Attenuation and recovery of pulmonary injury in rats following short-term,repeated daily exposure to ozone

Controlled human and epidemiology studies have demonstrated that during repeated exposure to ozone (O(3)) attenuation of lung function responses may occur. It is yet unknown whether inflammatory and biochemical effects in lower airways of humans, as observed upon single O(3) exposure, also show a diminutive response following repeated exposure to O(3). The aim of this study was to investigate inflammatory, permeability, and histopathological responses in lungs of rats following repeated daily O(3) exposure and to study the time course of attenuation and recovery of these effects using single O(3) challenges at various postexposure times. To aid in animal-to-human extrapolation, this study and a previously reported human study (Devlin et al., 1997) were designed with similar protocols. Wistar rats were exposed for 5 consecutive days to 0.4 ppm O(3) for 12 h/night. Subsequently, the time course of postexposure recovery was determined by a single challenge of 12 h to 0.4 ppm O(3) after a 5-, 10-, 15-, or 20-day recovery period. Bronchoalveolar lavage (BAL) examination and histopathology were performed 12 h after this O(3) challenge. To quantify the magnitude of the O(3) response, results were compared with a group exposed only once for 12 h to 0.4 ppm O(3) and sacrificed simultaneously. The results demonstrate that a single exposure of 0.4 ppm O(3) causes marked permeability and inflammatory responses in lower airways of rats, as evidenced by enhanced BAL fluid levels of proteins, fibronectin, interleukin (IL)-6, and inflammatory cells. However, 5 days of exposure to 0.4 ppm O(3) for 12 h/night resulted in a complete disappearance of these responses, resulting in BAL fluid values that were not different from those observed in unexposed controls. Postexposure analyses of pulmonary response to O(3) challenges demonstrated that these attenuated responses show a gradual recovery. The data indicate that with respect to BAL fluid levels of albumin, IL-6, and number of macrophages and neutrophils, the period for lung tissue to regain its full susceptibility and responsiveness to O(3) following a 5-day preexposure period is approximately 15-20 days. Remarkably, the total protein and fibronectin responses in BAL fluid still exhibited an attenuated response to an O(3) challenge at 20 days postexposure. Morphometry (number of BrdU-labeled cells in terminal bronchiolar epithelium, and number of alveolar macrophages) showed that after a recovery of 5-10 days following a 5-day preexposure the response to a challenge was identical to that after a single exposure. These results suggest that complete repair from lower airway inflammation caused by short-term, repeated exposure to O(3) may take longer than previously assumed.     

ATTENUATION AND RECOVERY OF PULMONARY INJURY IN RATS FOLLOWING SHORT-TERM,REPEATED DAILY EXPOSURE TO OZONE

Controlled human and epidemiology studies have demonstrated that during repeated exposure to ozone (O 3) attenuation of lung function responses may occur. It is yet unknown whether inflammatory and biochemical effects in lower airways of humans, as observed upon single O 3 exposure, also show a diminutive response following repeated exposure to O 3. The aim of this study was to investigate inflammatory, permeability, and histopathological responses in lungs of rats following repeated daily O 3 exposure and to study the time course of attenuation and recovery of these effects using single O 3 challenges at various postexposure times. To aid in animal-to-human extrapolation, this study and a previously reported human study (Devlin et al., 1997) were designed with similar protocols. Wistar rats were exposed for 5 consecutive days to 0.4 ppm O 3 for 12 h/night. Subsequently, the time course of postexposure recovery was determined by a single challenge of 12 h to 0.4 ppm O 3 after a 5-, 10-, 15-, or 20-day recovery period. Broncho-alveolar lavage (BAL) examination and histopathology were performed 12 h after this O 3 challenge. To quantify the magnitude of the O 3 response, results were compared with a group exposed only once for 12 h to 0.4 ppm O 3 and sacrificed simultaneously. The results demonstrate that a single exposure of 0.4 ppm O 3 causes marked permeability and inflammatory responses in lower airways of rats, as evidenced by enhanced BAL fluid levels of proteins, fibronectin, interleukin (IL)-6, and inflammatory cells. However, 5 days of exposure to 0.4 ppm O 3 for 12 h/night resulted in a complete disappearance of these responses, resulting in BAL fluid values that were not different from those observed in unexposed controls. Postexposure analyses of pulmonary response to O 3 challenges demonstrated that these attenuated responses show a gradual recovery. The data indicate that with respect to BAL fluid levels of albumin, IL-6, and number of macrophages and neutrophils, the period for lung tissue to regain its full susceptibility and responsiveness to O 3 following a 5-day preexposure period is approximately 15-20 days. Remarkably, the total protein and fibronectin responses in BAL fluid still exhibited an attenuated response to an O 3 challenge at 20 days postexposure. Morphometry (number of BrdU-labeled cells in terminal bronchiolar epithelium, and number of alveolar macrophages) showed that after a recovery of 5-10 days following a 5-day preexposure the response to a challenge was identical to that after a single exposure. These results suggest that complete repair from lower airway inflammation caused by short-term, repeated exposure to O 3 may take longer than previously assumed.     

OZONE-INDUCED DNA SINGLE STRAND BREAKS IN HUMAN AND GUINEA PIG LUNG CELLS IN VIVO

Ozone O3 has been postulated to induce DNA damage and has been shown to be mildly tumorigenic in some studies utilizing long-term rodent exposures. We investigated lung DNA damage induced by controlled O3 exposure in vivo in guinea pigs and human subjects. We specifically examined DNA single-strand breaks SSB using the single-cell gel electrophoresis assay. Guinea pigs were exposed for 2 h to air, 0.4 ppm O3, or 1.0 ppm O3, and lung cells were collected by bronchoalveolar lavage BAL and bronchial scraping within 1 h after exposure. Both the 0.4 and 1.0 ppm O3 exposures induced significant increases in SSB in both the BAL cells and tracheal cells as indicated by an increased cell DNA length in electrophoresized agarose gel. The increase in DNA SSB was a more sensitive biomarker of exposure compared to more traditional biomarkers BAL total protein and lactate dehydrogenase, alterations in BAL cell differential, which changed only at the 1.0 ppm exposure. In an initial study with human volunteers, BAL and bronchial epithelial cells were collected from human volunteers 1-2 h after an air or 0.4 ppm O3 exposure in vivo without exercise. BAL cells primarily macrophages and bronchial epithelial cells showed no change in DNA SSB compared to the air-exposed controls. In a second study, DNA SSB in bronchial epithelial cells and BAL cells collected from exercising subjects exposed to 0.4 ppm O3 were not altered by steroid prednisone, beclomethasone pre- treatment compared to placebo treatment. However the bronchial epithelial cell DNA SSB values in the O3-exposed, placebo-pretreated group were significantly increased compared to values air or O3 exposed, no exercise in the first study. The dosimetry of O3 deposi- tion in the guinea pig and human subjects appeared similar based on the amount of 18O derived from 18O-labeled O3 found in the BAL cell fraction. These data suggest that O3 exposure at 0.4 ppm induces DNA SSB in rodent and human lung cells, although the effect of exercise on the increase of human lung cell SSB is unclear. Formation of DNA SSB may be an indicator of the tumorigenic potential of O3. Additionally, DNA SSB can potentially be a good biomarker of O3 exposure in humans and animal model systems. 3     

INTERSPECIES DIFFERENCES IN TIME COURSE OF PULMONARY TOXICITY FOLLOWING REPEATED EXPOSURE TO OZONE

To compare the extent and time course of pulmonary injury and repair in 3 rodent species, rats, mice and guinea pigs were continuously exposed for 3, 7, 28, and 56 days to 400 and 800 mug O3/m3 (0.2 and 0.4 ppm). Recovery from 28 days of exposure was studied at 3, 7, and 28 days after exposure. Pulmonary injury and repair was studied at various time points by histology, electron microscopy, morphometry, and biochemistry. In all 3 species a concentration-related centriacinar inflammation occurred, with a maximum after 3 days of exposure. The number of alveolar macrophages and the pulmonary cell density in the centriacinar region increased progressively until 56 days of exposure, with the guinea pig the most sensitive species. Only the mouse displayed a concentration and exposure-time dependent hypertrophy of bronchiolar epithelium. After 56 days of exposure to 800 mug O3/m3 in the rat and the guinea pig, giant lamellar bodies in type II cells were present. Exposures for 3 and 7 days at near ambient ozone concentrations (400mug O3/m3) resulted in significantly elevated lung enzyme activities in the mouse, and in significant histological and morphometric changes in all 3 species. In rat and guinea pigs exposures for 56 days resulted in alveolar duct fibrosis. The highest biochemical response and the slowest recovery from ozone exposure were seen in the mouse. Histology, morphometry, and biochemistry revealed a total recovery from a 28-day exposure period in rats after 28 days, while in guinea pigs the ductular septa were still thickened and in mice all enzyme activities were still elevated in comparison with control values. In conclusion, the response of mice to ozone was evaluated as most severe, followed by those of guinea pigs and least in rats.     

Species comparison of acute inhalation toxicity of ozone and phosgene

A comparison of the concentration-response effects of inhaled ozone (O3) and phosgene (COCl2) in different species of laboratory animals was made in order to better understand the influence of the choice of species in inhalation toxicity studies. The effect of 4-h exposures to ozone at concentrations of 0.2, 0.5, 1.0, and 2.0 ppm, and to COCl2 and 0.1, 0.2, 0.5, and 1.0 ppm was determined in rabbits, guinea pigs, rats, hamsters, and mice. Lavage fluid protein (LFP) accumulation 18-20 h after exposure was used as the indicator of O3- and COCl2-induced pulmonary edema. All species had similar basal levels of LFP (250-350 mg/ml) when a volume of saline that approximated the total lung capacity was used to lavage the collapsed lungs. Ozone effects were most marked in guinea pigs, which showed significant effects at 0.2 ppm and above. Mice, hamsters, and rats showed effects at 1.0 ppm O3 and above, while rabbits responded only at 2.0 ppm O3. Phosgene similarly affected mice, hamsters, and rats at 0.2 ppm and above, while guinea pigs and rabbits were affected at 0.5 ppm and above. Percent recovery of lavage fluid varied significantly between species, guinea pigs having lower recovery than other species with both gases. Lavage fluid recovery was lower following exposure to higher levels of O3 but not COCl2. Results of this study indicate that significant species differences are seen in the response to low levels of O3 and COCl2. These differences do not appear to be related in a simple manner to body weight.     

Alteration of epithelial integrity, alkaline phosphatase activity, and fibronectin expression in lungs of rats exposed to ozone.

The deleterious effects of ozone (O3), an oxidant air pollutant, in the lung are dependent on dose and exposure duration and generally evolve with time postexposure. This study characterized the time sequence of epithelial injury and fibronectin expression in the lungs of rats exposed to O3. Bronchoalveolar lavage (BAL) fluid was analyzed for alkaline phosphatase and total protein as markers of epithelial injury and increased permeability, and fibronectin for its role in inflammation and lung injury. The results revealed a time-related increase in total protein in the BAL fluid following a 3-h exposure of rats to 1 ppm O3. The increased protein concentrations peaked at 12 h and then declined, but remained significantly higher than control at 24 h postexposure. A similar time-related significant increase also occurred for BAL fibronectin and alkaline phosphatase activity. However, the return of alkaline phosphatase levels to baseline prior to a comparable reduction in protein levels suggests repair of injured cells, but a delay in the formation of epithelial junctions that limit the transfer of serum proteins to air spaces. By cytochemistry, alkaline phosphatase activity was detected in association with lung type II epithelial cells and in BAL polymorphonuclear leukocytes (PMNs), but not in macrophages. While a significant increase in cytochemically detectable alkaline phosphatase resulted from the increase in PMN number following O3 exposure, mononuclear cells constituted the primary cell type responsible for fibronectin mRNA upregulation. While the cytochemical observations support the role of inflammatory cells in the injury process, the comparability of temporal changes in BAL protein, fibronectin, and alkaline phosphatase suggests a mechanistic role for fibronectin in lung injury.     

Increased susceptibility of hyperthyroid rats to ozone: early events and mechanisms

Previous studies demonstrated that ozone-induced lung damage and inflammation are much greater in hyperthyroid rats, compared to normal rats, at 18 h postexposure. The purpose of the present investigation was to study early events and mechanisms underlying the increased sensitivity to ozone in a hyperthyroid state. Specifically, the degree of lung epithelial cell barrier disruption, the antioxidant status of the extracellular lining fluid, and the release of inflammatory mediators were examined. To induce a hyperthyroid state, mature male Sprague-Dawley rats were implanted with time-release pellets containing thyroxine; control rats received placebo pellets. After 7 d, the animals were exposed to air or ozone (2 ppm, 3 h). Immediately following the end of the exposure, bronchoalveolar lavage (BAL) fluid and cells were harvested. BAL fluid albumin levels and total antioxidant status were examined. In addition, levels of prostaglandin E2 (PGE2), macrophage inflammatory protein (MIP)-2, MCP-1, and tumor necrosis factor (TNF)-alpha were determined in BAL fluid and in media samples following ex vivo culture of BAL cells harvested after in vivo inhalation exposures. The results of this study are consistent with the following hypotheses: (1) A marked increase in the permeability of the alveolar-capillary barrier is an early event following ozone exposure in a hyperthyroid state; however this does not appear to be due to overall changes in BAL fluid antioxidant potential. (2) Early increases in MIP-2, but not PGE2, are involved in the enhanced lung response to ozone in a hyperthyroid state. (3) Inflammatory mediator production (i.e., PGE2, MIP-2, MCP-1, and TNF-alpha) by alveolar macrophages plays a minimal role in the initial responses to ozone in a hyperthyroid state.     

ALTERATION OF EPITHELIAL INTEGRITY,ALKALINE PHOSPHATASE ACTIVITY,AND FIBRONECTIN EXPRESSION IN LUNGS OF RATS EXPOSED TO OZONE

The deleterious effects of ozone (O3), an oxidant air pollutant, in the lung are dependent on dose and exposure duration and generally evolve with time postexposure. This study characterized the time sequence of epithelial injury and fibronectin expression in the lungs of rats exposed to O3. Bronchoalveolar lavage (BAL) fluid was analyzed for alkaline phosphatase and total protein as markers of epithelial injury and increased permeability, and fibronectin for its role in inflammation and lung injury. The results revealed a time-related increase in total protein in the BAL fluid following a 3-h exposure of rats to 1 ppm O3. The increased protein concentrations peaked at 12 h and then declined, but remained significantly higher than control at 24 h postexposure. A similar time-related significant increase also occurred for BAL fibronectin and alkaline phosphatase activity. However, the return of alkaline phosphatase levels to baseline prior to a comparable reduction in protein levels suggests repair of injured cells, but a delay in the formation of epithelial junctions that limit the transfer of serum proteins to air spaces. By cytochemistry, alkaline phosphatase activity was detected in association with lung type II epithelial cells and in BAL polymorphonuclear leukocytes (PMNs), but not in macrophages. While a significant increase in cytochemically detectable alkaline phosphatase resulted from the increase in PMN number following O3 exposure, mononuclear cells constituted the primary cell type responsible for fibronectin mRNA upregulation. While the cytochemical observations support the role of inflammatory cells in the injury process, the comparability of temporal changes in BAL protein, fibronectin, and alkaline phosphatase suggests a mechanistic role for fibronectin in lung injury.     

Enhanced pulmonary inflammatory response to inhaled endotoxin in pregnant rats

Evidence suggests that pregnant animals are more sensitive than nonpregnant animals to the systemic administration of endotoxin. Studies were undertaken to assess whether an enhanced sensitivity of the pulmonary system to aerosolized endotoxin might exist during pregnancy. Pregnant Sprague-Dawley female rats (17 d of gestation) or age-matched virgin female rats were exposed to air or endotoxin (lipopolysaccharide) by inhalation for 3 h. At 18 h following exposure to endotoxin, lactate dehydrogenase activity levels in bronchoalveolar lavage (BAL) fluid samples from pregnant rats were 1.5-fold greater than those from endotoxin-exposed virgin rats. BAL polymorphonuclear leukocyte (PMN) numbers were also approximately twofold greater in pregnant rats than in virgins following the inhalation of endotoxin. The increases in BAL PMNs in pregnant rats following endotoxin exposure were observed just following exposure to endotoxin as well as at 18 h following exposure. These results indicate that an increased pulmonary inflammatory response to inhaled endotoxin occurs during pregnancy in rats. Additional findings suggest that these pregnancy-linked pulmonary responses to endotoxin cannot be explained by the following potential mechanisms: changes in the inhaled dose of endotoxin, or alterations in the responsiveness of alveolar macrophages to endotoxin. To our knowledge this is the first study that has evaluated pulmonary responses to inhaled endotoxin during pregnancy. Our finding that pregnancy is associated with an increased lung inflammatory response to aerosolized endotoxin raises the possibility that there may be a generalized enhancement of pulmonary responses to inhaled toxic agents during pregnancy.     

Assessment of early acute lung injury in rodents exposed to phosgene

Phosgene is a highly reactive oxidant gas used in the chemical industry. Phosgene can cause life-threatening pulmonary edema by reacting with peripheral lung compartment tissue components. Clinical evidence of edema is not usually apparent until well after the initial exposure. This study was designed to investigate early signs of acute lung injury in rodents within 45–60 min after the start of exposure. Male mice, rats, or guinea pigs were exposed to 87 mg/m³ (22 ppm) phosgene or filtered room air for 20 min followed by room air washout for 5 min. This concentration-time exposure causes a doubling of lung wet weight within 5 h. After exposure, animals were immediately anesthetized i.p., with pentobarbital. Bronchoalveolar lavage (BAL) was performed and fluid analyzed for total glutathione (GSH), lipid peroxidation thiobarbituric acid reactive substances (TBARS), and protein concentration. Lungs were perfused with saline to remove blood, freeze-snapped in liquid N₂, analyzed for tissue GSH, and TBARS. Lung edema was assessed gravimetrically by measuring tissue wet/dry (W/D) weight ratios and tissue wet weights (TWW). W/D and TWW were significantly higher in mice for phosgene vs air (P=0.001, P<0.0001, respectively), but not in rats or guinea pigs. Tissue TBARS was significantly higher in phosgene-exposed guinea pigs, P=0.027; however, BAL TBARS was higher in both rats and guinea pigs, P=0.013 and P=0.006, respectively. Tissue GSH was significantly lower in phosgene-exposed rats and guinea pigs but not mice, whereas BAL GSH was higher in rats, P<0.0001. There were significantly higher BAL protein levels in all phosgene-exposed species: mice, P<0.0001; rats, P<0.0001; and guinea pigs, P=0.002. Although there appears to be a species-specific biochemical effect of phosgene exposure for some biochemical indices, measurement of BAL protein in all three species is a better indicator of ensuing edema formation. Received: 30 June 1997 / Accepted: 5 January 1998     

Lung function changes in Sprague-Dawley rats after prolonged inhalation exposure to silver nanoparticles

The antimicrobial activity of silver nanoparticles has resulted in their widespread use in many consumer products. However, despite the continuing increase in the population exposed to silver nanoparticles, the effects of prolonged exposure to silver nanoparticles have not been thoroughly determined. Accordingly, this study attempted to investigate the inflammatory responses and pulmonary function changes in rats during 90 days of inhalation exposure to silver nanoparticles. The rats were exposed to silver nanoparticles (18 nm diameter) at concentrations of 0.7 x 10(6) particles/cm(3) (low dose), 1.4 x 10(6) particles /cm(3) (middle dose), and 2.9 x 10(6) particles /cm(3) (high dose) for 6 h/day in an inhalation chamber for 90 days. The lung function was measured every week after the daily exposure, and the animals sacrificed after the 90-day exposure period. Cellular differential counts and inflammatory measurements, such as albumin, lactate dehydrogenase (LDH), and total protein, were also monitored in the acellular bronchoalveolar lavage (BAL) fluid of the rats exposed to the silver nanoparticles for 90 days. Among the lung function test measurements, the tidal volume and minute volume showed a statistically significant decrease during the 90 days of silver nanoparticle exposure. Although no statistically significant differences were found in the cellular differential counts, the inflammation measurements increased in the high-dose female rats. Meanwhile, histopathological examinations indicated dose-dependent increases in lesions related to silver nanoparticle exposure, such as infiltrate mixed cell and chronic alveolar inflammation, including thickened alveolar walls and small granulomatous lesions. Therefore, when taken together, the decreases in the tidal volume and minute volume and other inflammatory responses after prolonged exposure to silver nanoparticles would seem to indicate that nanosized particle inhalation exposure can induce lung function changes, along with inflammation, at much lower mass dose concentrations when compared to submicrometer particles.     

Workshop summary: phosgene-induced pulmonary toxicity revisited: appraisal of early and late markers of pulmonary injury from animal models with emphasis on human significance

A workshop was held February 14, 2007, in Arlington, VA, under the auspices of the Phosgene Panel of the American Chemistry Council. The objective of this workshop was to convene inhalation toxicologists and medical experts from academia, industry and regulatory authorities to critically discuss past and recent inhalation studies of phosgene in controlled animal models. This included presentations addressing the benefits and limitations of rodent (mice, rats) and nonrodent (dogs) species to study concentration x time (C x t) relationships of acute and chronic types of pulmonary changes. Toxicological endpoints focused on the primary pulmonary effects associated with the acute inhalation exposure to phosgene gas and responses secondary to injury. A consensus was reached that the phosgene-induced increased pulmonary extravasation of fluid and protein can suitably be probed by bronchoalveolar lavage (BAL) techniques. BAL fluid analyses rank among the most sensitive methods to detect phosgene-induced noncardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum protein concentrations in BAL fluid occurred within 1 day after exposure, typically followed by a latency period up to about 15 h, which is reciprocal to the C x t exposure relationship. The C x t relationship was constant over a wide range of concentrations and single exposure durations. Following intermittent, repeated exposures of fixed duration, increased tolerance to recurrent exposures occurred. For such exposure regimens, chronic effects appear to be clearly dependent on the concentration rather than the cumulative concentration x time relationship. The threshold C x t product based on an increased BAL fluid protein following single exposure was essentially identical to the respective C x t product following subchronic exposure of rats based on increased pulmonary collagen and influx of inflammatory cells. Thus, the chronic outcome appears to be contingent upon the acute pulmonary threshold dose. Exposure concentrations high enough to elicit an increased acute extravasation of plasma constituents into the alveolus may also be associated with surfactant dysfunction, intra-alveolar accumulation of fibrin and collagen, and increased recruitment and activation of inflammatory cells. Although the exact mechanisms of toxicity have not yet been completely elucidated, consensus was reached that the acute pulmonary toxicity of phosgene gas is consistent with a simple, irritant mode of action at the site of its initial deposition/retention. The acute concentration x time mortality relationship of phosgene gas in rats is extremely steep, which is typical for a local, directly acting pulmonary irritant gas. Due to the high lipophilicity of phosgene gas, it efficiently penetrates the lower respiratory tract. Indeed, more recent published evidence from animals or humans has not revealed appreciable irritant responses in central and upper airways, unless exposure was to almost lethal concentrations. The comparison of acute inhalation studies in rats and dogs with focus on changes in BAL fluid constituents demonstrates that dogs are approximately three to four times less susceptible to phosgene than rats under methodologically similar conditions. There are data to suggest that the dog may be useful particularly for the study of mechanisms associated with the acute extravasation of plasma constituents because of its size and general morphology and physiology of the lung as well as its oronasal breathing patterns. However, the study of the long-term sequelae of acute effects is experimentally markedly more demanding in dogs as compared to rats, precluding the dog model to be applied on a routine base. The striking similarity of threshold concentrations from single exposure (increased protein in BAL fluid) and repeated-exposure 3-mo inhalation studies (increased pulmonary collagen deposition) in rats supports the notion that chronic changes depend on acute threshold mechanisms.     

Two-week inhalation toxicity of polymeric diphenylmethane-4, 4'-diisocyanate (PMDI) in rats: analysis of biochemical and morphological markers of early pulmonary response

The pulmonary response of Wistar rats to respirable polymeric diphenylmethane-4,4'-diisocyanate (PMDI) aerosol was examined in a 2-wk repeated nose-only inhalation exposure study. Exposure concentrations were 1.1, 3.3, and 13.7 mg PMDI/m(3) (6 h/day, 15 exposures). The level of 13.7 mg/m(3) was actually a combination of an initial target concentration of 10 mg/m(3) in wk 1, which was raised to 16 mg/m(3) in wk 2, due to a lack of signs suggestive of pulmonary irritation. An acute sensory irritation study on rats served as basis for selection of these concentrations. Shortly after the 2-wk exposure period, rats were subjected to pulmonary function and arterial blood gas measurements. Lungs were examined by light and transmission electron microscopy, and labeling indices in terminal bronchioles were measured. Bronchoalveolar lavage (BAL) was performed to assess various indicators of pulmonary inflammation, including neutrophil and macrophage numbers, protein, lactate dehydrogenase (LDH), gamma-glutamyltranspeptidase (gamma-GT), alkaline phosphatase (APh), acid phosphatase (ACPh), and beta-N-acetylglucosaminidase (beta-NAG). Phosphatidylcholine in BAL fluid and BAL cells was determined as aggregated endpoint suggestive of changes in pulmonary surfactant. Rats exposed to 3.3 and 13.7 mg/m(3) experienced concentration-dependent signs of respiratory tract irritation. Determination of arterial blood gases, lung mechanics, and carbon monoxide diffusing capacity did not demonstrate specific effects. Analysis of BAL fluid and BAL cells revealed changes indicative of marked inflammatory response and/or cytotoxicity in rats exposed to 13.7 mg/m(3), and the changes were characterized by statistically significantly increased activities of LDH, beta-NAG, and protein. Phospholipid concentrations were increased in rats exposed to 1.1 mg/m(3) and above (elevated levels of lipid material in alveolar macrophages demonstrated by polychrome stain) and 3.3 mg/m(3) and above (increased intracellular ACPh activity and intracellular phospholipids). In these groups, gamma-GT was statistically significantly increased. These findings suggest that changes in phospholipid homeostasis appear to occur at lower levels than those eliciting inflammation and cytotoxicity. Light and transmission electron microscopy suggest that exposure to 3.3 and 13. 7 mg/m(3) resulted in focal inflammatory lesions and an accumulation of refractile, yellowish-brownish material in alveolar macrophages with concomitant activation of type II pneumocytes. In the terminal bronchioles a concentration-dependent increase of bromodeoxyuridine (BrdU)-labeled epithelial cells was observed in all PMDI exposure groups. In summary, it appears that respirable PMDI aerosol interacts with pulmonary surfactant, which, in turn, may stimulate type II pneumocytes to increase their production of surfactant and to proliferate.     

Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats.

The aim of this study was to evaluate the acute lung toxicity of intratracheally instilled single-wall carbon nanotubes (SWCNT) in rats. The lungs of rats were instilled either with 1 or 5 mg/kg of the following control or particle types: (1) SWCNT, (2) quartz particles (positive control), (3) carbonyl iron particles (negative control), (4) phosphate-buffered saline (PBS) + 1% Tween 80, or (5) graphite particles (lung tissue studies only). Following exposures, the lungs of PBS and particle-exposed rats were assessed using bronchoalveolar lavage (BAL) fluid biomarkers and cell proliferation methods, and by histopathological evaluation of lung tissue at 24 h, 1 week, 1 month, and 3 months postinstillation. Exposures to high-dose (5 mg/kg) SWCNT produced mortality in ~15% of the SWCNT-instilled rats within 24 h postinstillation. This mortality resulted from mechanical blockage of the upper airways by the instillate and was not due to inherent pulmonary toxicity of the instilled SWCNT particulate. Exposures to quartz particles produced significant increases versus controls in pulmonary inflammation, cytotoxicity, and lung cell parenchymal cell proliferation indices. Exposures to SWCNT produced transient inflammatory and cell injury effects. Results from the lung histopathology component of the study indicated that pulmonary exposures to quartz particles (5 mg/kg) produced dose-dependent inflammatory responses, concomitant with foamy alveolar macrophage accumulation and lung tissue thickening at the sites of normal particle deposition. Pulmonary exposures to carbonyl iron or graphite particles produced no significant adverse effects. Pulmonary exposures to SWCNT in rats produced a non-dose-dependent series of multifocal granulomas, which were evidence of a foreign tissue body reaction and were nonuniform in distribution and not progressive beyond 1 month postexposure (pe). The observation of SWCNT-induced multifocal granulomas is inconsistent with the following: (1) lack of lung toxicity by assessing lavage parameters, (2) lack of lung toxicity by measuring cell proliferation parameters, (3) an apparent lack of a dose response relationship, (4) nonuniform distribution of lesions, (5) the paradigm of dust-related lung toxicity effects, (6) possible regression of effects over time. In addition, the results of two recent exposure assessment studies indicate very low aerosol SWCNT exposures at the workplace. Thus, the physiological relevance of these findings should ultimately be determined by conducting an inhalation toxicity study.     

TWO-WEEK INHALATION TOXICITY OF POLYMERIC DIPHENYLMETHANE-4,4'-DIISOCYANATE (PMDI) IN RATS: Analysis of Biochemical and Morphological Markers of Early Pulmonary Response

The pulmonary response of Wistar rats to respirable polymeric diphenylmethane-4,4'-diisocyanate (PMDI) aerosol was examined in a 2-wk repeated nose-only inhalation exposure study. Exposure concentrations were 1.1, 3.3, and 13.7 mg PMDI/m³ (6 h/day, 15 exposures). The level of 13.7 mg/m³ was actually a combination of an initial target concentration of 10 mg/m³ in wk 1, which was raised to 16 mg/m³ in wk 2, due to a lack of signs suggestive of pulmonary irritation. An acute sensory irritation study on rats served as basis for selection of these concentrations. Shortly after the 2-wk exposure period, rats were subjected to pulmonary function and arterial blood gas measurements. Lungs were examined by light and transmission electron microscopy, and labeling indices in terminal bronchioles were measured. Bronchoalveolar lavage (BAL) was performed to assess various indicators of pulmonary inflammation, including neutrophil and macrophage numbers, protein, lactate dehydrogenase (LDH), gamma-glutamyltranspeptidase (gamma-GT), alkaline phosphatase (APh), acid phosphatase (ACPh), and beta-N-acetylglucosaminidase (beta-NAG). Phosphatidylcholine in BAL fluid and BAL cells was determined as aggregated endpoint suggestive of changes in pulmonary surfactant. Rats exposed to 3.3 and 13.7 mg/m³ experienced concentration-dependent signs of respiratory tract irritation. Determination of arterial blood gases, lung mechanics, and carbon monoxide diffusing capacity did not demonstrate specific effects. Analysis of BAL fluid and BAL cells revealed changes indicative of marked inflammatory response and/or cytotoxicity in rats exposed to 13.7 mg/m³, and the changes were characterized by statistically significantly increased activities of LDH, beta-NAG, and protein. Phospholipid concentrations were increased in rats exposed to 1.1 mg/m³ and above (elevated levels of lipid material in alveolar macrophages demonstrated by polychrome stain) and 3.3 mg/m³ and above (increased intracellular ACPh activity and intracellular phospholipids). In these groups, gamma-GT was statistically significantly increased. These findings suggest that changes in phospholipid homeostasis appear to occur at lower levels than those eliciting inflammation and cytotoxicity. Light and transmission electron microscopy suggest that exposure to 3.3 and 13.7 mg/m³ resulted in focal inflammatory lesions and an accumulation of refractile, yellowish-brownish material in alveolar macrophages with concomitant activation of type II pneumocytes. In the terminal bronchioles a concentration-dependent increase of bromodeoxyuridine (BrdU)-labeled epithelial cells was observed in all PMDI exposure groups. In summary, it appears that respirable PMDI aerosol interacts with pulmonary surfactant, which, in turn, may stimulate type II pneumocytes to increase their production of surfactant and to proliferate.     

Workshop Summary: Phosgene-Induced Pulmonary Toxicity Revisited: Appraisal of Early and Late Markers of Pulmonary Injury From Animal Models With Emphasis on Human Significance

A workshop was held February 14, 2007, in Arlington, VA, under the auspices of the Phosgene Panel of the American Chemistry Council. The objective of this workshop was to convene inhalation toxicologists and medical experts from academia, industry and regulatory authorities to critically discuss past and recent inhalation studies of phosgene in controlled animal models. This included presentations addressing the benefits and limitations of rodent (mice, rats) and nonrodent (dogs) species to study concentration × time (C × t) relationships of acute and chronic types of pulmonary changes. Toxicological endpoints focused on the primary pulmonary effects associated with the acute inhalation exposure to phosgene gas and responses secondary to injury. A consensus was reached that the phosgene-induced increased pulmonary extravasation of fluid and protein can suitably be probed by bronchoalveolar lavage (BAL) techniques. BAL fluid analyses rank among the most sensitive methods to detect phosgene-induced noncardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum protein concentrations in BAL fluid occurred within 1 day after exposure, typically followed by a latency period up to about 15 h, which is reciprocal to the C × t exposure relationship. The C × t relationship was constant over a wide range of concentrations and single exposure durations. Following intermittent, repeated exposures of fixed duration, increased tolerance to recurrent exposures occurred. For such exposure regimens, chronic effects appear to be clearly dependent on the concentration rather than the cumulative concentration × time relationship. The threshold C × t product based on an increased BAL fluid protein following single exposure was essentially identical to the respective C × t product following subchronic exposure of rats based on increased pulmonary collagen and influx of inflammatory cells. Thus, the chronic outcome appears to be contingent upon the acute pulmonary threshold dose. Exposure concentrations high enough to elicit an increased acute extravasation of plasma constituents into the alveolus may also be associated with surfactant dysfunction, intra-alveolar accumulation of fibrin and collagen, and increased recruitment and activation of inflammatory cells. Although the exact mechanisms of toxicity have not yet been completely elucidated, consensus was reached that the acute pulmonary toxicity of phosgene gas is consistent with a simple, irritant mode of action at the site of its initial deposition/retention. The acute concentration × time mortality relationship of phosgene gas in rats is extremely steep, which is typical for a local, directly acting pulmonary irritant gas. Due to the high lipophilicity of phosgene gas, it efficiently penetrates the lower respiratory tract. Indeed, more recent published evidence from animals or humans has not revealed appreciable irritant responses in central and upper airways, unless exposure was to almost lethal concentrations. The comparison of acute inhalation studies in rats and dogs with focus on changes in BAL fluid constituents demonstrates that dogs are approximately three to four times less susceptible to phosgene than rats under methodologically similar conditions. There are data to suggest that the dog may be useful particularly for the study of mechanisms associated with the acute extravasation of plasma constituents because of its size and general morphology and physiology of the lung as well as its oronasal breathing patterns. However, the study of the long-term sequelae of acute effects is experimentally markedly more demanding in dogs as compared to rats, precluding the dog model to be applied on a routine base. The striking similarity of threshold concentrations from single exposure (increased protein in BAL fluid) and repeated-exposure 3-mo inhalation studies (increased pulmonary collagen deposition) in rats supports the notion that chronic changes depend on acute threshold mechanisms.     

Pulmonary toxicity screening studies in male rats with M5 respirable fibers and particulates

M5 fiber is a high-strength, high-performance organic fiber type that is a rigid rod material and composed of heterocyclic polymer fibers of type PIPD. The aim of this study was to evaluate the acute lung toxicity of intratracheally instilled M5 respirable fibers and particulates in rats. Using a pulmonary bioassay and bridging methodology, the acute lung toxicity of intratracheally instilled M5 particulates and that of its fibers were compared with a positive control particle type, quartz, as well as a negative control particle type, carbonyl iron particles. Moreover, the results of these instillation studies were bridged with data previously generated from inhalation studies with quartz and carbonyl iron particles, using the quartz and iron particles as the inhalation/instillation bridge material. For the bioassay experimental design, in the bronchoalveolar lavage studies, the lungs of rats were intratracheally instilled with 0.5 or 0.75 mg/kg of M5 particulate or 1 or 5 mg/kg of the following control or particle types: (1) M5 long fiber preparation, (2) silica-quartz particles, and (3) carbonyl iron particles. Phosphate-buffered saline (PBS)-instilled rats served as additional controls. Following exposures, the lungs of PBS and particle-exposed rats were assessed using bronchoalveolar lavage (BAL) fluid biomarkers, cell proliferation methods, and histopathological evaluation of lung tissue at 24 h, 1 wk, 1 mo and 3 mo post instillation exposure. The bronchoalveolar lavage results demonstrated that lung exposures to quartz particles, at both concentrations but particularly at the higher dose, produced significant increases vs. controls in pulmonary inflammation and cytotoxicity indices. Exposures to M5 particulate and M5 long fiber preparation produced transient inflammatory and cell injury effects at 24 h postexposure (pe) as well as at 24 h and 1 wk pe, respectively, but these effects were not sustained when compared to quartz-silica effects. Exposures to carbonyl iron particles and PBS resulted in only minor short-term and reversible lung inflammation, likely related to the effects of the instillation procedure. Histopathological analyses of lung tissues revealed that pulmonary exposures to M5 particulate and in particular, the M5 long fiber preparation in rats produced some inflammatory responses, observed up to 1 wk postexposure. These responses were often associated with the presence of M5 long fiber in the airways or in the proximal alveolar regions but appeared to be reversible at 1 and 3 mo postexposure. In contrast, pulmonary exposures to silica-quartz particles in rats produced a dose-dependent lung inflammatory response characterized by neutrophils and foamy (lipid-containing) alveolar macrophage accumulation and evidence of early lung tissue thickening consistent with the development of pulmonary fibrosis. Based on our results, we conclude the following: (1) It was very difficult to produce M5 fibers into a respirable fibrous form; these findings suggest that aerosol exposure concentrations of respirable fibrous M5 in the workplace are likely to be rather low. (2) The particulate and long fiber preparations of M5 that were tested produced a moderate amount of pulmonary inflammatory activity, more active than our negative control, carbonyl iron particles, but substantially less active in terms of inflammation, cytotoxicity, and fibrogenic effects than the positive control particle type, silica-quartz particles. Thus, based on the results of this study, we would expect that inhaled M5 respirable fibers have a low risk potential for producing adverse pulmonary effects.     

Effects of sulfuric acid mist inhalation on mucous clearance and on airway fluids of rats and guinea pigs

The responses of guinea pigs and rats to inhaled sulfuric acid aerosols were compared to define species differences and to determine the small-animal model most relevant to human exposures. Rats were exposed for 6 h to 1, 10, and 100 mg H2SO4/m3. Guinea pigs were exposed for 6 h to 1, 10, and 27 mg H2SO4/m3. Tracheal mucous clearance of guinea pigs was slowed 1 d after exposures to 1 mg H2SO4/m3. A tendency toward faster clearance was observed at high concentrations of H2SO4 for both guinea pigs and rats (statistically significant only for the rats). The speeding of mucous clearance was correlated with increases in airway sialic acid and also with the appearance of excess tracheal secretions, detected using scanning electron microscopy in both rats and guinea pigs. The responses of guinea pigs to sulfuric acid exposures were more similar to those reported for humans than were those of rats.     

Respiratory toxicity and immunotoxicity evaluations of microparticle and nanoparticle C60 fullerene aggregates in mice and rats following nose-only inhalation for 13 weeks

C60 fullerene (C60), or buckminsterfullerene, is a spherical arrangement of 60 carbon atoms, having a diameter of approximately 1?nm, and is produced naturally as a by-product of combustion. Due to its small size, C60 has attracted much attention for use in a variety of applications; however, insufficient information is available regarding its toxicological effects. The effects on respiratory toxicity and immunotoxicity of C60 aggregates (50?nm [nano-C60] and 1?μm [micro-C60] diameter) were examined in B6C3F1/N mice and Wistar Han rats after nose-only inhalation for 13 weeks. Exposure concentrations were selected to allow for data evaluations using both mass-based and particle surface area-based exposure metrics. Nano-C60 exposure levels selected were 0.5 and 2?mg/m³ (0.033 and 0.112 m²/m³), while micro-C60 exposures were 2, 15 and 30?mg/m³ (0.011, 0.084 and 0.167 m²/m³). There were no systemic effects on innate, cell-mediated, or humoral immune function. Pulmonary inflammatory responses (histiocytic infiltration, macrophage pigmentation, chronic inflammation) were concentration-dependent and corresponded to increases in monocyte chemoattractant protein (MCP)-1 (rats) and macrophage inflammatory protein (MIP)-1α (mice) in bronchoalveolar lavage (BAL) fluid. Lung overload may have contributed to the pulmonary inflammatory responses observed following nano-C60 exposure at 2?mg/m³ and micro-C60 exposure at 30?mg/m³. Phenotype shifts in cells recovered from the BAL were also observed in all C60-exposed rats, regardless of the level of exposure. Overall, more severe pulmonary effects were observed for nano-C60 than for micro-C60 for mass-based exposure comparisons. However, for surface-area-based exposures, more severe pulmonary effects were observed for micro-C60 than for nano-C60, highlighting the importance of dosimetry when evaluating toxicity between nano- and microparticles.     

Comparative study of ozone (O3) uptake in three strains of rats and in the guinea pig

Ozone uptake was assessed in awake, spontaneously breathing Fischer-344 Sprague-Dawley, and Long-Evans rats and Hartley guinea pigs to provide data on the dosimetry of O3 in small laboratory animals. This information is needed for extrapolation of O3 toxicity data from experimental animals to man. Breathing measurements and O3 exposure data were obtained using a head-out body plethysmograph connected to a nose-only exposure system. The fractional uptake of O3 was computed by measuring flow and O3 concentration both upstream and downstream from the nose. The quantity of O3 removed by the animal, O2 consumption, CO2 production, and tidal breathing measurements were automatically recorded once each minute. All animal types were exposed for 1 hr to 0.3 ppm O3. Other Fischer-344 rats were also exposed for 1 hr to 0.0 or to 0.6 ppm O3. Exposure concentrations of O3 had no significant effect on percentage O3 uptake in Fischer-344 rats. Results showed that percentage O3 uptake (47%) did not differ significantly among the three strains of rats nor between the rats and the guinea pigs. Similarly, percentage O3 uptake was independent of animal age, lung weight, and lung volume as well as normal variations encountered in the resting breathing measures. However, species-specific ventilation and O3 concentration were the primary determinants of the accumulated lung dose of O3 during the exposures.