Daniel Dziedzic

Cellular, biochemical and functional effects of ozone: new research and perspectives on ozone health effects.

Ozone, a toxic component of photochemical oxidant air pollution, has been the focus of considerable research efforts for several decades. In spite of this large body of work, questions remain as to the potential risks to human health represented by chronic low-level exposure to ozone. Newer studies in animals have provided fundamental information on the range of biochemical, functional and morphologic responses to ozone exposure. While the response to ozone exposure is extremely complex, some generalities have emerged which may aid attempts to apply the results of these studies to decisions regarding the protection of human health.

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.

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.

Feasibility Study of Using Brine for Carbon Dioxide Capture and Storage from Fixed Sources

Abstract A laboratory-scale reactor was developed to evaluate the capture of carbon dioxide (CO2) from a gas into a liquid as an approach to control greenhouse gases emitted from fixed sources. CO2 at 5–50% concentrations was passed through a gas-exchange membrane and transferred into liquid media—tap water or simulated brine. When using water, capture efficiencies exceeded 50% and could be enhanced by adding base (e.g., sodium hydroxide) or the combination of base and carbonic anhydrase, a catalyst that speeds the conversion of CO2 to carbonic acid. The transferred CO2 formed ions, such as bicarbonate or carbonate, depending on the amount of base present. Adding precipitating cations, like Ca++, produced insoluble carbonate salts. Simulated brine proved nearly as efficient as water in absorbing CO2, with less than a 6% reduction in CO2 transferred. The CO2 either dissolved into the brine or formed a mixture of gas and ions. If the chemistry was favorable, carbonate precipitate spontaneously formed. Energy expenditure of pumping brine up and down from subterranean depths was modeled. We conclude that using brine in a gas-exchange membrane system for capturing CO2 from a gas stream to liquid is technically feasible and can be accomplished at a reasonable expenditure of energy.

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.

IgG antibody and antibody–antigen complex imaging by scanning tunneling microscopy

We have examined two preparations of biomolecules labeled with colloidal gold markers using scanning tunneling microscopy. In the first preparation, we studied gold labeled IgG antibodies. In the second preparation, we studied unlabeled IgG antibodies which were complexed with gold labeled antigen (bovine serum albumin). Images of these noncrystalline biological specimens, obtained under atmospheric conditions, revealed features on a nanometer scale. The images show details of molecular organization in addition to the classical antibody configuration.

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.

An immunofluorescence study of T and B lymphocytes in ozone-induced pulmonary lesions in the mouse

Ozone is a photochemical oxidant which reacts with a variety of biological molecules. In experimental animals the toxicity of ozone results from damage to cells in the lung and is associated with the process of reactive repair and an influx of inflammatory cells. In this work we used an indirect immunofluorescence technique to study the effect of ozone on T and B lymphocytes with special reference to their presence in ozone-induced pulmonary lesions. BALB/c mice were exposed to ozone at a concentration of 0.7 ppm for 20 hr per day, 7 days per week, for 4 or 14 days. Control mice were housed in comparable chambers lacking ozone. Frozen sections of lung were prepared and stained for the surface markers Thy-1.2 (T lymphocyte) and sIgM (B lymphocyte). These experiments showed that T lymphocytes infiltrate the lung lesions during ozone inhalation, and increase their presence as ozone exposure continues. They were present in the centroacinar region and tended to occur in clusters within the ozone-induced lesions. In contrast, the infiltration of B lymphocytes was virtually nonexistent with few or no sIgM positive cells present in the lesions after either 4 or 14 days of exposure to ozone. These results show that T lymphocytes occur within the sites of ozone-induced damage and support that this cell type plays a role in the reactive host response to ozone inhalation.

Pulmonary response to ozone: Reaction of bronchus-associated lymphoid tissue and lymph node lymphocytes in the rat

The purpose of this work is to assess the effect of ozone, a reactive product of environmental photochemical oxidation, on lymphocytes of the lung. We exposed male Fischer rats to ozone at a concentration of 0.5 ppm for 20 hr/day for 1-14 days. Animals were treated with radioactive thymidine and were sacrificed at Day 1, 2, 3, 7, or 14 of exposure. Lungs and mediastinal lymph nodes were removed and prepared for histologic examination, evaluation of labeling indexes, and morphometric measurement. We examined two components of the lymphocyte response of the lung: the airway-related response, represented by the reaction of the bronchus-associated lymphoid tissue (BALT), and the deep lung-related response, represented by reaction of the mediastinal lymph node. Lymphocytes of both the BALT and the mediastinal lymph node showed elevated radioactive thymidine uptake; however, no evidence of cell death was observed at either site. The cells of the specialized epithelium covering the BALT (lymphoepithelium) showed increased vacuolization, indicating altered cellular function. The average size of BALTs was unchanged by ozone exposure. Under experimental conditions ozone can affect a variety of cells in the lung including bronchial epithelial cells, macrophages, and Type 1 cells. We have shown for the first time that in addition to these cells, the rat BALT also proliferates in response to ozone. In addition we confirm previous work in the mouse which shows that the mediastinal lymph node reacts as well. The airways can be affected by inflammation, can be targets of infection, and can respond to chemical irritants with bronchoconstrictive responses. They are an important target organ for hypersensitivity responses and are a primary site for pulmonary cancer formation. A role for lymphocytes has been implicated in each of these processes. Therefore, the clinical significance of ozone on BALT and mediastinal lymph node responses could be appreciable in terms of potentiation of, or protection against, such responses.

Effects of cyclosporine a on ozone-induced pulmonary lesion formation: Pharmacologic elimination of the T-lymphocyte regulatory response

To assess the involvement of T-lymphocytes in ozone-induced lung damage, CD-1 mice were exposed to air or 0.7 ppm ozone (1.37 mg O3/m3 air) in the presence and absence of the immunosuppressive drug cyclosporine A (CSA). Mice were thus divided into four treatment groups for both the 4 and 14 day exposure times: 1) AIR + VEH, 2) AIR + CSA, 3) O3 + VEH, and 4) O3 + CSA. Thy-1.2 positive cells (T-lymphocytes) per pulmonary lesion were determined and quantitative histomorphometric analysis of lesion volume was performed. By Day 14, the number of T-lymphocytes per lesion in O3 + VEH (vehicle) animals had increased to approximately 3.5 times that seen at Day 4. At 4 and 14 days of O3 + CSA treatment, the number of T cells per lesion was half that seen in O3 + VEH mice. At Day 4, lesion size and appearance were comparable in O3 + VEH and O3 + CSA animals, while at Day 14, O3 + CSA treatment resulted in larger and more cellular lesions that contained a greater proportion of polymorphonuclear cells, and the lesions extended further into the lung periphery. Inflammatory cells were observed in areas of epithelial cell proliferation and in alveolar spaces distal to the small airway terminus. After 14 days, lesion volume was approximately twice as great following O3 + CSA administration than with O3 treatment alone. These results are consistent with effects seen in another model of immunosuppression, the nude mouse, and they implicate a regulatory role for T-lymphocytes in the response to ozone.