Long-term responses of canine lungs to acidic particles.

Sixteen beagle dogs were housed in four large chambers under minimum restraint. They were exposed for 16 months to clean air and individual baseline data of markers were obtained. For 13 months, eight dogs were further exposed to clean air and eight dogs for 6 h/d to 1-microm MMAD (mass median aerodynamic diameter) acidic sulfate particles carrying 25 micromol H(+) m(-3) into their lungs. To establish functional responses (lung function, cell and tissue integrity, redox balance, and non-specific respiratory defense capacity), each exposed animal served as its own control. To establish structural responses, the eight non-exposed animals served as controls. Acidic particles were produced by nebulization of aqueous sodium hydrogen sulfate at pH 1.5. Only subtle exposure-related changes of lung function and structure were detected. A significant increase in respiratory burst function of alveolar macrophages points to a marginal inflammatory response. This can be explained by the significant production of prostaglandin E(2), activating cyclooxygenase-dependent mechanisms in epithelia and thus inhibiting lung inflammation. The non-specific defense capacity was slightly affected, giving increased tracheal mucus velocity and reduced in vivo dissolution of moderately soluble test particles. Hypertrophy and hyperplasia of bronchial epithelia were not observed, but there was an increase in volume density of bronchial glands and a shift from neutral to acidic staining of epithelial secretory cells in distal airways. The acidic exposure had thus no pathophysiological consequences. It is therefore unlikely that long-term inhalation of acidic particles is associated with a health risk.

Fate and toxic effects of inhaled ultrafine cadmium oxide particles in the rat lung.

Female Fischer 344 rats were exposed to ultrafine cadmium oxide particles, generated by spark discharging, for 6 h at a concentration of 70 microg Cd/m(3) (1 x 10(6)/cm(3)) (40 nm modal diameter). Lung morphology and quantification of Cd content/concentration by inductively coupled plasma (ICP)-mass spectrometry were performed on days 0, 1, 4, and 7 after exposure. Cd content in the lung on day 0 was 0.53 +/- 0.12 microg/lung, corresponding to 19% of the estimated total inhaled cumulative dose, and the amount remained constant throughout the study. In the liver no significant increase of Cd content was found up to 4 days. A slight but statistically significant increase was observed in the liver on day 7. We found neither exposure-related morphological changes of lungs nor inflammatory responses in lavaged cells. Another group of rats were exposed to a higher concentration of ultrafine CdO particles (550 microg Cd/m(3) for 6 h, 51 nm modal diameter). The rats were sacrificed immediately and 1 day after exposure. The lavage study performed on day 0 showed an increase in the percentage of neutrophils. Multifocal alveolar inflammation was seen histologically on day 0 and day 1. Although the Cd content in the lung was comparable between day 0 and day 1 (3.9 microg/lung), significant elevation of Cd levels in the liver and kidneys was observed on both days. Two of 4 rats examined on day 0 showed elevation of blood cadmium, indicating systemic translocation of a fraction of deposited Cd from the lung in this group. These results and comparison with reported data using fine CdO particles indicate that inhalation of ultrafine CdO particles results in efficient deposition in the rat lung. With regard to the deposition dose, adverse health effects of ultrafine CdO and fine CdO appear to be comparable. Apparent systemic translocation of Cd took place only in animals exposed to a high concentration that induced lung injury.

Comparison of predicted with observed biokinetics of inhaled plutonium nitrate and gadolinium oxide in humans.

The absorption kinetics to blood of plutonium and gadolinium after inhalation as nitrate and oxide in humans and animals has been studied. For each material, values describing the time dependence of absorption were derived from the studies in animals and used with the ICRP human respiratory tract model to predict lung retention and cumulative amounts to blood for the volunteers inhaling the same materials. Comparison with the observed behaviour in the volunteers suggests that absorption of plutonium and gadolinium is reasonably species independent, and that data obtained from animal studies can be used to assess their biokinetic behaviour in humans.

Effects of inhaled CdO particles on the sphingolipid synthesis of rat lungs.

Surfactant lipids of the alveolar space protect the lung from various environmental stimuli. We investigated the influence of ultrafine (UF) CdO particles inhalation on two key enzymes involved in lung sphingolipid metabolism, serine palmitoyltransferase (SPT), and sphingomyelinase (SMase). Rats inhaled either 0.63 mg UF-CdO/m(3) for 6 h (group 1), or 1.08 mg UF-CdO/m(3) 12 h/day for 10 days (group 2). Two corresponding control groups inhaled filtered clean air. Additional rats intratracheally instilled with lipopolysaccharide (LPS) were used as positive controls. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) of lung tissue showed a significant increase in the level of SPT mRNA (LCB2 subunit) expression in group 2 compared to the corresponding controls (p <.01). Group 1 and LPS were not statistically different from control. No alteration in the mRNA level of SMase was detected in any exposure group. The immunohistochemical analysis showed that SPT (LCB2 subunit) localization was stronger in the alveolar type II cells of group 2 lungs compared to the corresponding controls. These results were correlated with alterations in BALF cellular and biochemical parameters and lung morphology. Since SPT is the key enzyme for de novo sphingolipid synthesis in lung surfactant and SMase is responsible for sphingomyelin catabolism, we can postulate that high-dose UF-CdO exposure for 10 days induces an increase in sphingolipid synthesis in the type II cells of rat lungs that would not be promptly followed by its degradation.

Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low.

Recently it was speculated that ultrafine particles may translocate from deposition sites in the lungs to systemic circulation. This could lead to accumulation and potentially adverse reactions in critical organs such as liver, heart, and even brain, consistent with the hypothesis that ultrafine insoluble particles may play a role in the onset of cardiovascular diseases, as growing evidence from epidemiological studies suggests. Ultrafine (192)Ir radio-labeled iridium particles (15 and 80 nm count median diameter) generated by spark discharging were inhaled by young adult, healthy, male WKY rats ventilated for 1 h via an endotracheal tube. After exposure, excreta were collected quantitatively. At time points ranging from 6 h to 7 d, rats were sacrificed, and a complete balance of (192)Ir activity retained in the body and cleared by excretion was determined gamma spectroscopically. Thoracic deposition fractions of inhaled 15- and 80-nm (192)Ir particles were 0.49 and 0.28, respectively. Both batches of ultrafine iridium particles proved to be insoluble (<1% in 7 d). During wk 1 after inhalation particles were predominantly cleared via airways into the gastrointestinal tract and feces. This cleared fraction includes particles deposited in the alveolar region. Additionally, minute particle translocation of <1% of the deposited particles into secondary organs such as liver, spleen, heart, and brain was measured after systemic uptake from the lungs. The translocated fraction of the 80-nm particles was about an order of magnitude less than that of 15-nm particles. In additional studies, the biokinetics of ultrafine particles and soluble (192)Ir was studied after administration by either gavage or intratracheal instillation or intravenous injection. They confirmed the low solubility of the particles and proved that (1) particles were neither dissolved nor absorbed from the gut, (2) systemically circulating particles were rapidly and quantitatively accumulated in the liver and spleen and retained there, and (3) soluble (192)Ir instilled in the lungs was rapidly excreted via urine with little retention in the lungs and other organs. This study indicates that only a rather small fraction of ultrafine#10; iridium particles has access from peripheral lungs to systemic circulation and extrapulmonary organs. Therefore, the hypothesis that systemic access of ultrafine insoluble particles may generally induce adverse reactions in the cardiovascular system and liver leading to the onset of cardiovascular diseases needs additional detailed and differentiated consideration.

Respiratory mechanics in mice: strain and sex specific differences.

To assess the contribution of genetic background to respiratory mechanics, we developed a ventilator unit to measure lung function parameters in the mouse. We studied two commonly used inbred mice strains originating from Mus musculus domesticus (C57BL/6 and C3HeB/FeJ) and a third strain derived from Mus musculus molossinus [Japanese fancy mouse 1 (JF1)]. The ventilator allows for accurate performance of the different breathing manoeuvres required for measuring in- and expiratory reserve capacity, quasi-static and dynamic compliance, and airway resistance. In combination with a mass spectrometer for monitoring gas concentrations, single-breath manoeuvres were performed and He-expirograms obtained, from which dead space volume and slope of phase III were determined. From each strain and each sex, 10, 2-month old animals were studied immediately after being killed by an intraperitoneal overdose of xylazine and ketamine. C3HeB/FeJ and C57BL/6 exhibited comparable lung volumes. In male C3HeB/FeJ mice, e.g. vital capacity (VC) was 1072 +/- 79 microL, inspiratory reserve capacity 782 +/- 88 microL, and dead space volume at total lung inflation 216 +/- 18 microL. Lung volumes of JF1 were significantly lower (e.g. VC 611 +/- 53 microL, P < 0.01) even when normalized to body weight. In all three strains, specific lung volumes were significantly higher in females than in males, possibly explained by a higher oxygen demand during pregnancy and lactation, both of which fill most of their life times. Static compliance in C3HeB/FeJ was 64.3 +/- 5.4 microL cmH2O-1. It was smaller in C57BL/6 and JF1 mice, even when related to the lung volume. Analysis of the degree of genetic vs. non-genetic components of the phenotypic variation revealed that at least 80% of the total variation of lung volumes and static compliance in the mixed population is attributable to genetic differences between individuals. These differences will be verified in further studies by segregation and genetic linkage analysis.

Pulmonary and systemic distribution of inhaled ultrafine silver particles in rats.

The cardiovascular system is currently considered a target for particulate matter, especially for ultrafine particles. In addition to autonomic or cytokine mediated effects, the direct interaction of inhaled materials with the target tissue must be examined to understand the underlying mechanisms. In the first approach, pulmonary and systemic distribution of inhaled ultrafine elemental silver (EAg) particles was investigated on the basis of morphology and inductively coupled plasma mass spectrometry (ICP-MS) analysis. Rats were exposed for 6 hr at a concentration of 133 microg EAg m(3) (3 x 10(6) cm(3), 15 nm modal diameter) and were sacrificed on days 0, 1, 4, and 7. ICP-MS analysis showed that 1.7 microg Ag was found in the lungs immediately after the end of exposure. Amounts of Ag in the lungs decreased rapidly with time, and by day 7 only 4% of the initial burden remained. In the blood, significant amounts of Ag were detected on day 0 and thereafter decreased rapidly. In the liver, kidney, spleen, brain, and heart, low concentrations of Ag were observed. Nasal cavities, especially the posterior portion, and lung-associated lymph nodes showed relatively high concentrations of Ag. For comparison, rats received by intratracheal instillation either 150 microL aqueous solution of 7 microg silver nitrate (AgNO(3) (4.4 microg Ag) or 150 microL aqueous suspension of 50 microg agglomerated ultrafine EAg particles. A portion of the agglomerates remained undissolved in the alveolar macrophages and in the septum for at least 7 days. In contrast, rapid clearance of instilled water-soluble AgNO(3) from the lung was observed. These findings show that although instilled agglomerates of ultrafine EAg particles were retained in the lung, Ag was rapidly cleared from the lung after inhalation of ultrafine EAg particles, as well as after instillation of AgNO(3), and entered systemic pathways.

Agglomerates of ultrafine particles of elemental carbon and TiO2 induce generation of lipid mediators in alveolar macrophages.

Agglomerates of ultrafine particles (AUFPs) may cause adverse health effects because of their large surface area. To evaluate physiologic responses of immune cells, we studied whether agglomerates of 77-nm elemental carbon [(EC); specific surface area 750 m2/g] and 21 nm titanium dioxide (TiO(2) particles (specific surface area 50 m(2)/g) affect the release of lipid mediators by alveolar macrophages (AMs). After 60-min incubation with 1 microg/mL AUFP-EC (corresponding to 7.5 cm(2) particle surface area), canine AMs (1 x 10(6) cells/mL) released arachidonic acid (AA) and the cyclooxygenase (COX) products prostaglandin E(2) (PGE(2), thromboxane B(2), and 12-hydroxyheptadecatrienoic acid but not 5-lipoxygenase (5-LO) products. AUFP-TiO(2) with a 10-fold higher mass (10 microg/mL) than AUFP-EC, but a similar particle surface area (5 cm(2) also induced AMs to release AA and COX products. Agglomerates of 250 nm TiO(2) particles (specific surface area 6.5 m(2)/g) at 100 microg/mL mass concentration (particle surface area 6.5 cm(2) showed the same response. Interestingly, 75 cm(2)/mL surface area of AUFP-EC and 16 cm(2)/mL surface area of AUFP-TiO(2) additionally induced the release of the 5-LO products leukotriene B(4) and 5-hydroxyeicosatetraenoic acid. Respiratory burst activity of stimulated canine neutrophils was partially suppressed by supernatants of AMs treated with various mass concentrations of the three types of particles. Inhibition of neutrophil activity was abolished by supernatants of AMs treated with COX inhibitors prior to AUFP-incubation. This indicates that anti-inflammatory properties of PGE(2) dominate the overall response of lipid mediators released by AUFP-affected AMs. In conclusion, our data indicate that surface area rather than mass concentration determines the effect of AUFPs, and that activation of phospholipase A(subscript)2(/subscript) and COX pathway occurs at a lower particle surface area than that of 5-LO-pathway. We hypothesize a protective role of PGE(2) in downregulating potential inflammatory reactions induced by ultrafine particles.

A Morphologic Study on the Fate of Ultrafine Silver Particles: Distribution Pattern of Phagocytized Metallic Silver in Vitro and in Vivo.

The distribution pattern of inhaled particles is an important factor for the evaluation of health effects. In this study, we morphologically investigated the fate of agglomerated ultrafine particles in macrophages in vitro and in vivo. Metallic silver (Ag) was chosen as a test particle, since it can be easily produced and detected by elemental and morphologic analyses. Ultrafine Ag particles generated by an electric spark generator in an argon atmosphere were collected on PTFE filters. The particles were suspended in distilled water and adjusted to different concentrations (10 µg/ml to 1 mg/ml) with phosphate-buffered saline (PBS). For the in vitro study, 1774 macrophage cell suspensions (200,000 cells in 400 µl medium) were plated in small chambers. Six hours later, 100 µl of the silver-PBS suspension was added to each chamber. For the next 9 days, the chamber slides were examined daily with an inverted microscope in order to detect agglomerated particles in the cell. The medium was changed every day, and Ag in the medium was checked by inductively coupled plasma mass spectrometry (ICP-MS). On days 1, 3, 5, 7, and 9, cells in the chambers were fixed with 2.5% buffered glutaraldehyde and examined ultrastructurally. For the in vivo study using F344 rats, 50 µg Ag particles were instilled intratracheally. On days 1, 4, and 7 following instillation, rats were sacrificed and the lungs were examined morphologically. The Ag content in the lung, liver, and lung-associated lymph nodes was analysed by ICP-MS. In the in vitro study, the dose-dependent presence of agglomerated particles was observed in 1774 cells. The size and form of particles remained unchanged throughout the observation period. Electron microscopy with x-ray microanalysis showed that both single and agglomerated Ag particles were observed in the dilated phagolysosome of 1774 cells. In the in vivo study, focal accumulation of Ag-particle-laden alveolar macrophages was found. Ag particles were also observed in the alveolar wall. Ag content in the lung was constant between day 1 and day 7, indicating that no rapid particle translocation from the lung to other organs had taken place in this time period. In vitro and in vivo studies suggested that agglomerated Ag particles remained in targets for a given period of time-at least up to 7 days.

Anatomic localization of 24- and 96-h particle retention in canine airways.

Long-term retention of particles in airways is controversial. However, precise anatomic localization of the particles is not possible in people. In this study the anatomic location of retained particles after shallow bolus inhalation was determined in anesthetized, ventilated beagle dogs. Fifty 30-cm(3) boluses containing monodisperse 2.5-micron polystyrene particles (PSL) were delivered to a shallow lung depth of 81-129 cm(3). At 96 h before euthanasia, red fluorescent PSL were used; at 24 h, green fluorescent PSL and (99m)Tc-labeled PSL were used. Clearance of (99m)Tc-PSL was measured during the next 24 h. Sites of particle retention were determined in systematic, volume-weighted random samples of microwave-fixed lung tissue. Precise particle localization and distribution was analyzed by using gamma counting, conventional fluorescence microscopy, and confocal microscopy. Within 24 h after shallow bolus inhalation, 50-95% of the deposited (99m)Tc-PSL were cleared, but the remaining fraction was cleared slowly in all dogs, similar to previous human results. The three-dimensional deposition patterns showed particles across the entire cross-sectional plane of the lungs at the level of the carina. In these locations, 33 +/- 9.9% of the retained particles were found in small, nonrespiratory airways (0.3- to 1-mm diameter) and 49 +/- 10% of the particles in alveoli; the remaining fraction was found in larger airways. After 96 h, a similar pattern was found. These findings suggest that long-term retention in airways is at the bronchiolar level.

Health effects of sulfur-related environmental air pollution: the pulmonary surfactant system is not disturbed by exposure to acidic sulfate and neutral sulfite aerosols.

The goal of this study was to assess the impact of long-term exposure to environmental sulfur-related aerosols on the biochemical and biophysical properties of lung surfactant. Eight Beagle dogs were housed under clean air conditions for 450 days, followed by an exposure period of 400 days to 0.36 mg/m3 of sulfite (16.5 h/d) and to 5.66 mg/m3 of sulfate (6 h/d) equivalent to a pulmonary hydrogen burden of 15 mumol/m3. Other dogs kept in clean air for the whole study period were additional controls. Serial bronchoalveolar lavages (BALs) were analyzed for total phospholipid concentration, content and ratio of a surfactant-rich large aggregate (LA) fraction and a small aggregate (SA) fraction, in vitro surface area cycling of LAs into SAs as a measure of alveolar extracellular pulmonary surfactant aggregate metabolism, and surface activity of native and lipid-extracted LA. No significant changes over time and no differences between the clean air period and the exposure period were observed. Thus, long-term environmental exposure of dogs to the sulfur-related air pollution tested does not lead to alterations in the amount, extracellular metabolism, or surface-active properties of pulmonary surfactant.

Xenobiotic-metabolizing enzymes in the canine respiratory tract.

Airway epithelial surface is the primary target of airborne pollutants. To estimate the distribution of xenobiotic-metabolizing enzymes in the respiratory tract of dogs, epithelia from different airway sites of four animals were analyzed for metabolism of sulfite (sulfite oxidase) and formaldehyde (formaldehyde dehydrogenase and aldehyde dehydrogenase). In addition, glutathione S-transferases were assayed using several model substrates. Enzyme activities were compared with those found in liver parenchyma. The activity of sulfite oxidase was found to be comparable in nose, trachea, and proximal and medium bronchi, but appeared to be lower in lung parenchyma of most animals. In contrast, hepatic sulfite oxidase activity of these animals was substantially higher compared to that in airway epithelia. The activity of glutathione-dependent formaldehyde dehydrogenase (FDH) appeared to be highest in nose and lowest in distal bronchi, lung, and liver parenchyma. The distribution pattern of the glutathione-independent aldehyde dehydrogenase (AldDH) in the respiratory tract was different from that of FDH. Levels of AldDH were about 5- to 10-fold lower than those of FDH, suggesting that AldDH is of minor importance for pulmonary formaldehyde detoxification. With regard to ethanol detoxification by a class I alcohol dehydrogenase (ADH), no measurable enzyme activity could be detected at most respiratory sites contrary to the high activity found in liver parenchyma. Regarding glutathione S-transferases (GSTs), different distributions of enzyme activities were found in the large and small airways when using three substrates. The 1-chloro-2,4-dinitrobenzene (CDNB)-related activities in the cytosolic fraction of the upper (nose, trachea) and lower airways (proximal, medium and distal bronchi) were higher than those in the microsomal fraction. Interestingly, there was no difference between CDNB-related activities in the cytosolic and microsomal fraction of the liver. Highest cytosolic activities were found in the nose, and were comparable to those detected in the liver parenchyma. The cytosolic 1,2-dichloro-4-nitrobenzene (DCNB)-related activities in the nose, proximal bronchi, and lung parenchyma were appeared to be markedly higher than those in trachea and medium and distal bronchi, while the microsomal activities were not detectable at most respiratory sites. In contrast, distinctly higher activities were measured in both fractions of liver tissue. Cytosolic 1, 2-epoxy-3-(p-nitrophenoxy)-propane (EPNP)-related activities were present in upper and lower airways including lung parenchyma at comparable levels, while in liver tissue the mean activities were distinctly lower. No EPNP-related activities were found in the microsomal fractions. In conclusion, most xenobiotic-metabolizing enzymes investigated in this study could be detected in epithelia of various respiratory sites. The most outstanding result revealed higher levels of FDH activity in the nose and downstream to the medium bronchi in comparison to those found in the small airways, lung, and liver tissue. Similarly, the EPNP-related GST exhibited a distinctly higher activity at all respiratory sites compared to the activity in liver tissue, suggesting a different regulation of this enzyme in lung and liver.

Health effects of sulfur-related environmental air pollution. III. Nonspecific respiratory defense capacities.

Recently concern has been raised about health effects related to environmental sulfur and/or acidic aerosols. To assess long-term effects on respiratory lung function, 8 beagle dogs were exposed over a period of 13 mo for 16.5 h/day to 1.0 microm neutral sulfite aerosol with a particle associated sulfur(IV) concentration of 0.32 mg m(-3) and for 6 h/day to 1.1 microm acidic sulfate aerosol providing an hydrogen ion concentration of 15.2 micromol m(-3) for inhalation. Prior to exposure the dogs were kept under clean air conditions for 16 mo to establish physiological baseline values for each dog. A second group of eight dogs (control) was kept for the entire study under clean air conditions. Nonspecific defense mechanisms in the airways and in the peripheral lung were studied during chronic exposure of the combination of neutral sulfur(IV) and acidic sulfur(VI) aerosols. No functional changes of tracheal mucus velocity were found, in agreement with unchanged morphometry of the airways. However, the exposure resulted in changes of several alveolar macrophage (AM) mediated particle clearance mechanisms: (1) Based on in vivo clearance analysis and cultured AM studies using moderately soluble cobalt oxide particles, intracellular particle dissolution was significantly reduced since phagolysosomal proton concentration was decreased. We deduce exposure-related malfunction of proton pumps bound to the phagolysosomal membrane as a result of an increase of cytosolic proton concentration. (2) Based on in vivo clearance analysis using insoluble polystyrene particles, AM-mediated particle transport from the lung periphery toward ciliated terminal bronchioli and further to the larynx was significantly reduced. Activation of epithelial type II cells at the entrance of alveoli was inferred from observed type II cell proliferation at those alveolar ridges and enhanced secretion of alkaline phosphatase in the fluid of bronchoalveolar lavages. As a result, hypersecretion of chemotactic mediators by activated type II cells at these loci led to the observed decrease of particle transport toward ciliated bronchioli. (3) Based on in vivo clearance analysis using insoluble polystyrene particles, particle transport from the alveolar epithelium into interstitial tissues was increased and (4) particle transport to the tracheobronchial lymph nodes was significantly enhanced. Particle transport into interstitial tissues is the most prominent clearance pathway from the canine alveolar epithelium. We conclude that the deteriorated particle transport toward ciliated terminal bronchioli resulted in an enhanced particle transport across the epithelial membrane into interstitial tissues and the lymphatic drainage. The observed alterations in alveolar macrophage-mediated clearance mechanisms during chronic exposure of these air pollutants indicate an increased risk of health.

Health effects of sulfur-related environmental air pollution. II. Cellular and molecular parameters of injury.

Recently, concern has been raised about effects related to environmental sulfur and/or acidic aerosols. To assess long-term effects on nonrespiratory lung function, 8 beagle dogs were exposed over a period of 13 mo for 16.5 h/day to a neutral sulfite aerosol at a sulfur(IV) concentration of 0.32 mg m(-3) and for 6 h/day to an acidic sulfate aerosol providing a hydrogen concentration of 15.2 micromol m(-3) for inhalation. Prior to exposure the dogs were kept under clean air conditions for 16 mo to establish physiological baseline values for each animal. A second group of eight dogs (control) was kept for the entire study under clean air conditions. No clinical symptoms were identified that could be related to the combined exposure. Biochemical and cellular parameters were analyzed in sequential bronchoalveolar lavage (BAL) fluids. The permeability of the alveolo-capillary membrane and diethylenetriaminepentaacetic acid (DTPA) clearance was not affected. Similarly, oxidant burden of the epithelial lining fluid evaluated by levels of oxidation products in the BAL fluid protein fraction remained unchanged. Both the lysosomal enzyme beta-N-acetylglucosaminidase and the alpha-1-AT were increased (p <.05). In contrast, the cytoplasmic marker lactate dehydrogenase remained unchanged, indicating the absence of severe damages to epithelial cells or phagocytes. Various surfactant functions were not altered during exposure. Three animals showed elevated levels of the type II cell-associated alkaline phosphatase (AP), indicating a nonuniform response of type II cells. Significant correlations were found between AP and total BAL protein, but not between AP and lactate dehydrogenase, suggesting proliferation of alveolar type II cells. Absolute and relative cell counts in the BAL fluid were not influenced by exposure. Alveolar macrophages showed no alterations with regard to their respiratory burst upon stimulation with opsonized zymosan. The percentage of alveolar macrophages capable of phagocytozing latex particles was significantly decreased (p<.05), while the phagocytosis index was not altered. In view of the results of this and previous studies, we conclude that there is no synergism of effects of these two air pollutants on nonrespiratory lung functions. It is hypothesized that antagonistic effects of these air pollutants on phospholipase A2-dependent pathways account for compensatory physiological mechanisms. The results emphasize the complexity of health effects on lung functions in response to the complex mixture of air pollutants and disclose the precariousness in the risk assessment of air pollutants for humans.

Health effects of sulfur-related environmental air pollution. I. Executive summary.

The motivation of simulating real-world environmental exposure in a number of long-term studies with dogs was to address the question of whether or not perpetual inhalation of air pollutants can initiate diseases in healthy lungs and can thus contribute to the increasing prevalence of respiratory diseases in industrialized countries. The major conclusion of this article is that this question has to be answered in the negative for the simultaneous inhalation of the major constituents of combustion-related air pollution, particle-associated sulfur(IV), and particle-associated hydrogen ions. Over 13 mo, 8 healthy beagle dogs were exposed in 2 whole-body chambers daily for 16.5 h to 1 microm neutral sulfite [sulfur(IV)] particles at a mass concentration of 1.5 mg m-3 and for 6 h to 1.1 microm acidic sulfate particles carrying 15 micromol m-3 hydrogen ions into the canine lungs. This longitudinal study was characterized by repeated observations of individual respiratory response patterns. To establish baseline data the dogs were repeatedly examined preexposure while the chambers were ventilated over 16 mo with clean air. Each individual served thus as its own control. Another eight dogs served as additional controls. They were housed in 2 chambers ventilated with clean air over the entire study period of 29 mo. To assess response patterns, respiratory lung function tests were performed pre- and postexposure, segmental lung lavages were repeatedly performed to obtain epithelial lining fluid from the lungs for analysis of cell content, cell function, and biochemical indicators of lung injury, and radiolabeled test particles were used to study pathways of intrapulmonary particle elimination. At the end of the study, the lungs of all animals were morphologically and morphometrically examined. Functional and structural responses were finally compared to those observed previously as a result of a sole exposure of canine lungs to neutral sulfite particles over 10 mo (Heyder et al., 1992). Interactions between responses induced by neutral sulfite and acidic sulfate particles occurred, but antagonism rather than synergism was observed. The responses induced by sulfur(IV) were less pronounced, not detectable, or even reversed when hydrogen ions were also delivered to the lungs. On the other hand, responses not induced by the sole exposure to sulfur(IV) were observed: The activity of alkaline phosphatase was elevated and type II pneumocytes proliferated. It can, however, be concluded that long-term exposure of healthy lungs to particle-associated neutral sulfur(IV) and hydrogen ions at concentration close to ambient levels causes subtle respiratory responses but does not initiate pathological processes in the lungs. In other words, the perpetual inhalation of sulfur(IV) and hydrogen ions from the atmospheric environment presents no health risk to the healthy lungs. It is thus also very unlikely that respiratory diseases can be initiated by the inhalation of these pollutants.

Health effects of sulfur-related environmental air pollution. IV. Respiratory lung function.

Recently concern has been raised about health effects related to environmental sulfur and/or acidic aerosols. To assess long-term effects on respiratory lung function, 8 beagle dogs were exposed over a period of 13 mo for 16.5 h/day to 1-microm neutral sulfite aerosol with a particle-associated sulfur(IV) concentration of 0.32 mg m(-3) and for 6 h/day to 1.1-microm acidic sulfate aerosol providing an hydrogen ion concentration of 15.2 micromol m(-3) for inhalation. Prior to exposure the dogs were kept under clean air conditions for 16 mo to establish physiological baseline values for each dog. A second group of eight dogs (control) was kept for the entire study under clean air conditions. Before and at the end of exposure, respiratory lung function was evaluated in both groups in anesthetized and mechanically ventilated animals. Lung volumes as well as static and dynamic lung compliances were measured. Series dead-space volumes and slopes of the alveolar plateau for respiratory (O2, CO2) and inert test gases (He, SF6) were determined from single-breath washout tracings. Monodisperse 0.9-microm DEHS droplets were used to assess convective mixing in the lungs and to evaluate airway dimensions in vivo. Gas exchange across the alveolar-capillary layer was characterized by membrane diffusing capacity for carbon monoxide and alveolar-arterial pressure differences for respiratory gases. A bronchial challenge with carbachol was used to assess airway responsiveness. In comparison to the control group, dogs exposed to sulfur(IV) and acidic aerosol exhibited no significant changes in any respiratory lung function parameter. Also the responsiveness of the bronchial airways to carbachol was not affected. In view of the results obtained in this and previous studies, we conclude that anticipated synergistic effects of the two air pollutants on pulmonary lung function were not observed. It is hypothesized that antagonistic effects of the air pollutants on the activity of phospholipase A2 play an important role and account for counteracting physiological compensatory mechanisms. The results emphasize the complexity of health effects on lung function in response to the complex mixtures of ambient air pollutants and witness the precariousness in the risk assessment of air pollutants for humans.

Health effects of sulfur-related environmental air pollution. V. Lung structure.

The lungs of 8 male beagle dogs were examined morphologically and morphometrically after exposure for 13 mo to a respirable sulfur(IV) aerosol at a mass concentration of 1.53 mg m(-3) (16.5 h/day), and to an acidic sulfate aerosol carrying 15.2 micromol m(-3) hydrogen ions into the lungs (6 h/day). An additional eight dogs served as unexposed controls. Standard morphometric analyses of both the surface epithelia of the conducting airways and the alveolar region were performed. These analyses showed no difference between the exposure group and control group. However, there was a tendency to an increase in the volume density of bronchial glands in the exposure group. Five of eight exposed animals showed thickened ridges (knob-like structures) at the entrance to alveoli in the alveolar duct and alveolar sac. Transmission electron microscopy revealed that the thickening was mainly due to type II cell proliferation. As the previous experiment using sulfite aerosol only showed no alterations in the proximal alveolar regions, the changes observed may be considered as effects of acidic sulfate aerosol alone or in combination with sulfite. These findings suggest that sulfur aerosols have the potential to induce epithelial alterations in the proximal alveolar region, which is a primary target for air pollutants.

Viscoelastic properties of canine tracheal mucus: effects of structural inhomogeneities and storage.

Tracheal mucus is an inhomogeneous material and thus its viscoelastic properties may vary substantially within a mucus sample. In this study on canine tracheal mucus, we investigated this variation of mucus viscoelasticity and addressed the question whether or not the viscoelasticity is affected by storage of the mucus at low temperatures. Mucus samples were collected from 16 beagle dogs kept under clean air conditions. By means of magnetic rheometry, dynamic viscosity- and elasticity were determined either in the fresh state of these samples or after storage at -80 degrees C. Owing to inhomogeneities within the gel-structure of the mucus, local values of the viscoelastic parameters differed by more than an order of magnitude. Short storage periods (5 d) were found to decrease, long periods (100 d) to increase the viscosity as well as elasticity parameters of the mucus material. These results emphasize the fact that measured viscoelastic properties of mucus are average values depending on the measuring protocol. In addition, these values are modified by storage of the mucus at low temperatures.

Immunophenotyping of canine bronchoalveolar and peripheral blood lymphocytes.

The immunophenotype of canine lymphocytes obtained by bronchoalveolar lavage (BAL) was investigated and compared with that of peripheral blood leukocytes (PBL). Indirect immunofluorescence, generated by monoclonal antibodies (mAb) specific for canine CD5, CD4, CD8, CD45pan, CD45RA, MHCII and THY-1, was detected by flow cytometry. In comparison with PBL, in BAL there are fewer lymphocytes positive for CD45RA (75.4 +/- 12.6% vs. 42.3 +/- 9.4%; P < 0.05) and MHCI I (97.0 +/- 2.9% vs. 74.0 +/- 7.6%; P < 0.01), while there are more cells positive for CD8 (19.0 +/- 3.6% vs. 29.5 +/- 12.0%; P < 0.05). Thus there is a lower CD4/CD8 ratio in the cell compartment accessible by BAL (2.2 +/- 0.3 vs. 1.3 +/- 0.6; P < 0.005). The immunophenotype may be stable over time, as indicated by reexamination of cells obtained from one dog at four times over 1 year. Investigating the phenotype of lymphocytes from three different locations of the right lung, the cranial lobe lymphocytes show a lower CD4/CD8 ratio in comparison with PBL (1.81 +/- 0.35 vs. 1.12 +/- 0.31, n = 5; P < 0.02). In general, less MHCII positive lymphocytes (P < 0.001) and greater immunophenotype variability of results were found in these separate samples compared with pooled samples from these locations.

Isolation of a high-molecular mass glycoprotein from culture supernatant of an arthritogenic strain of the bacteria Erysipelothrix rhusiopathiae reacting with "inductive" monoclonal antibodies derived from rats with erysipelas polyarthritis.

A glycoprotein exhibiting a relative molecular mass of about 1000 kDa was purified to homogeneity from culture supernatant of arthritogenic bacteria (Erysipelothrix rhusiopathiae, strain T28) by ultrafiltration, ammonium sulfate precipitation, molecular mass exclusion, and ion exchange chromatography. Fractions obtained were analysed for their antigenic content by an enzyme linked immunosorbent assay (ELISA) using rabbit immune serum raised against this strain of Erysipelothrix rhusiopathiae. Distinct monoclonal antibodies obtained from rats suffering from erysipelas polyarthritis display a unique property by inducing very efficiently protective and regulatory mechanisms while being unable to generate classical "passive immunity". These "inductive" monoclonal antibodies recognize most likely linear epitopes on the purified glycoprotein. This makes it a prime source for analysing the target structure of these in vivo "inductive" antibodies.