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|Year : 2009 | Volume
| Issue : 43 | Page : 118--123
Chronic exposure of rats to occupational textile noise causes cytological changes in adrenal cortex
Maria Joao R Oliveira1, Mariana P Monteiro1, Andreia M Ribeiro1, Duarte Pignatelli2, Artur P Aguas1,
1 Department of Anatomy and UMIB (Unit for Multidisciplinary Biomedical Research) of ICBAS, Portugal
2 Institute of Histology Professor Abel Salazar, Medical Faculty, University of Porto, Porto, Portugal
Maria Joao R Oliveira
Instituto de Ciências Biomédicas Abel Salazar, Largo Prof. Abel Salazar, 2, 4099-003 Porto
Chronic exposure to industrial noise and its effects on biological systems. Occupational exposure to noise may result in health disorders. Our aim was to evaluate the effects of chronic exposure to high-intensity noise of textile industry cotton rooms on the adrenal morphology. The environmental noise of a cotton-mill room from a large textile factory of Northern Portugal was recorded and reproduced by an adopted electroacoustic setup in a sound-insulated animal room where the rats were housed. The sounds were reproduced at the original levels of approximately 92 dB, which was achieved by equalization and distribution of sound output in the room. Wistar rats were submitted to noise exposure, in the same time schedule as employed in textile plants. After one, three, five, and seven months, the adrenals were collected and analyzed by light microscopy. Analyzed by multivariate analysis of variance and post hoc Bonferroni correction for multiple comparisons of the means between the groups. Noise exposure induced time-dependent changes in adrenal cortex, with decrease of zona fasciculata (ZF) and increase of zona reticularis volumes, together with a significant depletion of lipid droplet density in ZF cells of exposed rats, in comparison to control rats. Chronic exposure of rats to textile industry noise triggers cytological changes in the adrenals that suggest the existence of a sustained stress response.
|How to cite this article:|
Oliveira MR, Monteiro MP, Ribeiro AM, Pignatelli D, Aguas AP. Chronic exposure of rats to occupational textile noise causes cytological changes in adrenal cortex.Noise Health 2009;11:118-123
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Oliveira MR, Monteiro MP, Ribeiro AM, Pignatelli D, Aguas AP. Chronic exposure of rats to occupational textile noise causes cytological changes in adrenal cortex. Noise Health [serial online] 2009 [cited 2022 Aug 13 ];11:118-123
Available from: https://www.noiseandhealth.org/text.asp?2009/11/43/118/50697
Noise of high intensity and low frequency is an environmental aggression that is present in a number of industrial textile plants of our days, particularly in industrial plants containing heavy machinery that produce high levels of noise pollution, namely in cotton rooms of textile outfits , and in aeronautical industry.  This type of noise is an occupational hazard that is liable to affect the health of industrial textile-plant workers. In fact, we have documented that chronic exposure of rats to noise taped at an industrial cotton room induces alterations in their respiratory epithelium, , a finding that is consistent with the reported increase in respiratory infections  and decrease in respiratory function in textile workers. , In general, chronic exposure to excessive noise pollution may lead to a systemic disorder, the so-called vibroacoustic disease, in susceptible individuals. 
Excessive occupational noise can be used as a model of chronic stress.  Furthermore, excessive noise exposure has been found to induce changes in adrenal function and morphology. Gesi and collaborators have documented time-dependent changes in male rats of the ultrastructure of zone fasciculate (ZF) of the adrenal cortex, and an increase of corticosterone release following chronic exposure (up to 21 days, 6 hours per day) to loud noise (100 dBA, 0-26 KHz).  In a different study by Soldani and collaborators, female rats exposed to noise for 7 or 21 days also displayed ultrastructural changes in the adrenal cortex, like diluted matrix, cristolysis of mitochondria, and swelling of smooth endoplasmic reticulum membranes, together with an increase of plasma corticosterone levels. These changes were most prominent after 21 days of exposure. 
Since there were no previous studies documenting the effects of more than 21 days of exposure to noise, similar to a daily period of exposure of textile workers, on the adrenal morphology, our aim was to evaluate adrenal morphology of rats exposed to a similar noise schedule up to seven months.
Materials and Methods
Animals and experimental groups
Forty adult male Wistar rats purchased from a commercial breeder (Charles River Laboratories, Barcelona, Spain) were used in this experiment. Animals were eight-weeks old at time of enrolment in the experiments and were housed in pairs in a plastic cage (42 × 27 × 16 cm) with a steel lid and unrestricted access to food (commercial chow) and tap water, in standard animal housing conditions.
The forty rats ( N = 40) were randomized into two equal groups to be submitted either to noise exposure or serve as unexposed controls. The 20 rats of each group were further allocated to four experimental subgroups and submitted to noise exposure, according to an occupationally simulated time schedule during the dark phase, which is the period of rodent activity similar to human daytime activity, (eight hours/day from 9 am to 5 pm; five days/week with weekends in silence), for different periods of noise-exposure, which ranged from 1-7 months. The different subgroups of noise-exposed rats were sacrificed after one, three, five, and seven months of exposure. The remaining 20 Wistar rats were used as age-matched controls.
The environmental noise of a cotton-mill room from a large textile factory of Northern Portugal was used as the paradigm of the occupational noise. The noise present in this cotton mill room was recorded and reproduced. An electroacoustic setup was adopted; it employed a PC-based system, with a DT2823 data acquisition and a SB live 5.1 cards, one B and K 4165 microphone with preamplifier, one 2-channel power amplifier, 16 monitor-type, and one subwoofer loudspeakers in bi-amplification. The software was designed using the LabVIEW system. Sound signals processing was done offline, applying LabVIEW and Matlab systems. This apparatus was capable of recording and reproducing the specified noise sounds while monitoring the saturation level in the amplitude dynamic range. A 99.7% dynamic range was preserved for all signals. Signal acquisition and processing methodologies were designed in order to carefully measure and preserve the sound characteristics. Total signals duration was one hour. Frequency and amplitude characterization of signals was done for all samples. Reproduction of sounds at the original levels of approximately 92 dB (with spectrum very near the original one) was achieved by equalization and distribution of sound output in the room. The spectrum of frequencies and intensities of the noise used in this study are documented in [Figure 1].
The recorded noise was then reproduced in a sound-insulated animal room, where the rats were to be exposed. The sound characterization and room equalization was done by means of a 35 filter bank composed by three low-frequency octave band band-pass filters and 32 one-third octave pass band filters for the upper bands. All filters had 50 dB selectivity. The average sound pressure level in the room, as well as the dispersion of values among cages was carefully controlled. The final sound pressure values that were obtained, measured with a quality calibrated sound-meter, were within a 3 dB tolerance relative to the original values, and the dispersion of values among cages was also inside a tolerance of 3 dB relative to the referenced average. The detailed spatial organization of the room where the rats were exposed to noise is illustrated in [Figure 2].
Adrenal morphologic studies
The different subgroups of noise-exposed rats were sacrificed after one, three, five, and seven months of exposure. The remaining 20 Wistar rats were used as age-matched controls.
At the end of the experiment, the rats were sacrificed by a lethal intravenous injection of sodium-pentobarbital (40 mg/kg), transcardially perfused with saline followed by paraformaldeide, and the left adrenal excised.
An equatorial coronal section passing by the adrenal hilus was made for all the adrenals and the fragments obtained were routinely processed for light microscopy, paraffin embedded and 3-µm slices were obtained. Slices in duplicate were dyed according to standard Hematoxylin and Eosin (H and E) and modified Masson tricromic techniques, to allow the discrimination between the different cortical layers.
Using a Zeiss optic microscope, on a 100 times magnification, the adrenal diameters (adrenal, medulla, zona glomerularis (ZG), ZF, and zona reticularis (ZR) widths), were measured with the aid of a 100-points ruler, corresponding each segment between consecutive points to 10 µm, on the magnification used. The diameters were then used to calculate the volumes using the formula for volume of a sphere.
Lipid droplets and nucleus density
To further characterize the morphologic changes, 20 pictures of the upper inner zone for each adrenal were taken with a Zeiss optic microscope with camera, Fujifilm 100 (Amplification: 1000×). The number of lipid droplets and nucleus per picture was counted as previously described  with the help of a transparent grid with 20 points, spaced 4 cm from each other that was superimposed on the photographs according to the Weibel method,  and the numerical density and relative area occupied with lipid droplets were calculated using the following formula: total points of lipid droplets or nucleus divided by total points of the grid inside the photograph. Data are presented as the average density of lipid droplets or nuclei.
Volumes of the different adrenal zones are expressed as a proportion of the total adrenal volume. Results are shown as means ± standard error of the mean (SEM), unless otherwise specified. Data were submitted to arcsin√(proportion) transformation to obtain normal distribution, and analyzed by multivariate analysis of variance after, with post hoc Bonferroni correction for multiple comparisons of the means between the groups, using the SPSS 14.0 software for Windows; P Adrenal volumes
Overall comparison of the volumes occupied by the different regions of the adrenal gland revealed that the chronic noise treatment of the rats caused a significant ( P P = 0.027) increase in the volume of the ZR domain of the adrenal cortex in the same noise-treated rats. In contrast, no significant difference was seen between noise-treated and control rats with regards to the volume of the adrenal ZG domain. Also, no significant difference was observed in volume of the adrenal medulla between the two groups of rats. A significant age-dependent decrease in the volume occupied by the adrenal FZ domain in noise-treated and control rats was also found ( P = 0.032). However, the total adrenal volume did not change significantly with age or between noise-treated and control rats [Table 1].
Lipid droplet density
Noise exposure caused significant ( P = 0.009) decreases in the density of lipid droplets present in ZF cells of the rat adrenal cortex [Figure 4]. The kinetics of lipid droplet density along the seven-month period of our study was different in control and noise-treated rats. In control rats, lipid droplet density showed an increase from one to three months (0.058 ± 0.012 to 0.110 ± 0.017), followed by a decrease from three up to seven months (0.110 ± 0.017 to 0.040 ± 0.007). In the age-matched noise-treated rats, there was also an initial (one-three months of exposure) increase in lipid droplet density (0.033 ± 0.007 to 0.071 ± 0.010) that was also followed (3-5 months of exposure) by a decrease (0.071 ± 0.010 to 0.018 ± 0.005); however, there was later on (5-7 months of exposure) an enhancement (0.018 ± 0.005 to 0.053 ± 0.005) in lipid droplet density. Comparison of the time-group interaction between the two groups of rats showed that the pattern of change within each of the two groups (noise-treated and control) was significantly different ( P = 0.041) [Figure 5], due to the opposite trends in lipid droplet density observed in rats with 5-7 months of exposure to noise. Morphometric analysis showed no significant differences in nuclear density between noise-treated and control rats.
The investigation shows that chronic exposure of rats to occupational noise of the textile industry, following a daily schedule similar to that of plant workers, triggered significant morphological changes in the adrenal cortex, namely a decrease in adrenal ZF volume that was derived, at least in part, from depletion of intracellular lipid droplets. In addition, the volume of adrenal ZR was enhanced in the noise-treated rats, and this may account for our finding that noise exposure resulted in no difference in the total volume of the adrenal gland between noise-treated and age-matched control rats. The noise-induced changes were located in the adrenal cortex inner zone (ACIZ) that comprises the ZR and the ZF domains. The ACIZ is the most sensitive area to adrenocorticotropic hormone (ACTH) stimulation, known to increase during stress conditions that activate the hypothalamus-pituitary-adrenal (HPA) axis.  Lipid droplet density decreases in the adrenal cortex when corticosterone secretion increases, viz ., after acute stress, , and it decreases when steroidogenesis is blocked.  Thus, changes in the lipid droplet content of adrenal cortex correlate with changes in steroidogenesis, since cholesterol, as the common precursor molecule for steroid hormones, is required in the first step of steroidogenesis; this molecule is mostly derived from the hydrolysis of cholesteryl esters present in lipid droplets of steroid-producing cells. ,, Therefore, variation in the lipid droplets density can act as surrogate marker for steroidogenic activity in the ZF. ,
The patterns of exposure to chronic stressful stimuli may modulate the adaptative response of the adrenal cortex, namely corticosterone synthesis and secretion. For instance, a permanent stressful stimuli results in an adaptative response in corticosterone secretion that is expressed by a return-to-normal of both ACTH and corticosterone values.  In contrast, a repetitive-type chronic stress may induce increase in corticosterone secretion that remains enhanced in spite of the return-to-normal levels of ACTH. , Interestingly, after seven months of noise exposure, the rats showed a reversal of the adrenal response to the noise stress since they displayed an increase in lipid droplets density when compared to controls, which suggests an escape phenomenon.
Our data indicate that occupational textile noise causes, during the first months of noise exposure, an intense activation of the HPA axis, due to discontinuous and repetitive stimuli triggering acute stress episodes. This interpretation is supported by the data of Koko and coworkers who reported, similarly to our findings, that acute heat stress exposure triggers a reduction of the adrenal ZF volume with depletion of the cell lipid droplets and enhancement in the adrenal ZR volume.  Chronic exposure of workers to textile noise is known to increase urinary cortisol excretion. , Adrenal androgens, such as, dehydroepiandrosterone-sulphate (DHEAS), which is secreted by the adrenal ZR, have also been reported to be enhanced following acute noise stress in humans.  In humans, the major androgen produced by adrenal ZR is DHEAS, which derives from sulfating of the DHEA precursor produced by the ZF.  Stressful stimuli are also able to activate the release of hormones that are independent of the HPA axis, such as prolactin, which has been shown to induce ZR hypertrophy.  Altogether, the morphological changes observed in the inner zone of the adrenal cortex suggest that there is a stress-driven activation of the HPA axis.
Textile noise is characterized by high intensity and a wide spectrum of wave lengths. It causes both psychological stress and physical vibration of body structures; the latter effect resulting from the impact of low-frequency sound waves on the body surface. Experimental studies have shown that rat tissues submitted to chronic noise undergo two major morphological changes: fibrosis of several viscera, such as lung  and stomach,  and of serous membranes, such as pleura  and pericardium;  as well as the alteration of cell membrane projections, such as cilia or microvilli. ,,,
We found here no evidence of fibrosis in the adrenal glands of rats submitted to textile noise. This may be due to the deep location of the adrenal glands that protects them from the vibration affecting the body surface of the rats. Our interpretation is consistent with the fact that noise-induced fibrosis has only been found in structures that are either close to the body surface, such as pleura,  or in body channels that are in continuity with the surface of the body, such as stomach, lung, and trachea. ,,,, Thus, the described alterations in the morphology of adrenal glands appear to be solely derived from the stress induced by the noise, with no participation of body vibration.
In conclusion, the present investigation offers morphologic evidence that chronic exposure to repetitive textile noise causes time-dependent changes in the adrenal cortex that involve enhancement in the volume of the ZR and decreased of ZF, together with depletion of adrenal lipid droplets that suggest stimulation of adrenal steroidogenesis of glucocorticoids and androgens.
We thank Mrs. Alexandrina Ribeiro, Mr. Emanuel Monteiro, Mr. António Costa e Silva, and Dr. Madalena Costa, for technical assistance. This work was funded by grants from FCT (POCTI/FEDER), Portugal.
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