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Health effects assessment of exposure to particles from wood smoke 6 Experimental studies
6.1 Human studiesOnly one study has been found with experimental exposure of human volunteers to wood smoke PM. The aim of this study was to examine whether short-term exposure to wood smoke affects markers of inflammation, blood haemostasis, and lipid peroxidation in healthy humans. Thirteen healthy persons (6 men and 7 women aged 20-56 years – mean 34 years) were exposed to wood smoke or clean air in an exposure chamber during two 4-hour sessions, 1 week apart (PM2.5 levels of 240-280 µg/m³; number PM concentration levels of 95,000-180,000/cm³) (Barregard et al. 2006). In addition to increased PM levels, the wood smoke also resulted in increased levels of VOCs (formaldehyde, acetaldehyde, 1,3-butadiene and aromatic hydrocarbons e.g. benzene) (Sällsten et al. 2006). Subjective symptoms were weak, with mild eye irritation after the wood smoke exposure. Blood and urine samples were analysed before and after exposure for markers of inflammation, coagulation and lipid peroxidation. There was a tendency toward an increase (about 10%) of high-sensitivity serum C-reactive protein (CRP), a marker of inflammation. Significant increases were found in serum amyloid A (an acute-phase protein that parallels CRP and considered to be an inflammatory cardiovascular risk factor) and in plasma factor VIII (considered as a marker both of inflammation and of haemostasis) and in the ratio factor VIII/von Willebrand factor indicating a slight effect on the balance of the coagulation factors. Moreover there was an increased urinary excretion of free 8-iso-prostaglandin2a indicating a temporary increase in free radical-mediated lipid peroxidation. The authors concluded that wood smoke particles, at levels that can be found in smoky indoor environments, seem to affect inflammation, coagulation, and possibly lipid peroxidation, factors that may be involved in the mechanisms whereby particulate air pollution affects cardiovascular morbidity and mortality. 6.2 Studies in experimental animalsIn general, the toxicological studies of air pollutants are short-term experimental studies. These studies have often analysed the early events rather than waiting for final disease, such as reduced respiratory system development, and they often focus on cells and biochemical systems rather than whole animals. Two approaches have generally been used to studying the adverse health effects of particulate matter in animals: 1) placing suspensions of the test substances in the nose or the trachea, or 2) inhalation of aerosols. Rodents have primarily been used in the studies to test the mechanisms of pollutant-induced lung and airway injury and as models for infection processes and the functioning of the immune system. Rats in particular appear to be susceptible to chronic inflammation, fibrous tissue development, and cancer from insoluble, non-cytotoxic particles, via a process believed to involve the overwhelming of normal particle removal mechanisms (particle overload). Dosage rates and tissue concentrations are key factors in determining toxicity. Many toxicological effects are related to inhalation exposure integrated over time, especially in those situations where exposure is of longer term. However, certain effects may be more related to peak exposures, such as local irritation in the upper and lower airways. Comparison between the effects in rodents and those in human beings can be rather difficult. This is due to anatomical and physiological differences, which can result in considerably lower concentrations in sensitive regions of the respiratory tract and the lungs of animals, compared to similar regions in humans. Furthermore, experimental animals used in toxicological studies are genetically very similar within specific strains, whereas human populations are heterogeneous. Thus, extrapolation of results from experimental animals to humans must also take strain and species differences into account. The effects of air pollutants may also be studied in isolated lungs from animals, cultures of various anatomical structures of the respiratory tract from animals, cultures of various cell types lining the respiratory system, and sub-cellular fractions of tissues and cells. Such in vitro studies are useful for characterising the mode of action / mechanisms for air pollutants and for studying biochemical aspects of qualitative and quantitative species differences in toxicity. Given the complex physiological and pathological reactions taking place in the intact organism when animals are exposed to air pollutants via inhalation, in vitro studies cannot in isolation be used for hazard characterisation purposes. 6.2.1 Particles in the general environment, an overview6.2.1.1 Non-carcinogenic effectsA brief overview of the non-carcinogenic effects of particles observed in studies of experimental animals is given in the following based on the AIRNET toxicology report (Dybing & Totlandsdal 2004). Toxicological studies in experimental animals suggest that PM can influence the functioning of the lung, the blood vessels and the heart. Much of the toxicological evidence indicating the potential of PM to induce toxicity arises from direct high dose administration of PM into airways and lungs. However, the few inhalation studies available also indicate that PM may induce toxicity and studies have shown dose-dependent PM-induced adverse effects, albeit at concentrations well above ambient exposure. Some studies have concluded that focusing not on mass but on the (reactive) surface area of PM is a better method of linking adverse effects with PM. The toxicity studies of PM have often been performed on specific fractions of PM, acidic aerosols, organic fractions, mixtures of particles and gaseous compounds, transition metals and particle charge while the ambient PM is a complex mixture. Not only particles of a certain chemical composition are responsible for the adverse effects of PM but the effects observed might depend very much on the chemical composition of PM. Thus, adverse health effects are unlikely to be related to a single PM fraction, but more likely to a complex, eventually synergistic interaction of multiple PM components with the respiratory tract and subsequent target organs. For example, inflammatory responses in the lung have been noted with chemically diverse PM mixtures. The effects have also been observed with particles of a presumed low intrinsic toxicity such as carbon black or metal oxides indicating that other conditions, e.g. surface area, may also play an important role in the induction and development of adverse health effects. There are, however, preliminary indications that primary, carbonaceous PM components may be more important for adverse health effects than secondary components like sulphates and nitrates. Experimental studies have supported the potential for combined gas-particle interactions such as fine carbon or diesel, acidic, or dispersed ambient particles combined with (in)organic gases or vapours (e.g. ozone, nitrogen dioxide, nitric acid, aldehydes). As an example, prolonged exposure (4 weeks) of rats to a mixture of carbon black, ammonium bisulphate and ozone showed more inflammatory effects than with the components individually. Diesel exhaust contains various respiratory irritants in the gas phase and in the particulate matter. Both can induce inflammatory responses in the airways and alveolar regions of the lung. Airway inflammation involves damage to epithelial cells, including lipid peroxidation of cell membranes by oxidizing gaseous pollutants such as nitrogen dioxide. Indirect effects of particles, resulting from phagocytosis, can include the formation and release of various mediators, including oxidants, such as superoxide anions and hydroxyl radicals, and cytokines. These mediators may play a role in focal loss or shortening of cilia, type II cell hypertrophy, and hyperplasia. The latter changes can lead to the hyperplastic lesions seen in animals exposed to diesel exhaust. Phagocytosis and subsequent clearance of particles by alveolar macrophages can be compromised by high particle burdens, which may also increase the access of particles to the interstitium, leading to focal fibrosis. (Henderson et al. 1988). Not only particles of a certain size are responsible for the adverse effects of PM. Both coarse and fine particles are capable of inducing toxicity. Recently, ultrafine particles (< 0.1 µm in diameter) have emerged as a possible cause of PM-associated adverse health effects. Toxicological studies indicate that ultrafine particles could produce serious health effects. Ongoing studies suggest that traffic-generated ultrafine particles, on an equal mass basis, are more potent compared to fine or coarse particles. However, studies using factory-produced ultrafine carbon black did not confirm this observation indicating an important role for the chemical composition on the surface of PM. Whether the ultrafine PM fraction, tested at environmentally relevant levels, is also toxic, remains more uncertain. In most studies, normal healthy animals have been used, but during the last years, animal models that resemble human diseases have gained more attention as tools for understanding how air pollution may affect the diseased and susceptible individual. For example, experiments have been carried out using models of asthma, COPD (Chronic Obstructive Pulmonary Disease), allergy, lung inflammation, increased blood pressure in the lung arteries, and general high blood pressure. There is now some support for the assumption that the presence of disease increases the susceptibility to PM pollutants. Some studies have also compared responses in ageing rodents with younger ones. 6.2.1.2 Carcinogenic effectsA review of the literature on the carcinogenic effects of particles and PAHs in animals is included in the WHO Environmental Health Criteria 171 (EHC 1996). The major findings from this review are summarised in the following. Carbon black is the powdered form of elemental carbon manufactured by vapour-phase pyrolysis of hydrocarbons. In this respect, it is partly similar to black soot such as that produced in wood burning. However, the content of carbon in soot is lower and soot usually contains much more PAH and other materials that can be extracted with organic solvents. (IARC 1996). The importance of pulmonary particle burden on lung tumour induction has been demonstrated clearly in long-term studies by inhalation in rats. Rats have similarly increased lung tumour incidences when exposed to diesel exhaust or carbon black particles by inhalation for 24 months. Similarly, carbon black particles practically devoid of PAHs induce pulmonary tumours after intra-tracheal instillation. The very large surface area of carbon black and of diesel exhaust particles after de-sorption of adsorbed organic compounds in vivo may be involved mechanistically in a carcinogenic effect. The tumour response to different types of carbon black particles instilled intra-tracheally has been shown to correlate well with their respective surface areas (Heinrich 1994). The correlation between particle surface area and lung tumour incidence was examined by evaluating published studies of inhalation of diesel and other particles. Tumour induction in rats was best correlated with the surface area of the particles retained in the lung rather than with the particle mass, particle volume, or number of particles, regardless of the PAH content. It was suggested that particle surface area and surface properties play a decisive role and that absorbed PAHs are not responsible for the tumour response in rats exposed to diesel exhaust. In the human situation, however, it could not be excluded that organic compounds and gas-phase components are also involved, since the human particulate lung burden is much lower than those achieved in rats after long-term inhalation. Inhalation of diesel engine exhaust can result not only in pulmonary tumours but also in inflammation and fibrosis and in a delay in alveolar pulmonary clearance. The mechanism by which tumours develop due to particle overload and its associated pathological and anatomical changes may be restricted to rats and may not occur under environmental conditions in humans, since the lung burdens of humans do not reach the levels that induce lung tumours in rats. This is of importance for quantitative risk assessment. Only occupational exposure to diesel exhaust may result in lung burdens near or at overload conditions, particularly if the lung is already compromised by exposure to other dusts. Retarded particle clearance in smokers has been reported; in these people, additional exposure to diesel exhaust may induce overload and associated toxic effects (Bohning et al. 1982). Not only diesel soot (particle size <50 nm) but also carbon black nearly completely devoid of organic compounds (Printex 90, particle size 10 nm) and ultrafine titanium dioxide (TiO2) particles (particle size 20 nm) caused lung tumours in female Wistar rats exposed by inhalation for 18 hours/day on five days per week for 24 months to a concentration at about 7.5 mg/m³. The tumour rate increased with increasing particle concentrations, independently of the type of particles inhaled. The authors concluded that the carcinogenic component of diesel exhaust is in the inner part of the diesel soot particle, the carbon core, and is not the relatively small amount of carcinogenic PAHs (Heinrich 1994). These results were confirmed in another two-year study, in which male and female rats were exposed by inhalation to various concentrations of carbon black and diesel exhaust. Lung tumours were observed with both particle types (Nikula et al. 1995). Diesel soot does not appear to have a specific carcinogenic effect in rats; rather, there is a non-specific effect of particles. Exposure of rats by inhalation to 2.6 mg/m³ of an aerosol of tar-pitch condensate with no carbon core but containing 50 µg/m³ benzo[a]pyrene and other PAHs for 10 months caused lung tumours at a rate of 39%. The same amount of tar-pitch vapour condensed onto the surface of carbon black particles at 2 and 6 mg/m³ resulted in tumour rates that were roughly two times higher (89 and 72%, respectively). Since exposure to 6 mg/m³ carbon black almost devoid of extractable organic material caused a lung tumour rate of 18%, the tumour rate of 72% seen after combined exposure to tar-pitch vapour and carbon black particles indicates a syn-carcinogenic effect of PAHs and carbon black. A possible mechanism involves an effect of deposition of PAHs (Heinrich et al. 1994). As the level of benzo[a]pyrene in the coal-tar pitch was about three orders of magnitude greater than those in diesel soot, PAHs may play a negligible role in the carcinogenicity of diesel soot in rats. The PAH profile in diesel soot is, however, quite different from that in coal-tar pitch, as diesel soot contains highly mutagenic, carcinogenic nitro-PAHs and other poorly characterized mutagens that are not present in coal-tar pitch or on some of the carbon black particles used in experimental studies (Nikula et al. 1995, Heinrich et al. 1994). Knaapen et al. (2004) reviewed the literature on particle induced lung cancer in order to elucidate the underlying mechanisms with focus on the role of reactive oxygen and nitrogen species. Both the particles themselves as well as particle-elicited events, such as activation of pathways of inflammation and proliferation, have been suggested to play a role in particle-induced genotoxicity, mutagenesis and carcinogenicity. The central hypothesis based on rat studies is that inflammation drives genotoxic events in airway epithelium as well as cell proliferation and tissue remodelling, which are processes that are all required for mutations and progression towards neoplastic lesions. Both particles and inflammatory cells can cause genetic damage as well as proliferative effects to target cells (Type II epithelial cells and Clara cells) through the production and release of oxidants. 6.2.2 Particles from wood smoke6.2.2.1 Non-carcinogenic effectsZelikoff et al. (2002) recently reviewed the health effects associated with exposure to wood smoke, in particular with focus on the immune system as a target. The information in this review is summarised in the following. Although health effects associated with exposure to whole wood smoke emissions are not as well studied as its individual components, a number of adverse health effects have been demonstrated. For example, exposure of laboratory animals to wood smoke effluents decreased ventilatory frequency and ventilatory response to CO2 (Wong et al. 1984), increased micro-vascular permeability and produced pulmonary oedema (Nieman et al. 1989), caused necrotising tracheobronchial epithelial cell injury (Thorning et al. 1982), possibly increased the lung cancer incidence in mice (Liang et al. 1988), increased levels of angiotensin-1-converting enzyme in the lungs (Brizio-Molteni et al. 1984), and compromised pulmonary macrophage-mediated immune mechanisms important in anti-microbial defence (Zelikoff et al. 1995a,b), most likely via alterations in the integrity of the macrophage surface membrane or cytoskeletal components (Fick et al. 1984, Loke et al. 1984). Only a limited number of studies have investigated the effects of whole wood smoke emissions on pulmonary immunity. It appears that host defence and/or immune cell function is depressed in a manner similar to that produced by many of the individual wood smoke constituents (Zelikoff et al. 2002). For example, a single inhalation exposure of rabbits to smoke from the pyrolysis of Douglas fir wood produced an increase in the total number of recovered pulmonary macrophages and a transitory decrease in macrophage adherence to glass (Fick et al. 1984). Moreover, this same exposure regime decreased macrophage uptake of the gram-negative bacterial pathogen Pseudomonas aeruginosa in the absence of an inflammatory response or changes in macrophage viability. In another study, a single inhalation exposure of Douglas fir-generated wood smoke altered the macrophage morphology and membrane ultra-structure (Loke et al. 1984). Inhaled wood smoke has also been reported to alter the chemotactic migration of broncho-pulmonary lavage human macrophages (Demarest et al. 1979). In studies by Zelikoff et al. (1995a,b), 3-month-old Sprague-Dawley rats were exposed repeatedly (1 hour/day, 4 days) to a single concentration of wood smoke (750 µg/m³ PM2.5) generated from red oak burned in a combustion furnace. At 3, 24, 72 and 120 hours following the final wood smoke exposure, rats were intra-tracheally instilled with the pneumonia-producing bacteria Staphylococccus aureus to assess effects upon pulmonary clearance. Inhalation of wood smoke progressively reduced the in vivo clearance/killing of S. aureus. Effects of inhaled wood smoke on intrapulmonary clearance appeared as early as 3 hours following the final exposure and persisted for up to 5 days; killing/clearance was reduced to 60% of control values after 3 hours and then progressively declined to 2% after 5 days. The authors stated that the results demonstrated that short-term repeated inhalation of wood smoke generated from the burning of a common hardwood used for home heating compromised pulmonary host resistance against an infectious, pneumonia-producing lung pathogen well after exposures ceased. In addition to the studies reviewed by Zelikoff et al. (2002), a few recent studies have investigated the effects of wood smoke. Reed et al. (2006) have summarised health effects in rats and mice of subchronic exposure to environmental levels of hardwood smoke (HWS) generated from an uncertified wood stove burning wood of mixed oak species. Animals were exposed by whole-body inhalation (6 hours/day, 7 days/week) for either 1 week or 6 months to clean air (control) or dilutions of whole emissions based on particulate (30 (low-level exposure, L), 100 (mid-level exposure low, ML), 300 (mid-level exposure high, MH), or 1000 (high-level exposure, H) µg/m³ total PM). The fractional abundance of the primary PM constituents showed that PM was composed of approximately 93% organic carbon mass at all exposure levels. In addition, there was approximately 5% elemental carbon and 2-3% metals and other elements. PM in the HWS exposure atmospheres had a mass median aerodynamic diameter of approximately 0.3 µm, with a small increase in particle size (0.25-0.35 µm median diameter) at the higher exposure levels. The dominant gases were carbon monoxide (maximum approximately 15 mg/m³, high-level exposure) and volatile organics (maximum approximately 3.5 mg/m³, high-level exposure). F344 rats (12 of each sex per group) were used for assessment of body weight, organ weight, histopathology, clinical chemistry and haematology parameters. Spontaneously hypertensive rats (SHR, 6 of each sex per group) were used for EKG, heart rate analyses and vessel histopathology. C57BL/6 mice (16 males per group) were used for assessment of bacterial clearance and inflammatory lung histopathology. Young strain A/J mice (10/20 of each sex per group) were used for assessment of body weight, organ weight, micronucleus formation, histopathology and carcinogenesis (20 of each sex per group). No exposure-related clinical abnormalities, mortality or effects on body weight were observed. Organ weights: Absolute liver weight was slightly decreased in H rats of both sexes at 1 week (maximum 8-9%) and relative liver weights in H female rats at 6 months (maximum 3%). Excised, fixed lung volume was increased (maximum 12%) in H female rats at 6 months. Lung weight was decreased (maximum 16%) in H female mice at 6 months. Spleen weight was increased in H female rats (maximum 9%) and ML/MH mice (maximum 53%) at 1 week. Thymus weight was decreased (maximum 17%) in H male rats at 1 week. Clinical chemistry: Blood urea nitrogen (BUN) was decreased in ML/MH/H female rats and in MH/H male rats at 1 week (maximum 35%) and in H female rats at 6 months (maximum 18%). Serum creatinine was decreased in MH/H male rats at 1 week (maximum 13%) and a negative trend was evident in female rats at 1 week. Alkaline phosphatase was decreased in ML/MH/H female rats and in H male rats at 1 week (maximum 23%) and in MH/H rats of both sexes at 6 months (maximum 38%). Aspartate aminotransferase was decreased in H male rats at 1 week (maximum 24%) and a negative trend was evident at 6 months. Other parameters (serum bilirubin, serum cholesterol, glucose, phosphorous and total protein) did not reflect coherent patterns. Haematology and clotting factors: Platelets were increased in ML/MH rats of both sexes and in H female rats at 1 week (maximum 21%). Total white blood cell counts were increased in H females at 1 week (maximum 27%) and a positive trend was observed in males at this time point. Eosinophils were decreased in ML/MH male rats at 1 week (maximum 39%). Lymphocyte counts were increased in L/ML/MH male rats at 6 months (maximum 13%) and a positive trend was evident in male rats at 1 week. Histopathology: The only exposure-related histopathological findings were in the lung. Minimal increases in alveolar macrophages and sparse brown-appearing macrophages were observed in rats. Very little accumulation of particulate matter was observed in the lungs of both rats and mice. Summary inflammatory scores of C57BL/6 mice instilled with bacteria were largely unaffected by exposure. Micronucleus: There were no statistically significant exposure-related effects on micro-nucleated reticulocyte counts in mice as well as no significant trends. Carcinogenesis: See section 6.2.2.2. Bacterial clearance: Bacterial clearance was unaffected by exposure at both the 1-week and 6-month intervals except for a slight downward trend in clearance at the 6-month high-level exposure. Cardio-vascular endpoints: No significant changes in any of the measured parameters were observed under any exposure condition. According to the authors, the results reported suggest that these realistic concentrations of HWS present little to small hazard with respect to clinical signs, lung inflammation and cytotoxicity, blood chemistry, haematology, cardiac effects, and bacterial clearance. However, the authors also noted that exposure to HWS generated in this study was reported to have mild effects in mouse and rat models of asthma (Barrett et al. 2006, Tesfaigzi et al. 2005) and mild effects on pulmonary lavage parameters (Seagrave et al. 2005), see below. Barrett et al. (2006) used two different models to characterise the effects of inhaled hard wood smoke (HWS) on allergic airway inflammation. In both models, male BALB/c mice were sensitised by injection with ovalbumin (OVA) and alum. In one model, mice were challenged by inhalation with OVA 1 day prior to exposure to HWS (30, 100, 300, or 1000 µg PM/m³) for 6 hours/day on 3 consecutive days. In the other model, mice were exposed by inhalation to OVA, rested for 11 days, were exposed to HWS for 3 consecutive days, and then were exposed to OVA immediately after the final HWS exposure. Broncho-alveolar lavage (BAL), and blood collection were performed about 18 hours after the last HWS or OVA exposure. In the first model, HWS exposure after the final allergen challenge led to a significant increase in BAL eosinophils only at the 300 µg PM/m³ level. In the second model, in contrast to the first model, changes in BAL cells did not reach statistical significance. There were no HWS-induced changes in BAL interleukin (IL)-2, IL-4, IL-13, and interferon (IFN)g levels in either model following OVA challenge. According to the authors, these results suggest that acute HWS exposure can minimally exacerbate some indices of allergic airway inflammation when a final OVA challenge precedes HWS exposure, but does not alter Th1/Th2 cytokine levels. Tesfaigzi et al. (2005) exposed Brown Norway rats immunized with ovalbumin to either filtered air or wood smoke at 1 mg PM/m³ for 70 days and challenged them with allergen during the last 4 days of exposure. Baseline values for dynamic lung compliance were lower while functional residual capacity was increased in rats exposed to wood smoke compared to rats exposed to filtered air. Interferon (IFN)g levels were reduced and interleukin (IL)-4 levels increased in the broncho-alveolar lavage fluid and blood plasma, inflammatory lesions in the lungs were 21% greater, and airway mucous cells/mm basal lamina were non-significantly increased in rats exposed to wood smoke compared to controls. According to the authors, these studies suggest that the pulmonary function was affected in rats by exposure to wood smoke and this decline was associated with only minor increases in inflammation of the lung. Seagrave et al. (2005) exposed groups of rats (12 rats of each sex per group) 6 hours/day, 7 days/week for 6 months to either clean air, diesel exhaust or hardwood smoke at 4 concentration levels between 30 and 1000 µg/m³ total PM. Lung lavage fluid was assayed for toxicity indicators, cytokines and glutathione. Lactate dehydrogenase, total protein, alkaline phosphatase, ß-glucuronidase, macrophage inflammatory protein-2, tumour necrosis factor-a, and total glutathione were affected by the exposure to diesel exhaust and/or hardwood smoke; however, no consistent response pattern was found in comparison between diesel exposure and hard wood exposure. Furthermore, some of the responses were found to be gender dependent with a tendency to greater effects in males compared to females. Several indicators were found not to follow a conventional linear dose-response relationship and responses at lower exposure levels were often more pronounced than at the highest exposure level where the responses were suppressed indicating the relevance of testing at relevant environmental exposure levels. The effects of hardwood smoke (HWS) inhalation (30, 100, 300, and 1000 µg/m³) on the systemic immune responses of A/J mice were evaluated after 6 months of daily (6 hours/day) whole-body exposures (Burchiel et al. 2005). HWS was generated from a conventional, uncertified wood stove. The dominant gases were vapour-phase hydrocarbons and carbon monoxide. PM in the wood smoke exposure atmospheres had a mass median aerodynamic diameter of approximately 0.3 µm, with a small increase in particle size (0.4 µm median diameter) at the higher exposure levels. The PM was composed primarily of organic carbon with approximately 3-10% Black Carbon and less than 1% of the transition metal elements (K, Ca, and Fe). HWS PAHs were enriched in the lower molecular weight compounds (naphthalene and methylated naphthalenes, fluorene, phenanthrene, and anthracene). Spleen cells from the mice were assessed for changes in cell number, cell surface marker expression (B, T, macrophage, and natural killer cells), and responses to B cell (LPS, endotoxin) and T cell (Con A) mitogens. HWS inhalation caused an increase in T cell proliferation in the 100 µg/m³ exposure group and produced a concentration-dependent suppression of T cell proliferation at concentrations >300 µg/m³. There were no effects on B cell proliferation, or in spleen cell surface marker expression. The results show that environmentally relevant concentrations of HWS may be immunosuppressive to the immune system of mice exposed during a 6-month period. In a study of sub-chronic exposure to low levels of wood smoke, minor but significant changes in the airways of rats were observed (Tesfaigzi et al. 2002). Brown Norway rats were whole-body exposed 3 hours/day, 5 days/week for 4 or 12 weeks to 1 or 10 mg/m³ wood smoke particles from pinus edulis. Control rats were exposed to air. The wood smoke was generated in a wood stove type generally used in the homes of Native American population in New Mexico, and consisted of fine particles (< 1 µm) that formed larger chains and aggregates having a size distribution of 63-74% in the < 1 µm fraction and 26-37% in the > 1 µm fraction. The particle-bound material was primarily composed of carbon and the majority of identified organic compounds consisted of sugar and lignin derivatives. Pulmonary function, specifically carbon monoxide-diffusing capacity and pulmonary resistance, was somewhat affected in the high-exposure group. Mild chronic inflammation and squamous metaplasia were observed in the larynx of the exposed groups. The severity of alveolar macrophage hyperplasia and pigmentation increased with smoke concentration and length of exposure, and the alveolar septae were slightly thickened. Exposure of rats in a nose-only chamber to smoke from burning Douglas fir (5, 10, 15, or 20 minutes of continuous inhalation) and 24-hour recovery time revealed acute inflammation of the airways with varying degrees of injury from loss of cilia, degeneration of epithelium and squamous metaplasia to sub-mucosal oedema (Bhattacharyya et al. 2004). These histological changes were reflected in variable expression of the secretory Muc5AC mucin gene and low expression of the membrane-associated Muc4 mucin gene. Park et al. (2004) evaluated the antioxidant status and the extent of pulmonary injury in sheep after graded exposure to wood smoke. Adult male sheep were received 0, 5, 10 or 16 units of cooled western pine bark smoke, corresponding to 0, 175, 350, and 560 seconds, respectively, of smoke dwell time in the airways and lung. Smoke was mixed at a 1:1 ratio with 100% oxygen to minimise hypoxia. Plasma and expired breath samples were collected before and at different time points (6, 12, 18, 24, 36, and 48 hours) after exposure. Lung and airway sections were evaluated histologically for injury and biochemically for indices of oxidative stress. Plasma thiobarbituric acid reactive substances (TBARS) were 66 and 69% higher than controls after moderate (10 units) and high (16 units) smoke exposure at 48 hours, whereas total antioxidant potential was not statistically different among groups at any time after exposure. Lung TBARS showed a dose-dependent response to smoke inhalation and were approximately 2-, 3- and 4-fold higher, respectively, than controls after exposure to 5, 10 and 16 units of smoke. Lung myeloperoxidase (MPO) activity was also higher in smoke-exposed animals, and MPO activity was markedly elevated (19- and 22-fold than controls in right apical and medial lobes) in response to severe (16 units) smoke exposure. Smoke exposure also induced a dose-dependent injury to tracheo-bronchial epithelium and lung parenchyma. The study showed that few indices of oxidative stress responded in a dose-dependent manner although most of the indices measured in the lung were affected by the highest dose of smoke (16 units), and it could not determined whether the oxidants are a cause or a consequence of the airway and lung injury associated with exposure to wood smoke. 6.2.2.2 Carcinogenic effectsIn the very recent study by Reed et al. (2006) described in detail in section 6.2.2.1, lung carcinogenesis measured as either the percentage of young strain A/J mice (20 of each sex per group) with tumours (incidence) or the number of tumours per tumour-bearing mouse (multiplicity) yielded no significant differences from the control group and there was no evidence of a progressive exposure-related trend. Animals were exposed to hardwood smoke (HWS) generated from an uncertified wood stove, burning wood of mixed oak species, by whole-body inhalation (6 hours/day, 7 days/week) for 6 months to clean air (control) or dilutions of whole emissions based on particulate (30, 100, 300, or 1000 µg/m³ total PM). Mumford et al. (1990b) studied mouse skin carcinogenicity of indoor coal and wood combustion emissions from homes in Xuan Wei in China where the lung cancer mortality rate is high. Indoor air particles (less than 10 microns) were collected from a central commune where the lung cancer mortality rate is high and smoky coal is the major fuel used, and also from a south-western commune where lung cancer mortality rate is low and where wood or smokeless coal are the major fuels used. The organic extracts of the indoor particles from smoky coal, smokeless coal and wood combustion were analysed for PAH and assayed for skin tumour initiation activity and complete carcinogenicity in SENCAR mice. The organic extract of the emission particles from smoky coal combustion was the most active in tumour initiation followed by smokeless coal and then wood. The organic extract of the particles from smoky coal combustion was shown to be a potent complete carcinogen, whereas the wood extract was relatively inactive as a complete carcinogen. Smokeless coal extract was not tested for complete carcinogenicity because of inadequate supply. Eighty-eight percent of the mice treated with the smoky coal extract showed carcinomas, averaging 1.1 carcinomas per tumour-bearing mouse at the end of the 77-week study. 6.3 In vitro studiesThe generation of free radicals by wood smoke and cellular injuries caused by these radicals have been investigated in cultured RAW 264.7 mouse macrophage cells (Leonard et al. 2000). The cells were exposed to liquid wood smoke generated by thermolysis of western bark (pine and fir) followed by bubbling the smoke through 10 ml saline. The wood smoke produced significant DNA damage and was also able to cause lipid peroxidation, activate the nuclear transcription factor (NFkB), and enhance the release of TNF-a from the cells. The results indicate that the free radicals generated by wood smoke are able to cause DNA and cellular damage and may act as a fibrogenic agent. The sister chromatid exchange (SCE) induction of emissions from an airtight horizontal baffled residential wood stove was investigated in Chinese Hamster Ovary (CHO) cells (Hytonen et al. 1983). The samples were taken under normal and starved air conditions, from burning birch and spruce separately. Both particle phase and vapour phase were collected. All samples induced a dose-related response in SCE both with and without a metabolic activation system (rat liver microsomal fraction). The burning conditions in the stove influenced the mutagenicity of the emissions more than the type of wood; the smoke from wood burning under starved air conditions was more than one order of magnitude more potent in inducing a significant SCE response. With all samples, the response in SCE induction was highest without metabolic activation. Smoke condensates of woods used for food preservation and aromatisation in Nigeria were tested for mutagenic activity using Salmonella typhimurium TA98 and TA100 (Asita et al. 1991). The woods were: white mangrove, red mangrove, mahogany, abura, alstonia and black afara. Cigarette tar was tested for comparison. The condensates induced dose-dependent increases in the number of His+ revertants mainly with S9 mix. With the exception of mahogany and cigarette smoke condensate, the smoke condensates induced more revertants/µg condensate in TA100 than in TA98. The number of revertants/µg condensate ranged between 0.04 and 0.9 for the wood smoke condensates and was 0.12 for the cigarette smoke in TA100. The range was between 0.1 and 0.3 for the wood smoke condensates and 0.18 revertants/µg condensate for cigarette smoke in TA98. The condensates contained varying concentrations of PAH and those with higher concentrations generally showed greater mutagenic activities. However, the order of mutagenic potency in the bacterial strains differed from the order of PAH concentrations, which were lower than the concentrations at which they have been reported to induce mutations. When 6 of the PAH were mixed in the concentrations in which they were found in the individual condensates, the mixtures did not induce mutation so that the contribution of the PAH to the mutagenic activities of the condensates could not be determined. Kubátova et al. (2004) used hot pressurised water for the fractionation at different temperatures of both the polar (low temperature) and non-polar fractions (high temperature) of diesel exhaust and wood smoke PM. Non-polar fractions from both PM sources showed strong cytotoxic responses (reduced viability) in mammalian cells with the strongest response from diesel PM. Also the polar fractions of both PM sources showed strong cytotoxic responses. The mid-polar fraction from wood-smoke PM showed a stronger response than the identical fraction from diesel PM where only limited cytotoxicity was observed. In a conference abstract, Klippel (2006) reported that PM from a wood stove operating under bad conditions contained substantially higher concentrations of PAH than diesel soot and also revealed increased toxicity in an in vitro test with lung cells from the Chinese hamster. Particles from an automatic wood furnace were less toxic than diesel soot. A recent study investigated and compared the genotoxicity and the ability to induce inflammatory mediators of nine different particle types from wood and pellets combustion, from tire-road wear and collected from an urban street and a subway station (Karlsson et al. 2006). All particles tested caused DNA damage in human lung cells (A549, Comet assay). The three types of particles from wood combustion (old-type boiler, modern boiler, pellets) showed similar genotoxic potency and there was no significant difference between them. The subway particles were most genotoxic of the particles tested and caused typically 4-5 times more DNA damage than the other particles, likely, according to the authors, due to redox-active iron. Of the wood particles, the only sample that caused a significant increase in cytokine release was the particles from the modern wood boiler; the most potent particles to induce cytokines were those collected from an urban street. 6.4 Summary, experimental studiesOnly one study (Barregard et al. 2006) has been found with experimental exposure (short-term) of human volunteers (13 individuals) to wood smoke PM. The study indicates that wood smoke particles, at levels that can be found in smoky indoor environments (PM2.5 levels of 240-280 µg/m³), seem to affect inflammation, coagulation, and possibly lipid peroxidation, factors that may be involved in the mechanisms whereby particulate air pollution affects cardiovascular morbidity and mortality. Toxicological studies in laboratory animals suggest that PM can influence the functioning of the lung, the blood vessels and the heart. Much of the toxicological evidence indicating the potential of PM to induce toxicity arises from direct high dose administration of PM into airways and lungs. The few inhalation studies available from experimental animals indicate that different types of PM may induce toxicity at relatively high levels. Not only particles of a certain size or chemical composition are responsible for the adverse effects of PM. Adverse health effects are unlikely to be related to a single PM fraction, but more likely to a complex, eventually synergistic interaction of multiple PM components with the respiratory tract and subsequent target organs. However, there are preliminary indications that primary, carbonaceous PM components may be more important for adverse health effects than secondary components like sulphates and nitrates. Both coarse and fine particles are capable of inducing toxicity; however, whether the ultrafine PM fraction, tested at environmentally relevant levels, is also toxic, remains more uncertain. Various animal studies have been used to elucidate the carcinogenicity of diesel exhaust. In all valid inhalation studies in rats, diesel exhaust was found to be carcinogenic at particle concentrations of > 2 mg/m³, corresponding to an equivalent continuous exposure of about 1 mg/m³. No carcinogenic effects were seen in hamsters or mice. Diesel exhaust, without the particulate fraction, administered by inhalation to rats did not show a carcinogenic potential. Long-term inhalation of carbon black, virtually devoid of PAH, resulted in lung tumours in rats. In studies using intra-tracheal instillation, both diesel exhaust particles and carbon black induced tumours; the surface area of the carbonaceous particles appeared to be correlated with the carcinogenic potency. It is not clear whether the carcinogenicity of diesel exhaust involves DNA-reactive or non-DNA-reactive mechanisms (or a combination). A recent review (Zelikoff et al. 2002) has addressed the adverse health effects associated with exposure to wood smoke in experimental animals. Although the effects associated with exposure to wood smoke are not as well studied as the effects of its individual components, a number of adverse health effects have been reported such as e.g. inflammation and damage to epithelial cells; inflammation has been seen both after single and repeated inhalation exposure of rats. In one study (Tesfaigzi et al. 2002), minor but significant changes in the airways of rats (mild chronic inflammation and squamous metaplasia in the larynx; alveolar macrophage hyperplasia and pigmentation, and slightly thickened alveolar septae) were observed following whole-body exposure (3 hours/day, 5 days/week for 4 or 12 weeks) to 1 or 10 mg/m³ wood smoke particles. Some studies investigating the effects of wood smoke emissions on pulmonary immunity are available. Studies in rats (exposed one hour/day for 4 days to wood smoke at 750 µg/m³ PM2.5) have indicated that host defence and/or immune cell function, leading to impairment of lung clearance, is depressed in a manner similar to that produced by many of the individual wood smoke constituents (Zelikoff et al. 1995a,b). Similarly, another study (Burchiel et al. 2005) has indicated that inhalation of wood smoke (6 hours/day for 6 months) may be immunosuppressive to the immune system of mice at environmentally relevant exposure levels. A very recent study (Reed et al. 2006) has summarised health effects of subchronic exposure to environmental levels (30-1000 µg/m³ total PM) of hardwood smoke in rats and mice exposed by inhalation for 6 months. The results reported suggest that these concentrations of hardwood smoke present little to small hazard with respect to clinical signs, lung inflammation and cytotoxicity, blood chemistry, haematology, cardiac effects, and bacterial clearance. However, exposure to hardwood smoke was reported to have mild effects in mouse and rat models of asthma (Barrett et al. 2006, Tesfaigzi et al. 2005) and mild effects on broncho-alveolar lavage parameters (Seagrave et al. 2005). In the very recent study (Reed et al. 2006), lung carcinogenesis measured as either the percentage of young mice with tumours (incidence) or the number of tumours per tumour-bearing mouse (multiplicity) yielded no significant differences from the control group and there was no evidence of a progressive exposure-related trend. A few in vitro studies with mammalian cells have shown that wood smoke may be associated with cytotoxicity, DNA and cellular damage, lipid peroxidation and cytokine release. Furthermore, wood smoke condensates have been shown to induce a dose-dependent increased mutagenicity in Salmonella typhimurium. The most recent study (Karlsson et al. 2006) demonstrated that particles from wood combustion (old-type boiler, modern boiler, pellets) caused DNA damage in human lung cells (A549, Comet assay); the three types of particles showed similar genotoxic potency. Overall, some of the available studies in experimental animals indicate that inhaled PM from wood smoke, similarly to PM in general, can cause adverse health effects in the respiratory tract and lungs such as inflammation, altered lung clearance, damage to epithelial cells, immuno-suppression, and hyperplastic lesions. However, a very recent study (Reed et al. 2006) summarising the health effects of subchronic exposure to environmental levels (30-1000 µg/m³ total PM) of hardwood smoke in rats and mice indicates that exposure to these concentrations present little to small hazard with respect to clinical signs, lung inflammation and cytotoxicity, blood chemistry, haematology, cardiac effects, and bacterial clearance, and carcinogenic potential. However, parallel studies demonstrated mild exposure effects on broncho-alveolar lavage parameters and in mouse and rats models of asthma.
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