Elaine C. Grose

Epithelial injury and interstitial fibrosis in the proximal alveolar regions of rats chronically exposed to a simulated pattern of urban ambient ozone

Abstract Electron microcopic morphometry was used to study the development of lung injury during and after chronic (78 weeks) exposure to a pattern of ozone (O3) designed to simulate high urban ambient concentrations that occur in some environments. The daily exposure regimen consisted of a 13-hr background of 0.06 ppm, an exposure peak that rose from 0.06 to 0.25 ppm, and returned to the background level over a 9-hr period, and 2-hr downtime for maintenance. Rats were exposed for 1, 3, 13, and 78 weeks. Additional groups of rats exposed for 13 or 78 weeks were allowed to recover in filtered clean air for 6 or 17 weeks, respectively. Rats exposed to filtered air for the same lengths of time were used as controls. Samples from proximal alveolar regions and terminal bronchioles were obtained by microdissection. Analysis of the proximal alveolar region revealed a biphasic response. Acute tissue reactions after 1 week of exposure included epithelial inflammation, interstitial edema, interstitial cell hypertrophy, and influx of macrophages. These responses subsided after 3 weeks of exposure. Progressive epithelial and interstitial tissue responses developed with prolonged exposure and included epithelial hyperplasia, fibroblast proliferation, and interstitial matrix accumulation. The epithelial responses involved both type I and type II epithelial cells. Alveolar type I cells increased in number, became thicker, and covered a smaller average surface area. These changes persisted throughout the entire exposure and did not change during the recovery pefiod, indicating the sensitivity of these cells to injury. The main response of type II epithelial cells was cell proliferation. The accumulation of interstitial matrix after chronic exposure consisted of deposition of both increased amounts of basement membrane and collagen fibers. Interstitial matrix accumulation underwent partial recovery during follow-up periods in air; however, the thickening of the basement membrane did not resolve. Analysis of terminal bronchioles showed that short-term exposure to O3 caused a loss of ciliated cells and differentiation of preciliated and Clara cells. The bronchiolar cell population stabilized on continued exposure; however, chronic exposure resulted in structural changes, suggesting injury to both ciliated and Clara cells. We conclude that chronic exposure to low levels of O3 causes epithelial inflammation and interstitial fibrosis in the proximal alveolar region and bronchiolar epithelial cell injury.

A comparative study of the effects of inhaled cadmium chloride and cadmium oxide: Pulmonary response

The effects of aerosols of cadmium chloride (CdCl2) and cadmium oxide (CdO) on pulmonary biochemical function were compared. Rats and rabbits were exposed to 0.25, 0.45, or 4.5 mg Cd/m3 for 2 h. Pulmonary toxicity was determined histologically and biochemically. Cadmium chloride and CdO showed a deposition response that was linearly related to the chamber concentration. Both compounds caused multifocal, interstitial pneumonitis 72 h after exposure, but the CdO lesion was more severe with proliferation of fibrocytic-like cells as well as pneumocytes. Comparing the two Cd compounds at the highest concentration (4.5 mg Cd/m3), the biochemical responses in the rat were similar. The majority of the effects occurred 72 h after exposure, with significant increases in lung weight, lung-to-body weight ratio, GSH reductase, GSH transferase, and G-6-PDH. However, GSH peroxidase was inhibited immediately after the CdO exposure. Cadmium oxide-related alterations in the parameters studied could easily be distinguished from those of CdCl2 at the exposure concentration of 0.45 mg Cd/m3. The response pattern in the rabbit resembled that of the rat. In both species Cd had a consistent inhibitory effect on pulmonary GSH peroxidase, even at the lowest concentration of 0.25 mg Cd/m3. Based on these findings, inhaled CdO appeared to be more toxic to the lung than inhaled CdCl2.

Response of ciliated epithelium to ozone and sulfuric acid.

The effects of ozone (O3), sulfuric acid (H2SO4), and their interaction on ciliary activity were investigated. Following in vivo exposure to various concentrations of O3 and H2SO4, ciliary activity of isolated tracheal ring cultures was microscopically determined under stroboscopic illumination. Assay of tracheal rings immediately after a 2-hr exposure to 880 μg H2SO4/m3 showed a significant decrease (P < 0.0001) in ciliary beating frequency from controls. Following 72 hr in vitro maintenance, there was still a significant depression (P < 0.01) in ciliary activity of treatment cultures. In vivo recovery studies indicated that ciliary activity had returned to the normal range 72 hr after exposure. Exposure to 196 μg O3/m3 for 3 hr resulted in no significant difference from controls in ciliary activity. Experiments designed to investigate the effects of a sequential exposure to O3 followed by H2SO4 indicated a significant decrease (P < 0.05) in the ciliary beating frequency of exposed animals which was less than that observed with H2SO4 alone. As indicated by the results of these studies, combined action experiments are extremely relevant in assessing the toxicity of environmental pollutants.

Alveolar epithelial cell injuries by subchronic exposure to low concentrations of ozone correlate with cumulative exposure

Electron microscopy morphometry has been used to study the effects of cumulative exposure of low levels of inhaled O3 on lung proximal alveolar tissue. Six-week-old Fisher 344 rats were exposed to O3 in two different subchronic low-level exposure patterns. The first was a 12 hr/day exposure for 6 weeks and included two O3 concentrations, 0.12 and 0.25 ppm. The second consisted of an exposure profile having a background level of 0.06 ppm with an exposure peak 5 days each week that went from 0.12 to 0.25 ppm and back to 0.12 ppm over a 9-hr period. Rats given the second exposure pattern were exposed for either 3 or 13 weeks. Changes in the volumes of alveolar epithelium were found to be consistent and reproducible markers for cell injury and/or response. Results from the first study indicated that the relative volume of the type I epithelium increased 13 and 23% over the control value (p less than 0.05) following exposures for 6 weeks to 0.12 and 0.25 ppm, respectively. The magnitude of the increases were clearly concentration related. Similarly, when a fixed exposure concentration was employed the relative volume of type I epithelium was found to increase in proportion to the exposure time. In the second exposure, increases of 9 and 33% in relative volume of type I epithelium were found respectively after 3 and 13 weeks of exposure. If the total exposure determined by the product of O3 concentration (including background) and exposure time is plotted against the relative volume of type I epithelium from both the 0.12 ppm (60.5 ppm-hr) and 0.25 ppm (126 ppm-hr) exposures and the 3-week (45.3 ppm-hrs) and 13-week (196.2 ppm-hr) exposures, a linear relationship between increases in type I cell volume and the concentration X time product is observed. The coefficient of correlation (r2) for the linear regression of the animal means is 0.72. Changes in the volume of Type II epithelial cell also correlate with the concentration X time product (r2 = 0.66). This suggests that epithelial cell reactions to low-level subchronic exposure of O3 are directly related to the cumulative oxidant concentration. The pattern of exposure did not appear to affect the resulting degree of injury. Furthermore, a low level of background exposure may contribute to the epithelial cell injuries.

Pulmonary host defense responses to inhalation of sulfuric acid and ozone

The effects of simultaneous exposure to ozone (O3) and sulfuric acid [H2SO4, 0.23 microns volume median diameter (VMD)] and a single exposure to ultrafine (less than 0.1 micron VMD) H2SO4 under various conditions were studied using the infectivity/mortality and the ciliary beating frequency model systems. A 3-h exposure to a combined aerosol of 196 micrograms O3/m3 and 483 or 241 micrograms H2SO4/m3 significantly increased the susceptibility of mice to a laboratory-induced respiratory infection. However, exposure to 543 micrograms ultrafine H2SO4/m3 for 2 h or 365 micrograms/m3 2 h/d for 5 d did not significantly affect this parameter. Upper airway response, as measured by changes in hamster tracheal ciliary beating frequency, was not affected by either a 3-h combined exposure to 196 micrograms O3/m3 and 847 micrograms H2SO4/m3 or a 2-h exposure to 458 micrograms ultrafine H2SO4/m3.

Evaluating the toxicity of urban patterns of oxidant gases. I. An automated chronic gaseous animal inhalation exposure facility

An automated animal inhalation exposure facility was designed to conduct chronic exposures of rodents to gaseous pollutants. The facility consisted of 3 walk-in chambers with modular cages to allow for exposure of up to a maximum of 480 mice or 240 adult rats. Critical parameters of operation, such as relative humidity, pollutant concentration, and temperature, were monitored continuously. Failure of the system to maintain parameters within specified limits activated the alarm system. Distribution of test gases in the chambers was evaluated for homogeneity, and the standard deviation was determined to be within +/- 5% of the target concentrations throughout the chamber. The exposure facility was successfully used to expose 720 rats to diurnal patterns of O3 (baseline of 0.06 ppm for 13 h with an exposure peak to 0.25 ppm over 9 h) and NO2 (baseline of 0.5 ppm for 16 h with an exposure peak to 1.5 ppm over 6 h) for a period of 18 mo.

Inhalation studies of Mt. St. Helens volcanic ash in animals. III. Host defense mechanisms.

The effects of inhalation exposure of mice or rats to 9.4 mg/m3 volcanic ash, 2.5 mg/m3 SO2, or both on host defense mechanisms were assessed. Cytologic changes in pulmonary lavage fluid included an increase in percentage polymorphonuclear leukocytes due to SO2 exposure and an increase in eosinophils due to ash. SO2 and ash also produced decreases in percentage alveolar macrophages. In the case of ash-exposed animals, this decrease was offset by an increase in lymphocytes. Total cell counts and viability were not affected by any of the exposures. Pulmonary clearance mechanisms were affected in that there were both decreased alveolar macrophage phagocytic capability following ash and ash + SO2 exposures and depressed ciliary beat frequency attributable to ash exposure. None of the inhalation exposures caused increases in susceptibility to an immediate or 24 hr postexposure aerosol challenge with Streptococcus. However, intratracheal instillation of both fine- and coarse-mode volcanic ash caused slight but significant increases in mortality due to bacterial challenge 24 hr after the instillation. The phytohemagglutinin-induced blastogenic response of splenic lymphocytes from exposed animals did not differ significantly from that of control lymphocytes, although the lipopolysaccharide-induced blastogenic response was enhanced. Ash exposure had no effect on susceptibility to murine cytomegalovirus. In summary, volcanic ash alone or in combination with SO2 had only minimal effects on certain host defense mechanisms.

Assessment of the hepatotoxicity of acute and short-term exposure to inhaled p-xylene in F-344 rats.

Due to the ubiquitous presence of p-xylene in air and the existing uncertainty regarding its hepatotoxic potential, we examined the effect of acute and short-term exposure to inhaled p-xylene on the liver. Male F-344 rats were exposed to 0 or to 1600 ppm p-xylene, 6 h/d, for 1 or 3 d. Exposure to inhaled p-xylene caused no histopathological evidence of hepatic damage and had little or no effect on the serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, ornithine carbamyl transferase, alkaline phosphatase, and total bilirubin. Exposure to p-xylene for 1 or 3 d resulted in an increase in relative liver weight on d 1 post-exposure. The concentration of hepatic cytochrome P-450 was increased by both p-xylene exposure regimens on d 1 postexposure and had returned to control levels by d 3 following the single p-xylene exposure and by d 2 following the 3-d exposure. These observations provide consistent evidence that acute and short-term exposure to 1600 ppm p-xylene by inhalation did not produce overt hepatotoxicity but resulted in a significant increase in the concentration of hepatic cytochrome P-450, the principal enzyme system involved in the metabolic biotransformation of xenobiotics.

Acute, Subchronic, and Chronic Exposure to a Simulated Urban Profile of Ozone: Effects on Extrapulmonary Natural Killer Cell Activity and Lymphocyte Mitogenic Responses

AbstractRats were exposed for 7, 3, 73, 52, or 78 wk to air or a simulated urban profile of O3 designed to mimic diurnal exposure patterns frequently seen in worst-case summer environments. Daily exposures consisted of a background level of 0.06 ppm for a period of 73 h, a broad exposure spike rising from 0.06 to 0.25 ppm and returning to 0.06 ppm over 9 h, and a 2 h downtime. Integration of the spike portion of the exposure pattern was equivalent to a 9-h square-wave exposure pattern of 0.79 ppm. Rats were exposed to this profile 5 days/wk; weekend exposures were to background levels only Spleens were removed and blood was drawn at the end of the exposure periods. Spleen cells were assessed for natural killer cell (NKC) activity and responses to T-cell mitogens, phytohemagglutinin and concanavalin A, and the B-cell mitogen, Salmonella typhimurium glycoprotein. Peripheral blood leukocytes (PSU were also assessed for responses to T-cell mitogens. Sections from spleen, femur (including bone marrow), thymus,...

The distribution and toxicological effects of inhaled methyl bromide in the rat

Sixty-day-old male CD rats were exposed by nose only to 14C-methyl bromide (55 ppm) for 3 min. The data indicated that the liver, lung, and kidney were the major organs of 14C distribution immediately after exposure. Up to 32 hr after exposure, the major routes of excretion were pulmonary (14CO2) and renal, with approximately 43% and 21% of the total inhaled radiolabel being eliminated, respectively. In separate experiments, 60-day-old CD male rats were exposed by whole body inhalation for 6 hr/day for 5 and 10 days to 30 ppm methyl bromide or filtered air. Glutathione (GSH) S-transferase and glucose-6-phosphate dehydrogenase (G-6-PDH) activities were increased in the lung. A decrease in GSH-reductase and GSH-S-transferase activities were found in the liver. Serum chemistries indicated a decrease in Cholinesterase, BUN, uric acid, and cholesterol, and leucine aminopeptidase exhibited an increase in activity. These data indicate that methyl bromide is quickly distributed to all tissues after inhalation and rapidly metabolized. A small percentage is cleared slowly and incorporated into metabolic pools, as evidenced by 25% of the initial dose of 14C-methyl bromide found in the rat 32 hr postexposure and the alterations found in hepatic enzyme activities.