Harold J. White

Functional and pathologic consequences of a 52-week exposure to 0.5 PPM ozone followed by a clean air recovery period

Male Fischer 344 rats were exposed to 0.5 ppm ozone for 20 hr/day, 7 days/week, for 52 weeks after which they were allowed to recover in clean filtered air for 12 weeks. Pulmonary function testing, which included measurements of lung volumes, expiratory air flows, and DLco, was performed before the initiation of exposure, after 26 and 52 weeks of exposure, and after the 12 week recovery. Control animals were tested at the same times but exposed only to clean filtered air. Another group, periodically sacrificed for histopathologic evaluation, was similarly exposed to ozone but allowed to recover in clean air for 24 weeks. The 52 weeks of ozone exposure produced small but statistically significant changes in several of the functional measurements when compared to clean air controls (FRC + 7.0%; RV + 11.2%; DLco - 7.3%). These measurements returned to control levels with 3 months of recovery. All other parameters showed no significantly different values between the 2 groups throughout the exposure and recovery periods. After both 6 and 12 months of ozone exposure, microscopic evaluation revealed a slight inflammatory response in the alveolar duct walls and septa of the immediately adjacent alveoli. This response included the accumulation of mononuclear cells and fibroblasts, thickening of alveolar septa, and a slight increase in macrophage population. With 6 months of recovery, the inflammation had all but disappeared. There remained only a slight dilation and thickening of an occasional alveolar duct and its adjacent alveoli. We conclude that the functional changes seen in the lungs in response to the ozone insult were the result of the observed inflammation in the distal areas of the lung, and the lesions produced were reversible to the extent that they could not be detected functionally after recovery.

Thymus and pulmonary lymph node response to acute and subchronic ozone inhalation in the mouse

Ozone is an oxidant gas which primarily injures the centroacinar portion of the lung. While the classical lesion of oxidant-mediated lung damage is relatively well described, the effect of this form of injury on the lymphocytic arm of the pulmonary defense system is less clear. In the present experiments we exposed CD-1 female mice to ozone at a level of 0.7 ppm for 20 hr per day for 1-28 days and observed the lymphocyte response in the pulmonary lymph nodes and the thymus. In the mediastinal lymph nodes we observed a marked hyperplastic response that was prominent in the paracortex and was characterized by the presence of blastic forms. In contrast, the thymus underwent an atrophic response characterized by cellular loss in the cortical region. Prior surgical adrenalectomy of ozone-exposed animals eliminated part, but not all of the thymic atrophy response, indicating that adrenal-mediated stress alone did not account for all of the observed effect. Thymectomy of animals prior to ozone exposure produced a 40% reduction in the mediastinal lymph node response, suggesting that a part of the node hyperplasia is thymus dependent. The results of these experiments indicate that lymphoid organs are altered following oxidant-mediated lung damage in the mouse. The changes are observed in the absence of exogenous antigenic stimulation and suggest that lymphoid cells are an integral aspect of the host response to high-level ozone inhalation.

Pulmonary functional and morphological changes induced by a 4-week exposure to 0.7 PPM ozone followed by a 9-week recovery period

Male Fischer-344 rats (control, C = 12; experimental, E = 11) were subjected to pulmonary-function testing procedures. The experimental group was then exposed to 0.7 ppm ozone for 28 d, 20 h/d, and both groups were tested at the termination of the exposure and after 4 and 9 wk of recovery in clean filtered air. Another group of animals (C = 6, E = 12) was similarly treated, but at each test point one-third were sacrificed for microscopic evaluation. When percent changes from preexposure values were compared to controls at each time point, the ozone exposure produced obstructive changes in the lung, including significant decreases (p less than 0.05) in forced expiratory flows (MEF25 40.3%, MEF10 70.7%), lung volumes (IC 22.5%, FVC 21.8%), and DLCO (20.7%); and a significant increase in functional residual capacity (FRC 61.1%). The total lung capacity (IC + FRC) was not significantly changed by the ozone. Microscopic examination revealed characteristic lesions in the region of terminal bronchioles and central acinar alveoli marked by peribronchiolar edema, bronchiolization of alveolar duct epithelium, and type II cell proliferation in involved alveoli with increased numbers of macrophages and a few leucocytes. Clearly discernable was a focal interalveolar-alveolar duct reaction made up of fibroblasts, a few inflammatory cells, and conspicuous mast cells, all embedded in a loose metachromatic matrix. After 4 wk of recovery, all measurements of lung volume and DLCO had returned to the values of the control group; however, even after 9 wk some of the measurements of lung flow (MEF25, MEF10) remained significantly although less depressed (27.9 and 40.1%, respectively). Histologically, after 4 wk recovery, there remained only a slight unevenly disturbed inflammatory reaction. In these foci there was often a residual, narrower, more condensed band of eosinophilic material, presumably collagen, that sometimes contained interspersed mast cells. After 9 wk, this collagen accumulation within the thickened wall of the alveolar duct could occasionally still be noted. These data suggest that the florid response seen at the end of exposure was related to the obstructive changes measured and that, with recovery, the residual central acinar-alveolar duct thickening may be responsible for the persistently diminished air flows.

T-cell activation in pulmonary lymph nodes of mice exposed to ozone.

As part of a project to assess the effect of ozone inhalation on cells of the mediastinal lymph nodes, groups of CD-1 female mice were exposed to ozone at 0.3, 0.5, and 0.7 ppm, 20 hr per day, 7 days per week for 1-28 days. The effect of ozone exposure on lymphoid cells was determined by studying mediastinal lymph nodes at various times of exposure. We found that lymphocyte numbers underwent a dose-dependent, four-phased change: cellular depletion (Days 1-2), followed by rapid hyperplasia (Days 3-4), incremental cell number reduction (Days 5-7), and a subsequent subacute phase of elevated lymphocyte numbers (Days 8-28). Using tritiated thymidine we determined that cells underwent a rapid burst of division by Day 3 of exposure and that mitosis subsequently declined to near baseline values by 2 weeks of exposure. Autoradiographic analysis of histologic sections revealed that the paracortical T-cell areas of the nodes were particularly involved. In addition to the increase in thymidine uptake, several morphologic changes were evident in affected cells including cellular reorganization, nuclear and cellular hypertrophy, and induction of a prominent nucleolus. By comparison, the B cells from ozone-exposed animals were virtually unaffected with respect to cell division or morphological alterations. Prior treatment of ozone-exposed animals with a monoclonal antibody that is cytotoxic for T cells eliminated the hyperplastic response. Since T cells seemed particularly affected by ozone inhalation, we studied immunologic aspects of T-cell reactivity. T-cell responsiveness to mitogenic stimulation with concanavalin A showed little alteration during the first days of exposure; however, by Day 14 an increase in reactivity was observed. This change indicated that functional lymphocyte stimulation occurred during ozone exposure. Thus, response to ozone inhalation involves an acute phase (Days 1-7) characterized by a hyperplastic increase in cell mass and a subacute phase (Days 8-28) characterized by functional changes in lymphocyte reactivity.

Response of t‐cell‐deficient mice to ozone exposure

The number, appearance, and functional reactivity of T-lymphocytes of mediastinal lymph nodes are altered during experimental ozone inhalation. The purpose of the present work is to determine how the lymph nodes and lungs of a mutant strain of animal, which lacks this type of cell, differ in their response to ozone exposure when compared with animals that possess a normal complement of lymphocytes. We exposed athymic nude (nu/nu) mice or heterozygous (nu/+) euthymic mice to 0.7 ppm ozone for 20 h/d for 7 or 14 d while maintaining control groups in clean air. At 7 d the lymph-node hyperplastic response normally seen in euthymic, ozone-exposed animals was greatly reduced in exposed athymic animals. By both 7 and 14 d, greater damage had occurred in the lungs of ozone-exposed, athymic animals than in similarly exposed euthymic animals. Lung wet weight divided by body weight, which was used as a general indicator of lung damage, increased by substantially more in athymic animals than in conventional animals. In a parallel manner, quantitative microscopic analysis, a more sensitive indicator, revealed a marked increase in the lung lesion volumes. Qualitative histologic analysis showed that the change in the response in the athymic animal was most prominent in the peripheral region of the lung extending from the alveolar duct to the alveoli, and was characterized by a greater acute inflammatory cell reaction. Possible mechanisms by which the T-cell could produce the observed effect include secretion of factors that enhance inherent resistance of the lungs target cells, or alterations in the way the inflammatory response to ozone-mediated damage occurs. The results support the idea that the mediastinal lymph node lymphocyte response is adaptive in nature and aids in protecting the lung from ozone-mediated effects.

Quantitation of ozone-induced lung lesion density after treatment with an interferon inducer or an antiinterferon antibody

Inhalation of ozone by experimental animals produces activation of lymphocytes in the mediastinal lymph node complex. Both the number and functional reactivity are affected, with evidence of blastic transformation in T cell but not B cell areas of the nodes. In the present work we determine the extent that modulation of a possible product of immune system activation, interferon, is capable of influencing the way that experimental animals respond to zone. Outbred CD-1 mice were treated with an interferon inducer, poly I:C, or with an anti-interferon antibody while being exposed to ozone at a concentration of 0.7 or 0.9 ppm for 20 h per day for 4 days. Interferon induction produced a significant reduction in lesion volumes in both exposure groups, while anti-interferon produced the opposite effect. Less alveolar damage was observed in interferon-induced, ozone-exposed animals than in animals exposed to ozone alone. In contrast, anti-interferon-treated, ozone-exposed animals showed larger lesions which extended to more peripheral structures and were more extensively infiltrated with neutrophilic leukocytes. These results show that interferon induction protects against zone-mediated lung damage. They also suggest that cells are activated during ozone inhalation which mitigate the effects of ozone on the lung by secretion of interferon.

The effect of ozone inhalation on metabolic functioning of vascular endothelium and on ventilatory function

The primary purpose of this research was to determine the effect of ozone inhalation on pulmonary vascular endothelium. Male Fischer-344 rats were exposed to 0.5 or 0.7 ppm ozone, 20 hr/day for 7 days. Lungs were excised and perfused with Krebs medium containing [14C]serotonin or [14C]hippurylhistidylleucine (HHL). When compared to controls, the animals exposed to the lower ozone concentration showed no statistically significant changes in serotonin removal. In contrast, the higher ozone concentration resulted in a 32% decrease (p less than 0.0001) in serotonin removal, but had no effect on HHL. Rats similarly exposed to 0.7 ppm ozone but allowed to recover for 14 days in clean air showed no decrease in serotonin removal compared to their controls. Animals exposed sequentially to 0.5 ppm ozone for 7 days and then to 0.7 ppm for 7 days showed no alteration in serotonin metabolism, suggesting the development of tolerance initiated by the lower dose. After 7 days exposure to 0.7 ppm ozone, lung ventilatory function measurements revealed small though significant decreases in several parameters. Electron microscopic evaluation of lung capillary endothelium from animals exposed to the 0.7 ppm ozone showed no changes. Positive control animals exposed to greater than 95% oxygen, 20 hr/day for 2 days showed a 23% decrease in serotonin removal (p less than 0.03) and a 12% decrease in HHL removal (p less than 0.017). These studies indicate that inhalation of ozone can induce functional alterations in the lung endothelium, and that this effect occurs at a dosage of ozone that produces minimal ventilatory changes and no observable endothelial ultrastructural changes.

Hepatic and splenic injury in dogs caused by direct impact to the heart.

The purpose of this study was to explore the possibility that nonpenetrating cardiac impact can directly result in hepatic and splenic injury through hemodynamic effects. Impact upon the anterior surface of the heart was produced in 34 open-chest anesthetized dogs. In 15 dogs the velocity of impact was 12 m/sec and in 19 dogs it was 18 m/sec. Pressure in the inferior vena cava transiently reached 194 +/- 25 mm Hg in the dogs impacted at 12 m/sec and 377 +/- 44 mm Hg in dogs impacted in 18 m/sec. Aortic pressure transiently reached 449 +/- 32 mm Hg in dogs impacted at 12 m/sec and 682 +/- 33 mm Hg in dogs impacted at 18 m/sec. Gross capsular lacerations of the liver occurred in six dogs (18%) following cardiac impact. All dogs showed hepatic congestion and most showed microscopic injury of liver cords, central veins, and portal tracts. Dogs that survived cardiac impact for the duration of observation (90 minutes) showed focal acute inflammation of liver triads and hepatic sinusoids, indicating a more subtle degree of injury. The spleens of eight dogs showed areas of grossly visible subcapsular hemorrhage. Subcapsular congestion of the spleen occurred in almost all dogs. This study shows therefore that nonpenetrating cardiac impact may result in hepatic and splenic injury. A likely mechanism may be the extraordinarily high venous pressure that develops at the instant of impact, although transient striking elevations of arterial pressure may also contribute.