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.

Effects of low levels of NO2 on terminal bronchiolar cells and its relative toxicity compared to O3.

This report describes structural changes occurring in the terminal bronchioles of rats exposed to low levels of NO2 continuously for 6 weeks. In addition, the relative susceptibility of epithelial cells to oxidants and the comparative toxicity of NO2 and O3 are discussed. Terminal bronchioles isolated from rats exposed 5 days/week to 2.0 ppm NO2 (plus two 1-hr daily spikes to 6.0 ppm) were found to have 19% less ciliated cells per unit area of epithelial basement membrane. The remaining ciliated cells had a reduced mean surface area (-29%). The shape of the Clara cell changed with reduced size of the dome protrusions but increased cell contact with the basement membrane. These data indicate that exposure to 2.0 ppm NO2 (+ spikes) for 6 weeks caused injuries to cilia and ciliated cells and possible Clara cell differentiation in the terminal bronchioles of adult rats. Exposures of adult or juvenile rats to 0.5 ppm NO2 (+ two 1-hr daily spikes 5 days/week to 1.5 ppm) did not cause morphologically measurable injuries in the terminal bronchioles. The severity of the concentration-dependent epithelial cell reactions to NO2 and O3 in adult rat terminal bronchioles were compared to those occurring in the proximal alveolar regions (PAR). Epithelial cells in the PAR appeared to be more susceptible to oxidant insult since both 0.5 ppm NO2 and 0.25 ppm O3 were found to cause epithelial injury only in the PAR. Comparison of epithelial reactions to 6-week exposures to either NO2 or O3 indicated that 0.25 ppm O3 caused four times as much increase in the number of type I epithelial cells as did 2 ppm (+spikes) NO2. Therefore, O3 could be 40 times more toxic than NO2 in the PAR on the basis of the inspired concentration and the focal response. On the other hand, there was no loss of ciliated cells following the 0.25 ppm O3 exposure. This suggests that the ratio of O3 to NO2 toxicity in the terminal bronchioles is considerably less than 10. The relative toxicity of the two oxidant gases appears to be site specific.

Effect of dietary vitamin E level on the biochemical response of rat lung to ozone inhalation.

We examined the effects of dietary vitamin E level on rat lung response to ozone (O3) inhalation. In one study, we fed 1-month-old Sprague-Dawley (SD) rats a test diet containing 0 or 50 IU vitamin E/kg for 2 months, and then exposed one-half of the animals from each dietary group to 0.8 ppm (1,568 micrograms/m3) O3 intermittently (8 hours daily) and the other half to room air for 7 days. After O3 exposure, we found significant increases in marker enzyme activities in rat lungs from both dietary groups relative to corresponding air-exposed controls, but the magnitude of increases was greater for the 0 IU than the 50 IU group. In another study, we fed 1-month-old SD rats a test diet containing 10, 50, or 500 IU vitamin E/kg for 2 months and then exposed one-half of the animals from each dietary group to 0.8 ppm (1,568 micrograms/m3) O3 continuously and the other half to room air for 4 days. The O3 exposure increased the metabolic activities in rat lungs from all three dietary groups relative to corresponding air-exposed controls, but the magnitude of increases was greater for the 10 IU than the 50 IU or 500 IU group, and the difference between the 50 IU and 500 IU groups was small. Because a greater increase in lung metabolism after O3 exposure is thought to be associated with a greater tissue injury, the results suggest that an absence of dietary vitamin E exacerbates lung injury from O3 inhalation, while its presence protects from injury. However, the magnitude of this protective effect does not increase proportionately with increased dietary vitamin E supplementation beyond a certain level.

Health risk analysis of human exposures to soil amended with sewage sludge contaminated with polychlorinated dibenzodioxins and dibenzofurans.

The risk of cancer to humans exposed to soil treated with wastewater/sewage treatment plant sludge, known to be contaminated with small amounts of polychlorinated dibenzodioxins and dibenzofurans (PCDDs and PCDFs), was evaluated. The particulate-bound PCDDs and PCDFs are found in trace amounts in the effluent from ground water pumping (dewatering) at an abandoned wood preservation facility. The water, which was sent to a water recovery plant, underwent primary and secondary treatment prior to discharge. The residual sludge was added to agricultural soil as a conditioner. The present analysis treats the extreme case of sludge applied near the home of a target individual, a lifetime resident, who is also a farm worker in the area of the application. The successive stages of infancy, childhood and adulthood are treated separately to assess the contributions of typical age-specific indoor and outdoor activities on exposure rates. Five toxicity rating schemes using so-called TCDD equivalents, and two unit risk slopes are applied to the chemical profile in sludge to determine the cancer potency of the soil contaminants. These risk estimates range from 1 X 10-8 to 3 X 10-7.

Effects of subchronic inhalation of low concentrations of nitrogen dioxide. I. The proximal alveolar region of juvenile and adult rats.

Inhalation of nitrogen dioxide (NO2) produces injury to the epithelium of terminal airways and the alveoli proximal to the airways. Techniques were devised to isolate alveolar tissue from this region for morphometric studies to define the extent of alveolar septal injury caused by NO2. One-day-old and six-week-old rats were exposed to either room air or 0.5 ppm NO2 for 23 hr per day 7 days per week for 6 weeks. An additional group of 6-week-old rats were exposed to 2.0 ppm NO2 for the same duration. Two daily hour spikes to three times the background concentrations (0.5 to 1.5 ppm and 2.0 to 6.0 ppm) were applied Monday through Friday. At the end of the exposure, rat lungs were fixed by intratracheally infusing buffered 2% glutaraldehyde. Pieces of lung tissue were embedded in large plastic blocks which were softened with heat and thin (0.3 mm) sliced. Terminal bronchioles and their corresponding proximal alveolar regions were identified from the thin plastic slices, removed, and glued to cylindrical EM blocks for thin sectioning. Morphometric analysis revealed that epithelial injury occurred in all exposed animals. The juvenile rats which had been exposed to 0.5 ppm NO2 since 1 day of age exhibited changes in the characteristics of type II epithelial cells. These cells spread to cover more alveolar surface and became thinner. Adult animals exposed to 0.5 and 2.0 ppm NO2 showed changes in alveolar macrophages and in the alveolar interstitium in addition to changes in the epithelium. Animals exposed to 0.5 ppm NO2 showed spreading and hypertrophy of type II epithelial cells. Those animals exposed to the higher concentration of NO2 had similar changes in type II epithelial cells and in addition showed an increase in type I cell number. The type I epithelial cells were smaller and covered less alveolar surface area than normal type I cells, suggesting a regenerating population of type I cells. These results suggest that prolonged exposure to low concentrations of NO2 can cause injury to the alveolar epithelium indicated initially by spreading and hypertrophy of type II cells followed by differentiation into type I cells to compensate and repair the injury. Adult rats were as sensitive or more sensitive to NO2 injury than were juvenile rats.

Influence of age on the biochemical response of rat lung to ozone exposure.

We have previously examined the influence of animal age on the pulmonary response to ozone (O3) in rats between 7 and 90 days of age (Elsayed et al., 1982a). In the present study, we expanded the age groups of rats, and examined in greater detail the relationship between animal age and pulmonary response to inhaled O3. We exposed 7 groups of specific pathogen free, male Sprague-Dawley rats, aged 24, 30, 45, 60, 90, 180, and 365 days, to 0.8 ppm (1568 micrograms/m3) O3 continuously for 3 days. After O3 exposure, we sacrificed the exposed rats and a matched number of controls from each age group, and analyzed their lungs for a series of physical and biochemical parameters, including glutathione metabolizing and NADPH producing enzyme activities. We observed that in control rats all the parameters increased as a function of age. However, the rate of increase was generally slower after age 60 days. After O3 exposure there was an increase in all the parameters for all age groups relative to their corresponding controls, but the extent of increase was significantly larger in rats 60 days and older than in younger rats. A regression of the difference in mean values between control and exposed animals for each parameter against age showed a linear correlation, indicating that the response was age-dependent. Since the magnitude of such increases is thought to reflect the degree of lung injury, the results suggest that O3 exposure causes greater lung injury in older rats than in younger rats. We tested this assumption by exposing rats from four different age groups (24, 45, 60 and 90 days) to a lethal dose of O3 (4 ppm or 7840 micrograms/m3 for 8 hours). The mortality rates were 50% and 83% for 24 and 45 day old rats, respectively, and 100% for 60 and 90 day old rats. The results of these studies further demonstrate that older rats are more susceptible to lung injury from O3 than younger rats.

Poor correlation between oxygen toxicity and activity of glutathione peroxidase.

To test the postulate that increased activity of the glutathione peroxidase system is required for the increased tolerance to oxygen toxicity that develops after several days of prior exposure to 85% oxygen we searched for a combination of increased tolerance but normal activity of the glutathione system. We exposed rats to 85% oxygen for 7 days and then placed them in air. After 0, 10, 20, and 30 days we estimated the potential role of an antioxidant system by measuring activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GR), glutathione peroxidase (GP), and the nonprotein sulfhydryl content (NPSH) of lung tissue. After 7 days in 85% oxygen (0 days in air) activities of G6PD, GR, and GP, were elevated above control values by 189%, 32%, and 126%, respectively, and NPSH was 146% higher. Twenty days later these activities and NPSH were not significantly different from those of control animals never exposed to 85% oxygen. We also tested these rats without increased enzyme activities for oxygen "tolerance" by exposing them, after 20 days of recovery in air, to 100% oxygen for 3 days and found that some were "tolerant" as judged by a mortality rate of only 42% compared with 100% in a group not previously exposed to oxygen. To determine if this degree of tolerance could be related to accelerated increases of enzyme activities during the exposures we measured the enzyme activities and NPSH of lungs at 12, 24, 36, 48 and 60 hr after the start of exposure to 100% oxygen in two groups: one preexposed to 85% oxygen 20 days earlier and the other not previously exposed to oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical changes in rat lungs after exposure to nitrogen dioxide.

Sixty-day-old male, specific pathogen-free rats were exposed continuously to 5 or 15 ppm NO2 for 1-7 d. Lung tissue from exposed and control rats was then analyzed for biochemical and enzymatic parameters. The exposure resulted in increased lung enzymatic activities, including elevated protein and DNA contents and nonprotein sulfhydryl levels. Biochemical and enzymatic parameters generally increased maximally after 4 d and remained elevated for up to 7 d of continued exposure. The magnitude of these increases was higher for 15 than for 4 ppm NO2. The increases in biochemical and enzymatic parameters may have occurred in response to NO2-induced lung injury.