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Acute effects of environmental tobacco smoke and dried dung smoke on lung histopathology in rabbits
Pathology (February 2006) 38(1), pp. 53–57
EXPERIMENTAL PATHOLOGY
Acute effects of environmental tobacco smoke and dried dung smoke on lung histopathology in rabbits FATMA FI˙DAN*, MEHMET UNLU*, MURAT SEZER*, ONDER SAHI˙N{, CI˙GDEM TOKYOL{ AND HI˙DI˙R ESME{ Afyon Kocatepe University Faculty of Medicine, Departments of *Pulmonary Medicine, {Pathology, and {Thoracic Surgery, Afyon, Turkey
Summary Aims: To compare the effects of cigarette smoke and dried dung smoke exposure on the histopathology of lungs. Methods: Three groups each with five rabbits were formed. The cigarette smoke group was exposed to cigarette smoke, the biomass group was exposed to dried dung smoke and the control group was exposed to dry air 1 hour daily for 1 month. At the end of 1 month, animals were sacrificed and lung tissues were examined histopathologically. Results: Histopathological evaluation of rabbits’ lungs revealed that intraparenchymal vascular congestion and thrombosis, intraparenchymal haemorrhage, respiratory epithelial proliferation, number of macrophages in the alveolar and bronchial lumen, alveolar destruction, emphysematous changes and bronchoalveolar haemorrhage scores were significantly increased in rabbits exposed to cigarette smoke compared with the control group. Respiratory epithelial proliferation, alveoli destruction and emphysematous change scores were significantly increased in rabbits exposed to dried dung smoke compared with the control group. Conclusion: Although less than the effects of cigarette smoke, dried dung smoke had severe histopathological effects on rabbits’ lungs. Key words: Cigarette smoke, dried dung, histopathology, lung, rabbit. Received 15 July, revised 14 September, accepted 17 September 2005
INTRODUCTION The hazardous effects of smoking tobacco have been recognised for centuries. The associations between tobacco smoke, bronchogenic carcinoma and obstructive airway diseases have been widely reviewed; the pathology and imaging characteristics of these disorders are well known. However, there is increasing awareness of the potential for cigarette smoke to cause pulmonary diseases other than lung cancer, chronic bronchitis and emphysema.1 Besides cigarette smoke, exposure to biomass combustion products may also play an important role in the aetiology of both acute and chronic respiratory diseases.2–5 Approximately 50% of the world’s population and up to 90% of rural households in developing countries still rely on unprocessed biomass fuels in the form of wood, dung and crop residues.6 These are typically burnt indoors in
open fires or poorly functioning stoves. As a result, there are high levels of air pollution to which women, especially those responsible for cooking, and their young children, are most heavily exposed.7 Biomass fuel is any material derived from plants or animals which is deliberately burnt by humans. Wood is the most common example, but the use of animal dung and crop residues is also widespread.8 Many of the substances in biomass smoke can damage human health. The most important are particles, carbon monoxide, nitrous oxides, sulphur oxides (principally from coal), formaldehyde, and polycyclic organic matter, including carcinogens such as benzo(a)pyrene.8 A health effect is determined not just by the pollution level but also, and more importantly, by the time people spend breathing polluted air, i.e., the exposure level. People in developing countries are commonly exposed to very high levels of pollution for 3–7 hours daily over many years.9 During winter in cold and mountainous areas, exposure may occur over a substantial portion of each 24-hour period.10 Many studies report that excessive exposure to biomass smoke has the same health risk as exposure to cigarette smoke.11 The risk of acute respiratory infections, chronic pulmonary disease, asthma, cancer (lung, nasopharyngeal and laryngeal cancer), pulmonary tuberculosis, low birth weight and infant mortality, and cataracts increases with biomass exposure.7 In Turkey, dried dung is used for heating, as well as cooking meals and bread. Women seem to be more often exposed to smoke because they are generally responsible for cooking. The aetiology of chronic pulmonary disease and associated cor pulmonale in women from rural areas who are non-smokers is thought to be the inhalation of noxious gases from burnt substances such as dried dung; however, it is yet unclear which physiopathological mechanisms are involved in this process.12 In our study, we aimed to compare the effects of cigarette smoke and dried dung smoke exposure on lung histopathology.
MATERIALS AND METHODS All procedures were carried out according to institutional approval. Adult male New Zealand White rabbits with weights ranging from 1.5 to 2 kg were used. There were three groups each with five rabbits. Animals were fed rabbit chow and water. The cigarette smoke (CS) group was exposed
ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia DOI: 10.1080/00313020500459615
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Pathology (2006), 38(1), February
FIDAN et al.
perivascular inflammation, intraparenchymal infiltration and fibrosis, intraparenchymal vascular congestion, thrombosis and haemorrhage, respiratory epithelial proliferation, nodular aggragates, number of macrophages in alveolar and bronchiolar lumen, pneumocyte type 2, alveoli destruction, emphysematous changes and bronchoalveolar haemorrhage. The level of change in each section was defined as follows: no changes (0), minimal (1+), mild (2+), modarate (3+), severe (4+), very severe (5+).
to cigarette smoke, the biomass (dried dung) group to dried dung smoke and the control group to dry air. After the 1-month period, all rabbits were sacrificed by administration of 100 mg/kg sodium pentothal intraperitoneally. Cigarette smoke exposure Rabbits in the CS group were placed in a (80 6 80 6 80 cm) continuous air flow chamber in a seperate room for 1 h per day, over a 1-month period with or without CS exposure, a modified method from Mays et al.13 A lit cigarette was placed in the chamber, and fresh air was delivered constantly into the chamber with a flow rate of 78 mL/min by a pump. Animals in the CS group were exposed to four cigarettes per day; although this is a low dose for humans, it is quite a high dose for rabbits. Each cigarette took about 15–20 min to burn. Carbon monoxide and oxygen levels in the chamber were measured. The carbon monoxide concentration in the chamber was 110¡41.8 ppm and the oxygen saturation was 19%. The temperature in the chamber was 27uC. This cigarette smoke exposure was well tolerated by the rabbits.
Statistical analysis Statistical analysis was carried out using the Statistical Package for Social Sciences SPSS 10.0 (SPSS Inc., USA). Appropriateness of data to normal ranges was controlled with the Shapiro–Wilk test. The Mann–Whitney U test was used to compare two groups, and the Kruskal–Wallis test was used to compare three groups. Data were expressed as mean¡standard deviation. p,0.05 was considered as statistically significant.
RESULTS The mean scores of histopathological changes in control, cigarette smoke and biomass groups are shown in Table 1. Histopathological evaluation of rabbit lungs revealed that intraparenchymal vascular congestion and thrombosis, intraparenchymal haemorrhage, respiratory epithelial proliferation, number of macrophages in the alveolar and bronchial lumen, alveolar destruction, emphysematous changes and bronchoalveolar haemorrhage scores were significantly increased in rabbits exposed to cigarette smoke compared with the control group. Respiratory epithelial proliferation, alveolar destruction and emphysematous changes scores were significantly increased in rabbits exposed to dried dung smoke compared with the control group. Histopathological findings are shown in Fig. 1.
Biomass (dried dung) smoke exposure Rabbits in the biomass group were placed in the same chamber for 1 h per day and exposed to dried dung smoke. Eighty grams of dried dung was burned in the reservoir of bellows. The smoke was initially fanned into the chamber with five strokes of the bellows, then allowed to leak freely into the chamber. This amount of dried dung kept burning for approximately 1 h. Carbon monoxide and oxygen levels in the chamber were measured. The carbon monoxide concentration in the chamber was 352¡25.3 ppm and the oxygen saturation was 21%. The temperature in the chamber was 22uC. This dried dung smoke exposure was well tolerated by the rabbits. Control group Animals in the control group were similarly placed in the chamber without CS or biomass smoke exposure and exposed to dry air only. Histological examination Lung tissue samples were fixed in 10% neutral buffered formaldehyde solution. After dehydration procedures, the samples were blocked in paraffin. Four-mm sections were cut by a microtome and stained with H&E. Mounted slides were examined under a light microscope (Nikon THP117; Nikon, Japan).
DISCUSSION Indoor air pollution is a major global public health threat requiring greatly increased efforts in research and policy making.7 Exposure to environmental tobacco smoke has been associated with the aetiology of a number of diseases, including asthma, coronary heart disease, cancers of the mouth, oesophagus, larynx, pharynx, lung, bladder and cervix, and fetal intrauterine growth restriction.16–18 These diseases, particularly the cancers, may be associated with long-term exposure. However, in our study, animals were
Semiquantitative evaluation of lung damage A semiquantitative evaluation of the lung histological damage was accomplished by scoring its degree of severity according to previously published methods.14,15 The scorer was not aware of the slides identity. We determined the presence and the degree of peribronchial inflammation, TABLE 1 Mean histopathological scores of the groups
Peribronchial inflammation Intraparenchymal infiltration Intraparenchymal fibrosis Intraparenchymal vascular congestion and thrombosis Intraparenchymal haemorrhage Respiratory epithelial proliferation Nodular aggragates Macrophages in alveolar and bronchiolar lumen Alveoli destruction Emphysematous changes Bronchoalveolar haemorrhage
Control
Cigarette
Dried dung
p*
p{
p{
1.4¡1.3 1.8¡1.1 1.6¡0.9 1.4¡1.3 1.2¡1.1 0.4¡0.9 0.0¡0.0 1.6¡0.9 0.4¡0.6 0.4¡0.6 0.0¡0.0
2.2¡0.5 1.6¡0.9 1.2¡1.1 4.2¡0.8 4.2¡0.8 2.2¡0.5 1.4¡1.3 4.2¡1.1 2.2¡0.8 2.0¡0.7 4.2¡1.3
2.4¡0.6 2.2¡0.5 0.4¡0.9 2.8¡0.5 1.2¡1.1 4.2¡1.1 0.0¡0.0 2.6¡0.9 2.2¡0.5 2.4¡0.6 0.0¡0.0
0.360 0.497 0.174 0.007 0.006 0.002 0.031 0.008 0.009 0.007 0.001
0.421 0.690 0.690 0.008 0.008 0.032 0.151 0.008 0.013 0.013 0.008
0.310 0.690 0.151 0.095 1.000 0.008 1.000 0.222 0.006 0.007 1.000
Figures in bold type are statistically significant. *Comparison among three groups (Kruskal–Wallis test). {Comparison between control and cigarette groups (Mann–Whitney U test). {Comparison between control and dried dung groups (Mann–Whitney U test).
EFFECTS OF SMOKE ON LUNG HISTOPATHOLOGY
55
Fig. 1 Histopathological findings. (A) Control (H&E, 6100). (B) Cigarette/dried dung. PI, peribronchial inflammation (H&E, 640). (C) Cigarette. VC, vascular congestion; VT, vascular thrombosis; black arrow, alveolar destruction (H&E, 6100). (D) Cigarette. BAH, bronchoalveolar haemorrhage; IPH, intraparenchymal haemorrhage (H&E, 6100). (E) Dried dung. REP, respiratory epithelial proliferation (H&E, 6200). (FI) Cigarette: (a) alveoli destruction (black arrow); (b) EC, emphysematous changes. (FII) Dried dung: (a) alveoli destruction (black arrow), (b) EC, emphysematous changes (H&E, 6100).
exposed to smoke for a short duration of time. Risk factors for chronic obstructive pulmonary disease associated with tobacco smoking include bronchial hyperreactivity, atopy and genetic susceptibility, all of which could apply to biomass smoke exposure. A predisposition to chronic obstructive pulmonary disease later in life may result from impaired lung growth in infancy, leading to reduced adult lung function. Exposure to tobacco smoke or biomass smoke during pregnancy and infancy may therefore increase the risk of such disease.7 Substantial deposition of carbon in the lung (anthracosis) occurred consistently in patients exposed to biomass. Necropsies of non-smoking women with cor pulmonale, most of whom were exposed to biomass smoke, revealed that all (18) had emphysema, 11
had bronchiectasis, five had chronic bronchitis and two had tuberculosis.19 Recently, it was also shown that there are relationships between traditional biomass combustion and the development of the chronic obstructive pulmonary diseases (COPD) in women who live in our rural areas.20,21 In some studies, pulmonary fibrosis, progressive massive fibrosis and pneumoconiosis were defined associated with biomass exposure, but in most of the cases airway disease was predominant. It is yet unknown through which mechanisms biomass smoke causes emphysema and airway disease. Oxidative stress induced by oxidised radicals released from inflammatory cells due to tobacco and biomass smoke exposure is thought to be responsible.22
56
Pathology (2006), 38(1), February
FIDAN et al.
In the present study, we aimed to simulate a house room in which family members are exposed to sidestream cigarette smoke or to dung smoke from an oven or a stove. A chamber was used, with adequate ventilation, which was quite big for rabbits. The mean level of indoor carbon monoxide (CO) for 24 hours due to biomass fuel usage was reported as 2–50 ppm in developing countries. This level was reported to increase to 10–500 ppm during cooking.23 In our study, mean carbon monoxide level in the glass chamber was measured as 352¡25.25 ppm with dried dung smoke. In a study by Yu et al.24 in which rats were exposed to environmental tobacco smoke, the CO concentration in the exposure chamber was measured as 112¡3 ppm. In our study, mean CO concentration in the chamber was 110¡41.8 ppm with tobacco smoke. We compared the histopathological changes in the lungs of rabbits exposed to cigarette smoke and dried dung smoke. We found that exposure to cigarette smoke in rabbits caused an increase in the scores of intraparenchymal vascular congestion and thrombosis, intraparenchymal haemorrhage, respiratory epithelial proliferation, number of macrophages in the alveolar and bronchial lumen, alveolar destruction, emphysematous changes and bronchoalveolar haemorrhage, histopathologically compared with the control group. Meshi et al.25 exposed juvenile female guinea pigs to cigarette smoke with five cigarettes for 40 minutes per day for 13–16 weeks. They found that smoke exposure caused enlargement in the air space and destruction in the centrilobular regions of alveolar tissue. Their findings are in agreement with ours and we consider that our findings are a result of acute cigarette smoke exposure. In our study, respiratory epithelial proliferation, alveolar destruction and emphysematous changes scores were significantly increased in rabbits exposed to dried dung smoke compared with the control group. These findings suggest that cigarette smoke influences more histopathological changes in the lungs than dried dung smoke. Sekhon et al.26 demonstrated in rats that cigarette smoke exposure rapidly causes increased levels of cell proliferation in the epithelium and walls of bronchioles, and in the walls of the associated pulmonary arteries. In another study, Li et al.27 exposed rats to 20 cigarettes per day for 5 days per week over a period of 6 weeks. They observed vasculitis and some haemorrhage in the lungs of the rats in the fourth week. At the end of 6 weeks they observed interstitial pneumonia and severe diffuse emphysema histopathologically in the lungs of the rats. The inflammatory process was characterised by alveolar septal thickening, septal infiltration by erythrocytes and chronic inflammatory cells (mostly macrophages), with a scattering presence of these cells within the alveolar space. A number of small calibre arteries also showed mild vasculitis, characterised by thickening of the artery wall (mostly the media), some periadventitial oedema with few scattered macrophages, and a modest reduction of the arterial and arteriolar lumen. Rubio et al.28 demonstrated in rats that small bronchi wall thickness was significantly increased due to cigarette smoke. Emphysema is another debilitating condition associated with chronic cigarette smoking.29
Although there are animal studies on wood smoke exposure, to our knowledge there are no animal studies on dried dung smoke exposure. After exposure to wood smoke for 3 hours per day for 3 months, guinea-pigs developed mild emphysema.30 Rats exposed intermittently to wood smoke for 75 minutes daily for 15 days had mononuclear bronchiolitis and mild emphysema; these conditions became more severe following exposure for 30 and 45 days.31 There is some uncertainty about the mechanisms of smoke causing emphysema and airway disease. Oxidative stress may be a component, as oxidising radicals are present in tobacco and biomass smoke and are released by inflammatory cells.22 In conclusion, although less than the effects of cigarette smoke, dried dung smoke had severe histopathological effects on the lungs of rabbits. Considering the relationship between biomass fuel usage and poverty, to provide a healthier indoor environment it seems important to take measures to improve socio-economical status. Address for correspondence: Dr F. Fidan, Hattat Karahisar Mah. 4. Sokak Kaya Apt D:7, Afyon, 03200, Turkey. E-mail: [email protected]
References 1. Desai SR, Ryan SM, Colby TV. Smoking-related interstitial lung diseases: histopathological and imaging perspectives. Clin Radiol 2003; 58: 259–68. 2. Behera D, Jindal SK. Respiratory symptoms in Indian women using domestic cooking fuels. Chest 1991; 100: 385–8. 3. Perez-Padilla R, Regalado J, Vedal S, et al. Exposure to biomass smoke and chronic airway disease in Mexican women. A case-control study. Am J Respir Crit Care Med 1996; 154: 701–6. 4. Albalak R, Frisancho AR, Keeler GJ. Domestic biomass fuel combustion and chronic bronchitis in two rural Bolivian villages. Thorax 1999; 54: 1004–8. 5. Bruce N, Neufeld L, Boy E, West C. Indoor biofuel air pollution and respiratory health: the role of confounding factors among women in highland Guatemala. Int J Epidemiol 1998; 27: 454–8. 6. World Resources Institute, UNEP, UNDP, World Bank. 1998–99 World Resources: A Guide to the Global Environment. Oxford: Oxford University Press, 1998. 7. Bruce N, Pe´rez-Padilla R, Albalak R. Indoor air pollution in developing countries: a major environmental and public health challenge. Bull World Health Org 2000; 78: 1078–92. 8. De Koning HW, Smith KR, Last JM. Biomass fuel combustion and health. Bull World Health Org 1985; 63: 11–26. 9. Engle PL, Hurtado E, Ruel M. Smoke exposure of women and young children in highland Guatemala: predictions and recall accuracy. Hum Organ 1997; 56: 408–17. 10. Norboo T, Yahya M, Bruce NG, Heady JA, Ball KP. Domestic pollution and respiratory illness in a Himalayan village. Int J Epidemiol 1991; 20: 749–57. 11. Chow KC. Cigarette smoking and oxidative damage in the lung. Ann N Y Acad Sci 1993; 686: 289–98. 12. Pandey MR. Prevalance of chronic bronchitis in a rural community of Hill regon of Nepal. Thorax 1978; 39: 331–6. 13. Mays BW, Freischlag JA, Eginton MT, et al. Ascorbic acid prevents cigarette smoke injury to endothelium-dependent arterial relaxation. J Surg Res 1999; 84: 35–9. 14. Molteni A, Ward WF, Ts’ao CH, et al. Prevention of monocrotalineinduced pulmonary fibrosis in the rat by administration of captopril and penicillamine. Proc Soc Exp Biol Med 1985; 180: 112–20. 15. Rotta AT, Gunnarsson B, Hernan LJ, et al. Partial liquid ventilation influences pulmonary histopathology in an animal model of acute lung injury. J Crit Care 1999; 14: 84–92. 16. Weiss S, Utell M, Sarnett J. Environmental tobacco smoke exposure and asthma in adults. Environ Health Perspect 1999; 107: 891–5. 17. Misra D, Nguyen RHN. Environmental tobacco smoke and low birthweight: a hazard in the workplace? Environ Health Perspect 1999; 107: 897–906. 18. Kawachi I, Colditz A. Workplace exposure to passive smoking and risk of cardiovascular disease: summary of epidemiologic studies. Environ Health Perspect 1999; 107: 847–51.
EFFECTS OF SMOKE ON LUNG HISTOPATHOLOGY
19. Padmavati S, Joshi B. Incidence and etiology of chronic cor pulmonale in Delhi: a necropsy study. Dis Chest 1964; 46: 457–63. 20. Demirtas N, Seyfikli Z, Topcu S. The relatinoships between traditional biomass combustion and development of COPD in women of Sivas area. (Turkish.) J Respir Dis 1999; 10: 148–55. 21. Arslan M, Akkurt I, Egilmez H, Atalar M, Salk I. Biomass exposure and the high resolution computed tomographic and spirometric findings. Eur J Radiol 2004; 52: 192–9. 22. Repine JE, Bast A, Lankhorst I. Oxidative stress in chronic obstructive pulmonary disease. Oxidative Stress Study Group. Am J Respir Crit Care Med 1997; 156: 341–57. 23. United States Environmental Protection Agency. Revisions to the National Ambient Air Quality Standards for Particles Matter. Fed Regist 1997; 62: 38651–701. 24. Yu M, Pinkerton KE, Witschi H. Short-term exposure to aged and diluted sidestream cigarette smoke enhances ozone-induced lung injury in B6C3F1 mice. Toxicol Sci 2002; 65: 99–106. 25. Meshi B, Vitalis TZ, Ionescu D, et al. Emphysematous lung destruction by cigarette smoke. The effects of latent adenoviral ınfection on
26. 27. 28. 29. 30. 31.
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the lung inflammatory response. Am J Respir Cell Mol Biol 2002; 26: 52–7. Sekhon HS, Wright JL, Churg A. Cigarette smoke causes rapid cell proliferation in small airways and associated pulmonary arteries. Am J Physiol 1994; 267: 557–63. Li T, Molteni A, Latkovich P, Castellani W, Baybutt RC. Vitamin A depletion induced by cigarette smoke is associated with the development of emphysema in rats. J Nutr 2003; 133: 2629–34. Rubio ML, Sanchez-Cifuentes MV, Ortega M, et al. N-acetylcysteine prevents cigarette smoke induced small airways alterations in rats. Eur Respir J 2000; 15: 505–11. Seagrave JC. Oxidative mechanisms in tobacco smoke-induced emphysema. J Toxicol Environ Health 2000; 61: 69–78. Juarez-Ceron B. Collagenolitic activity in a model of pulmonary emphysema induced by wood smoke. Mexico City: Metropolitan Autonomous University of Mexico, 1996. Thesis. Lal K, Dutta KK, Vachhrajani KD, et al. Histomorphological changes in lung of rats following exposure to wood smoke. Indian J Exp Biol 1993; 31: 761–4.
EXPERIMENTAL PATHOLOGY
Acute effects of environmental tobacco smoke and dried dung smoke on lung histopathology in rabbits FATMA FI˙DAN*, MEHMET UNLU*, MURAT SEZER*, ONDER SAHI˙N{, CI˙GDEM TOKYOL{ AND HI˙DI˙R ESME{ Afyon Kocatepe University Faculty of Medicine, Departments of *Pulmonary Medicine, {Pathology, and {Thoracic Surgery, Afyon, Turkey
Summary Aims: To compare the effects of cigarette smoke and dried dung smoke exposure on the histopathology of lungs. Methods: Three groups each with five rabbits were formed. The cigarette smoke group was exposed to cigarette smoke, the biomass group was exposed to dried dung smoke and the control group was exposed to dry air 1 hour daily for 1 month. At the end of 1 month, animals were sacrificed and lung tissues were examined histopathologically. Results: Histopathological evaluation of rabbits’ lungs revealed that intraparenchymal vascular congestion and thrombosis, intraparenchymal haemorrhage, respiratory epithelial proliferation, number of macrophages in the alveolar and bronchial lumen, alveolar destruction, emphysematous changes and bronchoalveolar haemorrhage scores were significantly increased in rabbits exposed to cigarette smoke compared with the control group. Respiratory epithelial proliferation, alveoli destruction and emphysematous change scores were significantly increased in rabbits exposed to dried dung smoke compared with the control group. Conclusion: Although less than the effects of cigarette smoke, dried dung smoke had severe histopathological effects on rabbits’ lungs. Key words: Cigarette smoke, dried dung, histopathology, lung, rabbit. Received 15 July, revised 14 September, accepted 17 September 2005
INTRODUCTION The hazardous effects of smoking tobacco have been recognised for centuries. The associations between tobacco smoke, bronchogenic carcinoma and obstructive airway diseases have been widely reviewed; the pathology and imaging characteristics of these disorders are well known. However, there is increasing awareness of the potential for cigarette smoke to cause pulmonary diseases other than lung cancer, chronic bronchitis and emphysema.1 Besides cigarette smoke, exposure to biomass combustion products may also play an important role in the aetiology of both acute and chronic respiratory diseases.2–5 Approximately 50% of the world’s population and up to 90% of rural households in developing countries still rely on unprocessed biomass fuels in the form of wood, dung and crop residues.6 These are typically burnt indoors in
open fires or poorly functioning stoves. As a result, there are high levels of air pollution to which women, especially those responsible for cooking, and their young children, are most heavily exposed.7 Biomass fuel is any material derived from plants or animals which is deliberately burnt by humans. Wood is the most common example, but the use of animal dung and crop residues is also widespread.8 Many of the substances in biomass smoke can damage human health. The most important are particles, carbon monoxide, nitrous oxides, sulphur oxides (principally from coal), formaldehyde, and polycyclic organic matter, including carcinogens such as benzo(a)pyrene.8 A health effect is determined not just by the pollution level but also, and more importantly, by the time people spend breathing polluted air, i.e., the exposure level. People in developing countries are commonly exposed to very high levels of pollution for 3–7 hours daily over many years.9 During winter in cold and mountainous areas, exposure may occur over a substantial portion of each 24-hour period.10 Many studies report that excessive exposure to biomass smoke has the same health risk as exposure to cigarette smoke.11 The risk of acute respiratory infections, chronic pulmonary disease, asthma, cancer (lung, nasopharyngeal and laryngeal cancer), pulmonary tuberculosis, low birth weight and infant mortality, and cataracts increases with biomass exposure.7 In Turkey, dried dung is used for heating, as well as cooking meals and bread. Women seem to be more often exposed to smoke because they are generally responsible for cooking. The aetiology of chronic pulmonary disease and associated cor pulmonale in women from rural areas who are non-smokers is thought to be the inhalation of noxious gases from burnt substances such as dried dung; however, it is yet unclear which physiopathological mechanisms are involved in this process.12 In our study, we aimed to compare the effects of cigarette smoke and dried dung smoke exposure on lung histopathology.
MATERIALS AND METHODS All procedures were carried out according to institutional approval. Adult male New Zealand White rabbits with weights ranging from 1.5 to 2 kg were used. There were three groups each with five rabbits. Animals were fed rabbit chow and water. The cigarette smoke (CS) group was exposed
ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia DOI: 10.1080/00313020500459615
54
Pathology (2006), 38(1), February
FIDAN et al.
perivascular inflammation, intraparenchymal infiltration and fibrosis, intraparenchymal vascular congestion, thrombosis and haemorrhage, respiratory epithelial proliferation, nodular aggragates, number of macrophages in alveolar and bronchiolar lumen, pneumocyte type 2, alveoli destruction, emphysematous changes and bronchoalveolar haemorrhage. The level of change in each section was defined as follows: no changes (0), minimal (1+), mild (2+), modarate (3+), severe (4+), very severe (5+).
to cigarette smoke, the biomass (dried dung) group to dried dung smoke and the control group to dry air. After the 1-month period, all rabbits were sacrificed by administration of 100 mg/kg sodium pentothal intraperitoneally. Cigarette smoke exposure Rabbits in the CS group were placed in a (80 6 80 6 80 cm) continuous air flow chamber in a seperate room for 1 h per day, over a 1-month period with or without CS exposure, a modified method from Mays et al.13 A lit cigarette was placed in the chamber, and fresh air was delivered constantly into the chamber with a flow rate of 78 mL/min by a pump. Animals in the CS group were exposed to four cigarettes per day; although this is a low dose for humans, it is quite a high dose for rabbits. Each cigarette took about 15–20 min to burn. Carbon monoxide and oxygen levels in the chamber were measured. The carbon monoxide concentration in the chamber was 110¡41.8 ppm and the oxygen saturation was 19%. The temperature in the chamber was 27uC. This cigarette smoke exposure was well tolerated by the rabbits.
Statistical analysis Statistical analysis was carried out using the Statistical Package for Social Sciences SPSS 10.0 (SPSS Inc., USA). Appropriateness of data to normal ranges was controlled with the Shapiro–Wilk test. The Mann–Whitney U test was used to compare two groups, and the Kruskal–Wallis test was used to compare three groups. Data were expressed as mean¡standard deviation. p,0.05 was considered as statistically significant.
RESULTS The mean scores of histopathological changes in control, cigarette smoke and biomass groups are shown in Table 1. Histopathological evaluation of rabbit lungs revealed that intraparenchymal vascular congestion and thrombosis, intraparenchymal haemorrhage, respiratory epithelial proliferation, number of macrophages in the alveolar and bronchial lumen, alveolar destruction, emphysematous changes and bronchoalveolar haemorrhage scores were significantly increased in rabbits exposed to cigarette smoke compared with the control group. Respiratory epithelial proliferation, alveolar destruction and emphysematous changes scores were significantly increased in rabbits exposed to dried dung smoke compared with the control group. Histopathological findings are shown in Fig. 1.
Biomass (dried dung) smoke exposure Rabbits in the biomass group were placed in the same chamber for 1 h per day and exposed to dried dung smoke. Eighty grams of dried dung was burned in the reservoir of bellows. The smoke was initially fanned into the chamber with five strokes of the bellows, then allowed to leak freely into the chamber. This amount of dried dung kept burning for approximately 1 h. Carbon monoxide and oxygen levels in the chamber were measured. The carbon monoxide concentration in the chamber was 352¡25.3 ppm and the oxygen saturation was 21%. The temperature in the chamber was 22uC. This dried dung smoke exposure was well tolerated by the rabbits. Control group Animals in the control group were similarly placed in the chamber without CS or biomass smoke exposure and exposed to dry air only. Histological examination Lung tissue samples were fixed in 10% neutral buffered formaldehyde solution. After dehydration procedures, the samples were blocked in paraffin. Four-mm sections were cut by a microtome and stained with H&E. Mounted slides were examined under a light microscope (Nikon THP117; Nikon, Japan).
DISCUSSION Indoor air pollution is a major global public health threat requiring greatly increased efforts in research and policy making.7 Exposure to environmental tobacco smoke has been associated with the aetiology of a number of diseases, including asthma, coronary heart disease, cancers of the mouth, oesophagus, larynx, pharynx, lung, bladder and cervix, and fetal intrauterine growth restriction.16–18 These diseases, particularly the cancers, may be associated with long-term exposure. However, in our study, animals were
Semiquantitative evaluation of lung damage A semiquantitative evaluation of the lung histological damage was accomplished by scoring its degree of severity according to previously published methods.14,15 The scorer was not aware of the slides identity. We determined the presence and the degree of peribronchial inflammation, TABLE 1 Mean histopathological scores of the groups
Peribronchial inflammation Intraparenchymal infiltration Intraparenchymal fibrosis Intraparenchymal vascular congestion and thrombosis Intraparenchymal haemorrhage Respiratory epithelial proliferation Nodular aggragates Macrophages in alveolar and bronchiolar lumen Alveoli destruction Emphysematous changes Bronchoalveolar haemorrhage
Control
Cigarette
Dried dung
p*
p{
p{
1.4¡1.3 1.8¡1.1 1.6¡0.9 1.4¡1.3 1.2¡1.1 0.4¡0.9 0.0¡0.0 1.6¡0.9 0.4¡0.6 0.4¡0.6 0.0¡0.0
2.2¡0.5 1.6¡0.9 1.2¡1.1 4.2¡0.8 4.2¡0.8 2.2¡0.5 1.4¡1.3 4.2¡1.1 2.2¡0.8 2.0¡0.7 4.2¡1.3
2.4¡0.6 2.2¡0.5 0.4¡0.9 2.8¡0.5 1.2¡1.1 4.2¡1.1 0.0¡0.0 2.6¡0.9 2.2¡0.5 2.4¡0.6 0.0¡0.0
0.360 0.497 0.174 0.007 0.006 0.002 0.031 0.008 0.009 0.007 0.001
0.421 0.690 0.690 0.008 0.008 0.032 0.151 0.008 0.013 0.013 0.008
0.310 0.690 0.151 0.095 1.000 0.008 1.000 0.222 0.006 0.007 1.000
Figures in bold type are statistically significant. *Comparison among three groups (Kruskal–Wallis test). {Comparison between control and cigarette groups (Mann–Whitney U test). {Comparison between control and dried dung groups (Mann–Whitney U test).
EFFECTS OF SMOKE ON LUNG HISTOPATHOLOGY
55
Fig. 1 Histopathological findings. (A) Control (H&E, 6100). (B) Cigarette/dried dung. PI, peribronchial inflammation (H&E, 640). (C) Cigarette. VC, vascular congestion; VT, vascular thrombosis; black arrow, alveolar destruction (H&E, 6100). (D) Cigarette. BAH, bronchoalveolar haemorrhage; IPH, intraparenchymal haemorrhage (H&E, 6100). (E) Dried dung. REP, respiratory epithelial proliferation (H&E, 6200). (FI) Cigarette: (a) alveoli destruction (black arrow); (b) EC, emphysematous changes. (FII) Dried dung: (a) alveoli destruction (black arrow), (b) EC, emphysematous changes (H&E, 6100).
exposed to smoke for a short duration of time. Risk factors for chronic obstructive pulmonary disease associated with tobacco smoking include bronchial hyperreactivity, atopy and genetic susceptibility, all of which could apply to biomass smoke exposure. A predisposition to chronic obstructive pulmonary disease later in life may result from impaired lung growth in infancy, leading to reduced adult lung function. Exposure to tobacco smoke or biomass smoke during pregnancy and infancy may therefore increase the risk of such disease.7 Substantial deposition of carbon in the lung (anthracosis) occurred consistently in patients exposed to biomass. Necropsies of non-smoking women with cor pulmonale, most of whom were exposed to biomass smoke, revealed that all (18) had emphysema, 11
had bronchiectasis, five had chronic bronchitis and two had tuberculosis.19 Recently, it was also shown that there are relationships between traditional biomass combustion and the development of the chronic obstructive pulmonary diseases (COPD) in women who live in our rural areas.20,21 In some studies, pulmonary fibrosis, progressive massive fibrosis and pneumoconiosis were defined associated with biomass exposure, but in most of the cases airway disease was predominant. It is yet unknown through which mechanisms biomass smoke causes emphysema and airway disease. Oxidative stress induced by oxidised radicals released from inflammatory cells due to tobacco and biomass smoke exposure is thought to be responsible.22
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Pathology (2006), 38(1), February
FIDAN et al.
In the present study, we aimed to simulate a house room in which family members are exposed to sidestream cigarette smoke or to dung smoke from an oven or a stove. A chamber was used, with adequate ventilation, which was quite big for rabbits. The mean level of indoor carbon monoxide (CO) for 24 hours due to biomass fuel usage was reported as 2–50 ppm in developing countries. This level was reported to increase to 10–500 ppm during cooking.23 In our study, mean carbon monoxide level in the glass chamber was measured as 352¡25.25 ppm with dried dung smoke. In a study by Yu et al.24 in which rats were exposed to environmental tobacco smoke, the CO concentration in the exposure chamber was measured as 112¡3 ppm. In our study, mean CO concentration in the chamber was 110¡41.8 ppm with tobacco smoke. We compared the histopathological changes in the lungs of rabbits exposed to cigarette smoke and dried dung smoke. We found that exposure to cigarette smoke in rabbits caused an increase in the scores of intraparenchymal vascular congestion and thrombosis, intraparenchymal haemorrhage, respiratory epithelial proliferation, number of macrophages in the alveolar and bronchial lumen, alveolar destruction, emphysematous changes and bronchoalveolar haemorrhage, histopathologically compared with the control group. Meshi et al.25 exposed juvenile female guinea pigs to cigarette smoke with five cigarettes for 40 minutes per day for 13–16 weeks. They found that smoke exposure caused enlargement in the air space and destruction in the centrilobular regions of alveolar tissue. Their findings are in agreement with ours and we consider that our findings are a result of acute cigarette smoke exposure. In our study, respiratory epithelial proliferation, alveolar destruction and emphysematous changes scores were significantly increased in rabbits exposed to dried dung smoke compared with the control group. These findings suggest that cigarette smoke influences more histopathological changes in the lungs than dried dung smoke. Sekhon et al.26 demonstrated in rats that cigarette smoke exposure rapidly causes increased levels of cell proliferation in the epithelium and walls of bronchioles, and in the walls of the associated pulmonary arteries. In another study, Li et al.27 exposed rats to 20 cigarettes per day for 5 days per week over a period of 6 weeks. They observed vasculitis and some haemorrhage in the lungs of the rats in the fourth week. At the end of 6 weeks they observed interstitial pneumonia and severe diffuse emphysema histopathologically in the lungs of the rats. The inflammatory process was characterised by alveolar septal thickening, septal infiltration by erythrocytes and chronic inflammatory cells (mostly macrophages), with a scattering presence of these cells within the alveolar space. A number of small calibre arteries also showed mild vasculitis, characterised by thickening of the artery wall (mostly the media), some periadventitial oedema with few scattered macrophages, and a modest reduction of the arterial and arteriolar lumen. Rubio et al.28 demonstrated in rats that small bronchi wall thickness was significantly increased due to cigarette smoke. Emphysema is another debilitating condition associated with chronic cigarette smoking.29
Although there are animal studies on wood smoke exposure, to our knowledge there are no animal studies on dried dung smoke exposure. After exposure to wood smoke for 3 hours per day for 3 months, guinea-pigs developed mild emphysema.30 Rats exposed intermittently to wood smoke for 75 minutes daily for 15 days had mononuclear bronchiolitis and mild emphysema; these conditions became more severe following exposure for 30 and 45 days.31 There is some uncertainty about the mechanisms of smoke causing emphysema and airway disease. Oxidative stress may be a component, as oxidising radicals are present in tobacco and biomass smoke and are released by inflammatory cells.22 In conclusion, although less than the effects of cigarette smoke, dried dung smoke had severe histopathological effects on the lungs of rabbits. Considering the relationship between biomass fuel usage and poverty, to provide a healthier indoor environment it seems important to take measures to improve socio-economical status. Address for correspondence: Dr F. Fidan, Hattat Karahisar Mah. 4. Sokak Kaya Apt D:7, Afyon, 03200, Turkey. E-mail: [email protected]
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