Livestock Production Science 2005;94:213-24. 20. Samson J, Sheeladevi R, Ravindran R. Oxidative stress in brain and antioxidant activity of Ocimum sanctum in noise exposure. Neurotoxicology 2007;28:679-85. 21. Li H, Han M, Guo L, Li G, Sang N. Oxidative stress, endothelial
dysfunction and inflammatory response in rat heart to NO2 inhalation exposure. Chemosphere 2011;82:1589-96. 22. Blum A. Heart failure — New insights. Isr Med Assoc J 2009;11:105-11. 23. Babisch W. Health aspects of extra-aural noise research. Noise Health 2004;6:69-81. 24. Prasher D. Is there evidence that environmental noise is immunotoxic? Noise Health 2009;11:151-5. 25. Selander J, Bluhm G, Theorell T, Pershagen G, Babisch W, Seiffert I, et al. Saliva cortisol and exposure to aircraft noise in six European countries. Environ Health Perspect 2009;117:1713-7. 26. Belojevic G, Paunovic K, Jakovljevic B, Stojanov V, Ilic J, Slepcevic V, et al. Cardiovascular effects of environmental noise: Research in Serbia. Noise Health 2011;13:217-20. 27. Maschke C. Cardiovascular effects of environmental noise: Research in Germany. Noise Health 2011;13:205-11. 28. Lercher P, Botteldooren D, Widmann U, Uhrner U, Kammeringer E. Cardiovascular effects of environmental noise: Research in Austria. Noise Health 2011;13:234-50. 29. Bluhm G, Eriksson C. Cardiovascular effects of environmental noise: Research in Sweden. Noise Health 2011;13:212-6. 30. Yasuda N, Nakamura K. Heterogeneity of corticotropin-releasing factor (CRF). Jpn J Physiol 1997;47:147-59. 31. Lenzi P, Frenzilli G, Gesi M, Ferrucci M, Lazzeri G, Fornai F, et al. DNA damage associated with ultrastructural alterations in rat myocardium after loud noise exposure. Environ Health Perspect 2003;111:467-71. 32. Frenzilli G, Lenzi P, Scarcelli V, Fornai F, Pellegrini A, Soldani P, et al. Effects of loud noise exposure on DNA integrity in rat adrenal gland. Environ Health Perspect 2004;112:1671-2. 33. McIntosh LJ, Sapolsky RM. Glucocorticoids increase the accumulation of reactive oxygen species and enhance adriamycin-induced toxicity in neuronal culture. Exp Neurol 1996;141:201-6. 34. Brown-Grant K, Pethes G. The response of the thyroid gland of the guinea-pig to stress. J Physiol 1960;151:40-50. 35. Spreng M. Possible health effects of noise induced cortisol increase. Noise Health 2000;2:59-64.
How to cite this article: Gannouni N, Mhamdi A, Tebourbi O, El May M, Sakly M, Rhouma KB. Qualitative and quantitative assessment of noise at moderate intensities on extra-auditory system in adult rats. Noise Health 2013;15:406-11. Source of Support: Nil, Conflict of Interest: None declared.
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Abstract Noise has long been realized as an environmental stress causing physiological, psychological and behavioral changes in humans. The aim of the present study was to determinate the effect of chronic noise at moderate intensities on both glandular and cardiac function and oxidative status. Our problem comes from working conditions in call centers where operators are responsible for making simple and repetitive tasks. One wishes to ascertain the effects of moderate sound levels on rats exposed to the same noise levels during similar periods to those experienced by call center operators. Male Wistar rats were exposed to 70 and 85 dB(A) to an octave-band noise (8-16 kHz) 6 h/day for 3 month. Corticosterone levels, oxidative status and functional exploration of adrenal and thyroid glands and cardiac tissue were determined. Exposure to long-term noise for different intensities (70 and 85 dB(A)) resulted in increased corticosterone levels, affected various parameters of the endocrine glands and cardiac function. Markers of oxidative stress (catalase, superoxide dismutase and lipid peroxidation) were increased. These results imply that long-term exposure to noise even at moderate levels may enhance physiological function related to neuroendocrine modulation and oxidative imbalance. In these data, the physiological changes occur during the different sounds suggests the concept of allostatic load or homeostatic response of the body. Keywords: Corticosterone, dilated veins, glandular dysfunction, moderate intensities, noise stress, oxidative stress
Introduction During daily life, people are exposed to potentially hazardous noise levels related to work environment, urban traffic, household appliances, discos and the like.[1,2] Extra-auditory effects of noise have been related to psychophysiologic stress and the involvement of the pituitaryadrenocortical axis.[3,4] According to World Health Organization (WHO), environmental noises cause stress and have direct consequences on the psychological and physiological health: Hearing loss, sleep disorders, cardiovascular problems, increased levels of stress hormones with effects on metabolism and the immune Access this article online Quick Response Code:
Website: www.noiseandhealth.org DOI: 10.4103/1463-1741.121236 PubMed ID: ***
Noise & Health, November-December 2013, Volume 15:67, 406-11
system. Romuald Abadie and Jean-Paul Bernadat consultantstrainers by CS3 have considered that when we living in houses exposed to more than 60 dB could be responsible for hormonal stimulation (adrenaline and cortisone) while our body did not need it. This release of hormones grows 38% all 5 dB between 45 and 65 dB.[5] The biological effects of noise does not only reduce auditory effects due narrow interconnection of nerve pathways and the acoustic nerve messages attain to other nerve centers, which can cause specific reactions in biological functions or other physiological systems as those related to hearing.
Methods Animals Male Wistar rats weighing 200-250 g (Institut of Pasteur, Tunisia) were used for the experiments. Animals were housed in the animal facility, fed ad libitum and kept under closely controlled environmental conditions (12 h light: Dark cycle, lights on between 07:00 and 19:00 h; room temperature 21°C). Animals were treated in accordance with the European Convention (1986) for the protection and use of 406
Gannouni, et al.: Effect of noise on extra-auditory system in rats
vertebrate animals. All possible efforts were made to reduce animal suffering and minimize the number of animals used. Noise exposure Two groups were exposed in this study for 3 months (9 rats of each group), the acoustic exposure was done using the audio software Audacity 1.3.12 (Unicode). The noise level was set at 70 dB(A) and 85 dB(A) to an octave-band noise (8-16 kHz) by the use of two loud speakers installed at a distance of 10 cm to each cage. The noise level was monitored using an Integrating Sound Level Meter with Class 1 accuracy-Type 2238 Bruel and Kjaer. The signals duration was 6 h/day and repeated automatically after selecting start and end of noise. Before starting exposure sessions, the sound was passed through a band-pass filter. During noise exposure, noise levels were controlled using a sound level meter, a preamplifier and a condenser microphone positioned at the level of the animal’s head. Control rats (N = 9) were placed in the same kind of cage without being exposed to noise when the background sound level was 32-35 dB(A). In order to avoid circadian variation, all animals were sacrificed by decapitation and the blood samples were obtained always between 7:00 and 8:00 A.M. Determination of corticosterone levels The assay[6] is to put in a buffer medium of labeled hormone and cold hormone in the presence of corticosteroid binding globulin. Briefly, 500 µl of ethanol was added to 100 µl of plasma assay. After agitation and centrifugation at 3000 rpm for 10 min, the supernatant was aspirated and evaporated under a stream of air. The same protocol is used for the standard range (0, 1, 2, 4, 8, 16, 32 and 64 ng/100 µl), using 100 µl of plasma adrenalectomized rats. After evaporation, 1 ml of 3H-corticosterone (specific radioactivity 82 Ci/mmol) was added and incubation 7 mn at 45°C. For determination of non-specific binding, 0.5 ml of charcoal-dextran solution was added followed by 10 mn incubation in ice and centrifugation at 3000 tr/mn. Aliquot of supernatant fraction was taken for scintillation counting (µβ2Perkin Elmer 2450 Microplate counter).
The superoxide dismutase (SOD) activity was assessed by the addition of bovine catalase and epinephrine. For catalase estimation, standard hydrogen peroxide (0.3 M) was provided as substrate and the catalase activity was terminated at intervals of 0, 15, 30, 60, 90, 120, 150, 180 s by the addition of sodium-potassium phosphate buffer, pH 7.[7] Lipid peroxidation was measured by estimating malonyldialdehyde, an intermediary product of lipid peroxidation, using thiobarbituric acid.[8] Bovine serum albumin (20 µl/ml) obtained from Sigma chemical, was used as standard for protein estimation.[9] Statistical analysis The results are expressed as mean ± standard error of mean the data collected were statistically analyzed using Student’s t-test. A value of P ˂ 0.05 was considered to be significant.
Results Noise induced corticosterone levels and altered adrenal gland During noise exposure, marked differences in endocrine hormone activation were observed. The corticosterone level was higher significantly (P < 0.05) in both exposed animals to 70 dB(A) and 85 dB(A) compared with the control [Figure 1]. However, no significant increase in the animals exposed to 85 dB(A) compared with animals exposed to 70 dB(A). The structural disorganization in the adrenal gland was shown in Figures 2 and 3 at both sound levels. Noise protocol 70 dB(A) showed morphological alterations at the various adrenocortical zonae [Figure 2]. The first layers of the zona fasciculate are eosinophils, not containing lipids and showed the absence of arranged cords [Figure 2b]. Figure 2c shows that the abnormal segregation between the glomerulosa and the fasciculata, the fibroblasts which develop and form a capsule. However, Figure 2d shows that the cortex was
Exploration methods of heart, adrenal and thyroid function The tissue was rapidly removed, washed for several times with 0.1 M phosphate buffer saline (pH 7.4), fixed in 10% formalin for 24 h at real time, dehydrated by graded ethanol and embedded in paraffin. During experiment, sections (5-6 µm thick) were deparaffinized with xylene, stained with hematoxylin and eosin, after that observed by light microscopy. Measurement of various markers of oxidative stress A total of 400 mg of heart were homogenized in phosphate buffer, followed by centrifugation at 10,000tr/10 mn. 1 ml of aliquot is reserved for the determination of protein. 407
Figure 1: Plasma corticosterone concentration in noise exposure. Significant differences compared with control are indicated by asterisks: *P < 0.05 Noise & Health, November-December 2013, Volume 15
Gannouni, et al.: Effect of noise on extra-auditory system in rats
completely disorganized and zona reticularis was located between two layers of zona fasciculata. Animals exposed to 85 dB(A) showed same changes [Figure 3], cell damage at both areas glomerulosa and fasciculate were showed in Figure 3a and glomerulosa was disarranged and thin. The adrenocortical zonae showed in Figure 3b was small and specially the zona reticularis. Architecture change of the cells of the zona fasciculate was demonstrated in Figure 3c. In contrast, Figure 3d shows that zona fasciculate contains a lot of lipids. Noise induced oxidative stress and injury in cardiac cell The noise induced oxidative stress is estimated by analyzing the levels of free radical scavenging enzymes and lipid peroxidation. The noise exposure showed a significant
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(P < 0.05) increase in lipid peroxidation levels in the exposed group to 70 dB(A) when compared with the control group [Figure 4]. However, higher significantly lipid peroxidation in group exposed to 85 dB(A). The catalase levels [Figure 5] showed significantly (P < 0.05) elevated in both noise exposed groups when compared with controls. The SOD levels were significantly (P < 0.05) increased in the entire exposed group after noise exposure [Figure 6]. Increased lipid peroxidation and SOD levels depend of noise intensity. This finding was not demonstrated to the catalase level. Histological sections of heart tissue of animals exposed to noise, showed the presence of highly inflamed areas containing lymphocytes, macrophages and polynucleaire in the pericardium groups exposed to 70 dB(A) [Figure 7a]. Lots of dilated veins in the periphery [Figure 7b and c] were observed for both noise exposure to 70 dB(A) and 85 dB(A).
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Figure 2: Morphological evaluation of adrenal gland in rats exposed to noise. (a) (×40): Control group, (b-d) (×40): Group exposed to noise at 70 dB(A). Normal adrenal gland characterized by two concentric layers: Adrenal cortex and adrenal medulla, the cells of both layers are grouped in cords along capillaries. After noise exposure to 70 dB(A), adrenal cortex characterized by disorganization of each zona. ZG: Zona glomerulosa, ZF: Zona fasciculata, ZR: Zona reticularis, M: Medulla, Eo: Eosinophils, blue arrows: Abnormal segregation between zona glomerulosa and zona fasciculata
Figure 4: Lipid peroxidation after noise exposure. The results are expressed as mean ± standard error of mean. *Significance compared with control, **Higher significance compared with control Noise & Health, November-December 2013, Volume 15
Figure 3: Morphological evaluation of adrenal gland in rats exposed to noise. (a-d) (×100): Animals exposed to 85 dB(A). (a) The cells of zona glomerulosa and zona fasciculata lose their arrangement in cords. (b) Decrease in thickness of adrenal cortex. (c) Structural alteration in cells of zona fasciculata. (d) Zona fasciculata rich in lipid vesicle. ZF: Zona fasciculata, C: Capsule, M: Medulla, AC: Adrenal cortex, Eo: Eosinophils, LV: Lipid vesicle
Figure 5: Catalase after noise exposure. The results are expressed as mean ± standard error of mean. **Higher significance compared with control 408
Gannouni, et al.: Effect of noise on extra-auditory system in rats
Figure 6: Superoxide dismutase after noise exposure. The results are expressed as mean ± standard error of mean. **Higher significance compared with control
Noise induced morphological changes in thyroid gland After noise exposure, the histological sections of thyroid glands showed animals exposed to 70 dB(A) had colloids filled with inflammatory cells compared with the controls [Figure 8a and b]. Complete disorganization of the thyroid gland [Figure 8c and d] showed that colloids of animals exposed to 85 dB(A) are damaged and replaced by connective tissue.
Discussion Noise is one of the most widespread sources of environmental stress in living environment.[10] WHO has declared noise to be an international health problem. The present study showed that moderate intensity 70 dB(A) and 85 dB(A) induced in long-term an increased corticosterone level followed by alteration on the adrenal gland. Exposure to noise stress is known to activate the hypothalamicpituitary-adrenal (HPA) axis in different species.[11-13] The concentration of corticosterone was elevated in stressed animals between 3 and 6 h post-noise stress, confirming the activation of the HPA axis.[14] The few experiments on chronic noise exposure (>1 week) also showed a significant activation of the HPA axis although a habituation was observed to repeated noise sessions.[15] Increased levels of cortisol were reported in persons, who were experimentally exposed to aircraft noise with maximal levels of 55-65 dB(A)[16] during sleep at home. Our results show that long-term exposure to noise may lead to chronic increase of above the normal range. The effects of chronic exposure of rats to intensities noise of the call center were evaluated on both adrenal cortex structure and on plasma corticosterone levels. Cellular examination showed a marked involvement of each zona of the adrenal cortex. 409
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Figure 7: Morphological evaluation in the heart of rats exposed to noise. (a) (×100): Control group. (b) (×100): Animal exposed to 70 dB(A) showed inflamed area of pericardium and dilated veins. (c) (×100) and (d) (×40): Animals exposed to 85 dB(A) showed a lot of dilated veins in the periphery of pericardium
Our findings indicate that each zona of the adrenal cortex show a differential reaction to noise stress. In particular, we observed various alterations, the most frequent consisting of disorganization of zona fasciculata. This zona, producing cortisol (controlled by adrenocorticotropic hormone) showed clearly after noise exposure affected cells, they are not arranged in cords as in control, but become anarchically arranged due to the effect of noise. Similarly, corticosterone plasma levels significantly increased over the time of application of noise stimulus. These morphological changes seem to be in accord with the increased corticosterone levels. These results indicate that prolonged exposure to noise, whatever the sound intensity, induced structural and functional modifications in the adrenal gland. This finding has also been described in rats exposed to loud noise stimulation of 100 dB.[17,18] Moderate noise stress induced structural alterations of the adrenal cortex and medulla in association with noise density, indicating that noise dose-over a period of time and frequency of noise events may have an impact on adrenal morphology with possible consequences to endocrine dysfunction.[19] The association between call center noise and cardiovascular disorders were showed in this study. Oxidative stress, dilated veins and inflammatory responses in the heart of rats treated with different intensities of noise was investigated. Marked oxidative stress was induced as evaluated by antioxidants activity and increasing of lipid peroxidation. One consequence of noise exposure is increased production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide and hydroxyl radicals. In this study, the lipid peroxidation level increased markedly after chronic Noise & Health, November-December 2013, Volume 15
Gannouni, et al.: Effect of noise on extra-auditory system in rats
The increased activity of all these enzymes in chronic noise exposure may be a protective mechanism.
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Figure 8: Morphological evaluation of thyroid gland in rats exposed to noise. (a) (×40): Control group showed normal thyroid with colloids of different size (c: Colloid). (b) (×40): Animal exposed to 70 dB(A) showed damage cells of thyroid gland with inflammatory reaction. (c and d) (×100): Animals exposed to 85 dB(A). Thyroid gland is completely disorganized when the colloids are filled with inflammatory cells (c) and pathological effect demonstrated in (d), few intact colloids and the other replaced by connective tissue
noise exposure following by the significant increase in SOD and catalase levels, which is in agreement with the findings of Samson.[20] The significant increase observed in SOD and catalase levels in chronic noise exposure is an indicator of a relative increase in the superoxide radical production. The increased SOD activity is therefore an indication that the heart’s antioxidant machinery is activated in response to excessive generation of free radicals. The damage mechanisms were possibly associated with oxidative stress,[21] which is a common denominator in many aspects of heart-related diseases.[22] Histological detection of heart tissue showed damage cellular related to the creation of oxidative stress and increased levels of corticosterone. Some newer publications report significantly increased cortisol levels and changes in cardiovascular parameters in subjects exposed to noise.[23-30] The results of epidemiological studies on traffic noise and the cardiovascular risk can be explained theoretically by noise-induced chronic increase of cortisol.[31] The negative effects of noise on cell structure and function were supposed to be, at least in part, mediated by the increase of reactive oxygen species (ROS). The involvement of ROS might play a causal role in the induction and persistence of genetic damage related to loud noise exposure also in extra-auditory organs.[32] Elevation of the corticosterone level accelerates the generation of free radicals.[33] However, in the present study corticosterone, lipid peroxidation, SOD and catalase levels were significantly increased in animals exposed to chronic noise at moderate intensities. Noise & Health, November-December 2013, Volume 15
The relationship of noise stress at different intensities identified in call center and thyroid gland was investigated in this study however, the thyroid gland is one of the largest endocrine glands. The thyroid gland controls how quickly the body uses energy, makes proteins and controls how sensitive the body is to other hormones. The responses to various stress stimuli report that certain forms of stress such as noise exposure altered the thyroid activity in the guinea-pig and it has been observed in other species.[34] The thyroid allows the perception of sound and especially those of the human voice, which it allows understanding of the symbolism and fixing our language. It proceeds to the establishment of our verbal images and all the feelings resulting from human speech. Our study showed structural disorders in the thyroid gland after noise exposure for both noise levels. These facts indicate that endocrine noise effects may play a role in widespread functional disorders or even diseases.[35]
Conclusions The results of the present study indicate that exposure of rats to occupational call center noise causes morphological alterations at endocrine gland. Furthermore, it has been shown that noise exposure induces increase of corticosterone concentration and also affects the morphology of the adrenal gland and heart tissue. Oxidative stress and glandular changes occur during the different sound levels. All the results, implicate that chronic noise at moderate intensity sound exerted tissue injuries and damage mechanisms. The noise does not necessarily have to be strong to be annoying; it also depends on the duration of exposure. Finally, the data from this study suggests two concepts; the concept of allostasis and ischemia-reperfusion phenomena. Allostasis concept results in resilience of the organism to stress and their repetition in time. Address for correspondence: Dr. Noura Gannouni, Imm Elezz Villa, J11 Radès Méliane, Radès 2040, Tunis, Tunisia. E-mail: [email protected]
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Gannouni, et al.: Effect of noise on extra-auditory system in rats 5. Abadie R, Bernadat JP. Le Bruit, Source de stress. Actualité 445. Available from: http://www.gereso.com/service-public/le-bruit-sourcede-stress.html. [Last cited on 2008 Jan 01]. 6. Murphy BE. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J ClinEndocrinolMetab 1967;27:973-90. 7. Aebi H. Catalase. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis, Vol. 2. NY: Academic Press; 1974. p. 673-84. 8. Devasagayam TP, Tarachand U. Decreased lipid peroxidation in the rat kidney during gestation. Biochem Biophys Res Commun 1987;145:134-8. 9. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75. 10. Wallenius MA. The interaction of noise stress and personal project stress on subjective health. J Environ Psychol 2004;24:167-77. 11. Engeland WC, Miller P, Gann DS. Pituitary-adrenal and adrenomedullary responses to noise in awake dogs. Am J Physiol 1990;258:R672-7. 12. van Raaij MT, Dobbe CJ, Elvers B, Timmerman A, Schenk E, Oortigiesen M, et al. Hormonal status and the neuroendocrine response to a novel heterotypic stressor involving subchronic noise exposure. Neuroendocrinology 1997;65:200-9. 13. Evans GW, Lercher P, Meis M, Ising H, Kofler WW. Community noise exposure and stress in children. J Acoust Soc Am 2001;109:1023-7. 14. Mazurek B, Haupt H, Joachim R, Klapp BF, Stöver T, Szczepek AJ. Stress induces transient auditory hypersensitivity in rats. Hear Res 2010;259:55-63. 15. Armario A, Castellanos JM, Balasch J. Adaptation of anterior pituitary hormones to chronic noise stress in male rats. Behav Neural Biol 1984;41:71-6. 16. Maschke C, Ising H, Arndt D. Nocturnal traffic noise and health: Results from laboratory and field studies. Federal Health Gazette 1995;4:130-7. 17. Pellegrini A, Soldani P, Gesi M, Lenzi P, Natale G, Paparelli A. Effect of varying noise stress duration on rat adrenal gland: An ultrastructural study. Tissue Cell 1997;29:597-602. 18. Soldani P, Gesi M, Lenzi P, Natale G, Fornai F, Pellegrini A, et al. Longterm exposure to noise modifies rat adrenal cortex ultrastructure and corticosterone plasma levels. J Submicrosc Cytol Pathol 1999;31:441-8. 19. Kanitz E, Otten W, Tuchscherer M. Central and peripheral effects of repeated noise stress on hypothalamic-pituitary-adrenocortical axis in pigs. Livestock Production Science 2005;94:213-24. 20. Samson J, Sheeladevi R, Ravindran R. Oxidative stress in brain and antioxidant activity of Ocimum sanctum in noise exposure. Neurotoxicology 2007;28:679-85. 21. Li H, Han M, Guo L, Li G, Sang N. Oxidative stress, endothelial
dysfunction and inflammatory response in rat heart to NO2 inhalation exposure. Chemosphere 2011;82:1589-96. 22. Blum A. Heart failure — New insights. Isr Med Assoc J 2009;11:105-11. 23. Babisch W. Health aspects of extra-aural noise research. Noise Health 2004;6:69-81. 24. Prasher D. Is there evidence that environmental noise is immunotoxic? Noise Health 2009;11:151-5. 25. Selander J, Bluhm G, Theorell T, Pershagen G, Babisch W, Seiffert I, et al. Saliva cortisol and exposure to aircraft noise in six European countries. Environ Health Perspect 2009;117:1713-7. 26. Belojevic G, Paunovic K, Jakovljevic B, Stojanov V, Ilic J, Slepcevic V, et al. Cardiovascular effects of environmental noise: Research in Serbia. Noise Health 2011;13:217-20. 27. Maschke C. Cardiovascular effects of environmental noise: Research in Germany. Noise Health 2011;13:205-11. 28. Lercher P, Botteldooren D, Widmann U, Uhrner U, Kammeringer E. Cardiovascular effects of environmental noise: Research in Austria. Noise Health 2011;13:234-50. 29. Bluhm G, Eriksson C. Cardiovascular effects of environmental noise: Research in Sweden. Noise Health 2011;13:212-6. 30. Yasuda N, Nakamura K. Heterogeneity of corticotropin-releasing factor (CRF). Jpn J Physiol 1997;47:147-59. 31. Lenzi P, Frenzilli G, Gesi M, Ferrucci M, Lazzeri G, Fornai F, et al. DNA damage associated with ultrastructural alterations in rat myocardium after loud noise exposure. Environ Health Perspect 2003;111:467-71. 32. Frenzilli G, Lenzi P, Scarcelli V, Fornai F, Pellegrini A, Soldani P, et al. Effects of loud noise exposure on DNA integrity in rat adrenal gland. Environ Health Perspect 2004;112:1671-2. 33. McIntosh LJ, Sapolsky RM. Glucocorticoids increase the accumulation of reactive oxygen species and enhance adriamycin-induced toxicity in neuronal culture. Exp Neurol 1996;141:201-6. 34. Brown-Grant K, Pethes G. The response of the thyroid gland of the guinea-pig to stress. J Physiol 1960;151:40-50. 35. Spreng M. Possible health effects of noise induced cortisol increase. Noise Health 2000;2:59-64.
How to cite this article: Gannouni N, Mhamdi A, Tebourbi O, El May M, Sakly M, Rhouma KB. Qualitative and quantitative assessment of noise at moderate intensities on extra-auditory system in adult rats. Noise Health 2013;15:406-11. Source of Support: Nil, Conflict of Interest: None declared.
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