Oliveira, Maria João R. BSc, MSc; Pereira, António S. MD, PhD; Guimarães, Laura BSc, MSc; Freitas, Diamantino PhD; Carvalho, António P. O. PhD; Grande, Nuno R. MD, PhD; Águas, Artur P. MD, PhD
From the Department of Anatomy (Dr Oliveira, Dr Pereira, Dr Grande, Dr Águas) and Population Studies (Dr Guimarães), ICBAS, Abel Salazar Institute for Biomedical Sciences, UMIB and IBMC; Engineering Faculty (Dr Freitas, Dr Carvalho), Polo da Asprela, University of Porto, Portugal, European Union.
Address correspondence to: Maria João R. Oliveira, MSc, Department of Anatomy, ICBAS/UP Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal; e-mail: email@example.com.
Copyright © by American College of Occupational and Environmental Medicine
Noise has become a common feature of working environments of modern man. 1–4 Textile industries, in particular, use machinery that exposes its operators to high levels of noise pollution. We have shown before that in other working environment where noise pollution is prevalent, eg, jet engine repair rooms, systemic disorders may occur, particularly with regards to nervous and respiratory diseases;5–8 we have reproduced before this pathology in experimental models using rodents. 9–12
Here, we have investigated the effect of chronic exposure of rats to the type of noise that occurs in cotton mill rooms of a modern textile plant. This type of noise is distinct from that of jet engine repair rooms. Our research was aimed at changes of the respiratory epithelium of the trachea caused by noise exposure of the animals. To reach this goal, we have used high resolution “en face” views of the luminal surface of the rat trachea obtained by scanning electron microscopy (SEM). Random SEM micrographs of the samples were made to quantify the relative areas occupied by the different cell types that are characteristic of the rat trachea.
Our data document the elective susceptibility of ciliated cells to noise aggression. They also suggest that an adaptative response of the respiratory epithelium occurs. This response balances the loss of ciliated cells with an increase in the area of the tracheal surface that becomes covered by serous cells after the animals are exposed to noise. We propose that textile noise represents a potential health hazard that may damage the respiratory lining of operators working in cotton mill rooms.
Materials and Methods
Animals and Experimental Groups
We have used 45 adult male Wistar rats that were obtained from a local breeder (Gulbenkian Institute of Science, Oeiras, Portugal). All animals had unrestricted access to food (commercial chow) and water, and were treated in accordance with the European Union laws on animal protection (86/609/EC). Standard house conditions were used and they involved keeping two rats in a plastic cage (42 × 27 × 16 cm) with a steel lid. No signs of infection or inflammation were seen in the histological slides of organs of the rats.
Thirty-five of the animals were divided in 7 experimental groups that were submitted to increasing lengths of noise exposure, ranging from 1 to 7 months, according to an occupationally simulated time schedule (8 hours/day; 5 days/week with weekends in silence). The several groups of noise-exposed rats were sacrificed monthly (from 1 up to 7 months).
The remaining 10 Wistar rats were used as controls and sacrificed either at the beginning or at end of the study, ie, with the same age of rats submitted to 1 or 7 months of noise treatment.
The rats were sacrificed by a lethal intravenous injection of sodium-pentobarbital (40 mg/kg) and the cervical trachea was excised and processed for SEM. Each trachea was divided in halves along its saggital line. The samples were then fixed in a solution of 3% glutaraldehyde in 0.1M phosphate buffer, pH 7.2, washed in several changes of 5% sucrose in 0.1 M phosphate buffer, pH 7.2, dehydrated, critical point-dried and coated with gold-palladium. 13,14 Observations of the samples by SEM (JEOL JSM-35C, Japan) were performed at an accelerating voltage of 10 kV.
Quantification of Density of Epithelial Cells of Rat Trachea
Different densities of the major epithelial cells of the trachea are seen on the areas of the airway that are located over or in-between the cartilage rings, our quantitative analysis was carried out, for each sample, in separate for the two epithelial domains of the tracheal lining.
Ciliated and serous cells are the major cell types of the tracheal epithelium of the rat, together they cover more than 90% of the inner surface of the airway; this is in accordance with data from other workers. 15,16
To evaluate the relative area of the tracheal surface that was occupied by ciliated cells, serous cells, brush cells and other unidentified cells, random SEM micrographs of the samples were obtained at a magnification of ×1000, as we have done before. 12 Twenty micrographs were made of each sample; a total area of 0.22 mm2 of the epithelium surface of the trachea was used for quantitative analysis of each sample. The relative area occupied by each cell type, was determined with the help of a transparent grid of 320 points, spaced 1 cm from each other, that was superimposed on the printed micrographs. The numerical values of the relative area of ciliated and nonciliated cells of the tracheal epithelium were calculated using the following formula: total points of ciliated cells/total points of the grid inside the micrograph. The data are presented as the average proportion of area that ciliated, serous, brush, and other unidentified cells occupied on the whole tracheal epithelium.
All values are reported as mean ± SE. Computing the partial correlation coefficient assessed the relationship between the area occupied by ciliated and serous cells, after controlling for age, duration of exposure, and localization over or in-between trachea rings. Because a strong correlation (r = −0,95, P < 0.001) between the relative areas occupied by ciliated and serous cells has been found, we only analyzed differences in the relative area occupied by ciliated cells located in regions in-between or over the cartilage rings of the trachea. Differences between the experimental groups in the proportion of area occupied by ciliated cells were compared using least-squares analyses of variance. Arcsine transformation of the data MATHwas used because of non-normality. Statistical significance was accepted for P < 0.05. Statistical procedures were carried out on LSMLMW. 17
Textile industry is a major component of the economy of Northern Portugal. We have visited several cotton-mill rooms of textile plants in our region, and we have chosen one of these factories as the paradigm of environmental noise occurring in these type of plants.
Recording and reproduction of the noise present in the cotton mill room of this factory, was performed with an electro-acoustic set-up that used a PC based system, with a DT2823 data acquisition and a SB Live 5.1 cards, one B&K 4165 microphone with preamplifier, one 2-channel power amplifier, 16 monitor-type and 1 sub-woofer loudspeakers in bi-amplification. The software was designed using the LabVIEW system. Sound signals processing was done offline, applying LabVIEW and Matlab systems. Our 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 to carefully measure and preserve the sound characteristics. Total signals duration was 1 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 is documented in graph 1 (Figure 5).
Figure. Graph 1 (Fig...Image Tools
The recorded noise was then reproduced in a noise-insulated animal room, where the rats were to be exposed to it. The sound characterization and room equalization was done by means of a 35 filter bank composed by 3 low-frequency octave band band-pass filters and 32 1/3 octave pass band filters for the upper bands. All filters have 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 soundmeter, 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 Fig. 1.
We have used the SEM to examine the ultrastructure of the tracheal epithelium of Wistar rats after chronic exposure of the animals to noise recorded in a cotton-mill room. The cellular composition of the respiratory epithelium of the trachea was quantified and compared with that of control animals. Figure 2 shows a SEM micrograph of a ciliated cell surrounded by a number of serous cells containing secretory vesicles under their cell surfaces. We found no significant differences between these two differently-aged control groups of rats with regards to the relative area occupied on the inner tracheal surface by ciliated, serous, and others cells of the epithelium.
We have found that the exposure to the textile-type noise caused a significant decrease in the density of ciliated cells in both cilia-rich and cilia poor domains of the tracheal epithelium of the rats. This loss of ciliated cells was balanced by enhancement in the area occupied on the epithelium by serous cells. The phenomenon is illustrated in Figs. 3 and 4 showing the distribution of ciliated and serous cells on the tracheal lumen of control and noise-treated animals. The disorganized appearance of cilia of cells of noise-treated rats contrasted with the parallel distribution of cilia observed in samples of control trachea (Figs. 3 and 4). Quantitative comparison of these cell densities in the two domains of the trachea is depicted in graph 2 (Figure 6); here, the data compare control rats with those exposed to noise for 1 or 7 months. We also observed that the areas of the tracheal lining located in-between the cartilage rings (cilia-poor areas) showed a higher degree of loss of ciliated cells than the regions of the epithelium located over the rings.
The noise-induced alterations of the tracheal lining were observed all along the course of the exposure of the rats (from 1 to 7 months), but lacked any significant change throughout this 7-months period of treatment with regards to the degree of the cellular alterations that we have quantified. This finding indicated that after being established, (ie, after 1 month of noise exposure) the cellular change of the tracheal epithelium is kept at the same level, ie, with no quantitative evidence of either amelioration or aggravation of the proportion between ciliated and serous cells (graph 3, Figure 7).
Figure. Graph 3 (Fig...Image Tools
Examination of the rats by veterinary doctors, along the 7 months of the experiment, revealed no clinical alterations in noise-exposed rats in comparison with control animals.
The herein investigation demonstrates that the cellular composition of rat tracheal epithelium is altered if the animals are kept in an environment that reproduces the same noise found in a cotton mill room of a modern textile plant. We have submitted our rats to 40 hours/week schedule of noise that is similar to the exposure time of textile workers that operate the machinery of the cotton mill room. Ciliated cells of the rat trachea were found to be particularly vulnerable to the noise aggression. In fact, the area of the trachea that was covered by ciliated cells was significantly reduced and the tracheal lining of noise-treated rats showed an increased area of serous cells. This epithelial change was installed early, ie, it was already seen at 1 month of noise treatment of the rats, but was not aggravated by continued exposure of the animals to noise for as long as 7 months. This is the first report documenting alterations of the cellularity of the respiratory epithelium caused by exposure of animals to workplace noise present in a modern cotton mill room.
It is well established that workers of cotton mill rooms may develop different health disorders. 18–21 Among these disorders are respiratory diseases because of chronic inhalation of cotton dusts. 18 Other pathogenic cofactors, such as cigarette smoking, have been identified before as having important contributions to respiratory diseases found among cotton mill workers. 19–21 Our investigation adds workplace noise as a putative contributor for the pathogenesis of respiratory disease among textile industry workers. In fact, the relative loss of ciliated cells that we have experimentally observed in the rat trachea, is likely to be associated with a decrease in the clearance capacity of the respiratory lining. Our previous studies on the effects of a different type of aggressive noise had already suggested that the ciliated cell is the main target for cellular damage induced by noise on the respiratory epithelium. 11,12
We have recently observed that the inner lining of the rat trachea presents different densities of ciliated and serous cells in areas of the epithelium that are located either over or in-between the cartilage rings (data not published). Thus, we have separated here the two epithelial domains of the tracheal lining in our quantitative analysis of the density of cell types counted in SEM micrographs of the samples. This discrimination allowed us to conclude that the noise-induced loss of ciliated cells was more intense in the region of the tracheal epithelium located in-between cartilage rings. It should be underlined that this area of the epithelium already has a lower density of ciliated cells than the over the ring regions of the airway.
In conclusion, the present investigation offers the first experimental evidence that textile-type noise alters the composition of the respiratory epithelium of rodents. It also points to the need to perform human studies to determine whether individuals chronically exposed to this type of workplace aggression will show evidence of respiratory impairment.
The authors are very grateful to Dr Daniela Silva and Prof. Carlos M. Sá (CIMUP, UP) for expert help with scanning electron microscopy. We thank Mr António Costa e Silva, Mrs Alexandrina Ribeiro, and Mr Emanuel Monteiro for technical assistance, Mr Duarte Monteiro for technical assistance and artwork, Mr Humberto Andrade Fonseca and Mr Hugo Silva Santos for the help in the setting up of animal room, for noise exposure, Dr Pinto de Almeida who allowed us to use the textile facilities to tape the cotton-mill noise and Mr José Aurélio Mexedo for photography. This work was funded by grants from IDICT, Portugal.
1. Melamed S, Bruhis S. The effects of chronic industrial noise exposure on urinary cortisol, fatigue and irritability: a controlled field experiment. J Occup Environ Med. 1996; 38: 252–256.
2. Kamal AA, Mikael RA, Faris R. Follow-up of hearing thresholds among forge hammering workers. Am J Ind Med. 1989; 16: 645–658.
3. Alves-Pereira M. Noise-induced extra-aural pathology: a review and commentary. Aviat Space Environ Med. 1999; 70: A7–21.
4. McDonald AD, MacDonald JC, Armstrong B, Cherry NM, Nolin AD, Robert D. Prematurity and work in pregnancy. Br J Ind Med. 1988; 45: 56–62.
5. Nicholas JS, Lackland DT, Dosemeci M, et al. Mortality among US commercial pilots and navigators. J Occup Environ Med. 1998; 40: 980–985.
6. Castelo Branco NAA, Rodriguez E. The Vibroacoustic Disease – An emerging pathology. Aviat Space Environ Med. 1999; 70: A1–6.
7. Reis Ferreira JM, Couto AR, Jalles-Tavares N, Castelo Branco MSN, Castelo Branco NAA. Airway flow limitation in patients with Vibroacoustic Disease. Aviat Space Environ Med. 1999; 70: A63–69.
8. Gomes LMP, Martinho Pimenta AJF, Castelo Branco NAA. Effects of occupational exposure to low frequency noise on cognition. Aviat Space Environ Med. 1999; 70: A115–118.
9. Grande NR, Águas AP, Pereira AP, Monteiro E, Castelo Branco NAA. Morphological changes in rat lung parenchyma exposed to low frequency noise. Aviat Space Environ Med. 1999; 70: A70–77.
10. Pereira AS, Grande NR, Monteiro E, Castelo Branco MSN, Castelo Branco NAA. Morphofunctional study of rat pleural mesothelial cells exposed to low frequency noise. Aviat Space Environ Med. 1999; 70: A78–85.
11. Pereira AS, Águas AP, Grande NR, Mirones J, Monteiro E, Castelo Branco NAA. The effect of chronic exposure to low frequency noise on rat tracheal epithelia. Aviat Space Environ Med. 1999; 70: A86–A90.
12. Oliveira MJR, Pereira AP, Castelo Branco NAA, Grande NR, Águas AP. In utero and postnatal exposure of Wistar rats to low frequency/high intensity noises depletes the tracheal epithelium of ciliated cells. Lung. 2002; 179: 225–232.
13. Zagalo C, Grande NR, Santos JM, Monteiro E, Brito J, Águas AP. Tracheal transplantation: cytological changes studied by scanning and transmission electron microscopy in the rabbit. Laryngoscope. 2001; 111: 657–662.
14. Peão MND, Águas AP, Sá CM, Grande NR. Anatomy of Clara cell secretion: surface changes captured by scanning electron microscopy. J Anat. 1993; 183: 377–388.
15. Plopper CG, Mariassy AT, Wilson DW, Alley JL, Nishio SJ, Nettesheim P. Comparison of nonciliated tracheal epithelial cells in six mammalian species: ultrastructure and population densities. Exp Lung Res. 1983; 5: 281–294.
16. Jeffery PK, Reid L. New observations of the rat airway epithelium: a quantitative and electron microscopic study. J Anat. 1975; 120: 295–320.
17. Harvey WR. Mixed model least-squares and maximum likelihood program. User’s Guide for LSMLMW. Ohio University, OH, USA; 1985.
18. Cloutier MM, Guernsey L. Byssinosis: role of polymer length on the effect of tannin on the airway beta-adrenergic receptor. Lung. 1998; 176: 393–401.
19. Raza SN, Fletcher AM, Pickering CA, Niven RM, Faragher EB. Respiratory symptoms in Lancashire textile weavers. Occup Environ Med. 1999; 56: 514–519.
20. Simpson JC, Niven RM, Pickering CA, Fletcher AM, Oldham LA, Francis HM. Prevalence and predictors of work related respiratory symptoms in workers exposed to organic dust. Occup Environ Med. 1998; 55: 668–672.
21. Fishwick D, Fletcher AM, Pickering CA, Mc LNR, Faragher EB. Lung function in Lancashire cotton and man made fibre spinning mill operatives. Occup Environ Med. 1996; 53: 46–50.