Brian K. Tarkington

Interaction of nitrogen dioxide with human plasma Antioxidant depletion and oxidative damage

Nitrogen dioxide (NO*2) is often present in inhaled air and may be generated in vivo from nitric oxide. Exposure of human blood plasma to NO*2 caused rapid losses of ascorbic acid, uric acid and protein thiol groups, as well as lipid peroxidation and depletions of α‐tocopherol, bilirubin and ubiquinol‐10. No increase in protein carbonyls was detected. Supplementation of plasma with ascorbate decreased the rates of lipid peroxidation. α‐tocopherol depletion and loss of uric acid. Uric acid supplementation decreased rates of lipid peroxidation but not the loss of α‐tecopherol. We conclude that ascorbic acid, protein ‐SH groups, uric acid and α‐tocopherol may be important agents protecting against NO*2 in vivo. If these antioxidants are depleted, peroxidation of lipids occurs and might contribute to the toxicity of NO*2.

Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae)

To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4(+) lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA.

Repeated episodes of ozone inhalation amplifies the effects of allergen sensitization and inhalation on airway immune and structural development in Rhesus monkeys

Twenty-four infant rhesus monkeys (30 days old) were exposed to 11 episodes of filtered air (FA), house dust mite allergen aerosol (HDMA), ozone (O3), or HDMA + O3 (5 days each followed by 9 days of FA). Ozone was delivered for 8 h/day at 0.5 ppm. Twelve of the monkeys were sensitized to house dust mite allergen (Dermatophagoides farinae) at ages 14 and 28 days by subcutaneous inoculation (SQ) of HDMA in alum and intraperitoneal injection of heat-killed Bordetella pertussis cells. Sensitized monkeys were exposed to HDMA aerosol for 2 h/day on days 3-5 of either FA (n = 6) or O3 (n = 6) exposure. Nonsensitized monkeys were exposed to either FA (n = 6) or O3 (n = 6). During the exposure regimen, parameters of allergy (i.e., serum IgE, histamine, and eosinophilia), airways resistance, reactivity, and structural remodeling were evaluated. Eleven repeated 5-day cycles of inhaling 0.5 ppm ozone over a 6-month period had only mild effects on the airways of nonsensitized infant rhesus monkeys. Similarly, the repeated inhalation of HDMA by HDMA-sensitized infant monkeys resulted in only mild airway effects, with the exception of a marked increase in proximal airway and terminal bronchiole content of eosinophils. In contrast, the combined cyclic inhalation of ozone and HDMA by HDMA sensitized infants monkeys resulted in a marked increase in serum IgE, serum histamine, and airways eosinophilia. Furthermore, combined cyclic inhalation of ozone and HDMA resulted in even greater alterations in airway structure and content that were associated with a significant elevation in baseline airways resistance and reactivity. These results suggest that ozone can amplify the allergic and structural remodeling effects of HDMA sensitization and inhalation.

Ozone and nitrogen dioxide exposure: murine pulmonary defense mechanisms.

Since ozone and nitrogen dioxide impair pulmonary resistance to infection by inhibiting the function of the alveolar macrophage, we investigated the effect of combinations of these gases to determine if they interacted biologically in a synergetic, indifferent, or antagonistic manner. Mice were exposed to atmospheres of ozone and nitrogen dioxide for 17 hours prior to, or four hours after, infection with aerosols of Staphylococcus aureus labeled with radioactive phosphorus (32P). Animals infected and then exposed to various oxidant combinations manifested bactericidal dysfunction only when the level of one of the pollutants approximated its individual threshold value. Similar results were obtained when mice were exposed to pollutants for 17 hours before infection. Hence, the pulmonary consequence of exposure to ozone and nitrogen dioxide is equivalent to the injury that would be expected from each individual oxidant.

An in vitro system for studying the effects of ozone on mammalian cell cultures and viruses

A unique in vitro system was developed for exposing mammalian cell cultures, viruses, or both to ozone under conditions mimicking those of the respiratory tract. The system used borosilicate glass roller culture bottles equipped with specially designed caps to permit the flow of humidified gas (ozone or filtered air) through the rotating vessels. The system was designed to allow two test cultures and one control culture to be simultaneously exposed to different precisely defined concentrations of ozone. The input and exhaust concentrations of ozone were sequentially monitored with an ultraviolet photometric ozone analyzer. The system was used to determine the reactivity of ozone with several tissue culture media at different flow rates. The reaction rate of ozone with media was shown to be a function of the input concentration and increased as the gas flow rate was increased. Input ozone concentrations measured during 48-hr test exposures remained stable, yielding standard deviations of less than 4%. Exposure of Madin-Darby bovine kidney cells to ozone concentrations of 0.16 and 0.64 ppm for 24 hr resulted in a slight but significant decrease in the synthesis of RNA as measured by the incorporation of [3H]uridine into TCA-precipitable material. The synthesis of protein and DNA was not significantly affected by identical treatments. Vesicular stomatitis virus exposed to ozone at concentrations of 0.16 and 0.64 ppm for 12 hr showed marked loss of biological function when compared to identical controls exposed to filtered air. The feasibility of using enveloped viruses as a model for investigation of ozone-induced damage to cellular membranes and membrane proteins is discussed.

Differential expression of stress proteins in nonhuman primate lung and conducting airway after ozone exposure.

The presence of seven stress proteins including various heat shock proteins [27-kDa (HSP27), 60-kDa (HSP60), 70-kDa (HSP70) and its constitutive form HSC70, and 90-kDa (HSP90) HSPs] and two glucose-regulated proteins [75-kDa (GRP75) and 78-kDa (GRP78) GRPs] in ozone-exposed lungs of nonhuman primates and in cultured tracheobronchial epithelial cells was examined immunohistochemically by various monoclonal antibodies. Heat treatment (42°C) resulted in increased HSP70, HSP60, and HSP27 and slightly increased HSC70 and GRP75 but no increase in GRP78 in primary cultures of monkey tracheobronchial epithelial cells. Ozone exposure did not elevate the expression of these HSPs and GRPs. All of these HSPs including HSP90, which was undetectable in vitro, were suppressed in vivo in monkey respiratory epithelial cells after ozone exposure. Both GRP75 and GRP78 were very low in control cells, and ozone exposure in vivo significantly elevated these proteins. These results suggest that the stress mechanism exerted on pulmonary epithelial cells by ozone is quite different from that induced by heat. Furthermore, differences between in vitro and in vivo with regard to activation of HSPs and GRPs suggest a secondary mechanism in vivo, perhaps related to inflammatory response after ozone exposure.

In vitro exposure of tracheobronchial epithelial cells and of tracheal explants to ozone

An in vitro system for exposing respiratory epithelial cells or explant tissues to ozone has been developed and characterized. This system is designed to generate and monitor consistent, reproducible levels of ozone, over a range of concentrations, in a humidified atmosphere, and to allow an exposure time of 24 h or longer. Based on chemical analysis, highly reproducible concentrations of ozone are delivered throughout the chamber, with a coefficient of variation of < 5% between five replicate vials exposed to 0.5 ppm of ozone for 50 min. The viability of cultured human tracheobronchial epithelial cells, as measured by the ability to oxidize a vital dye, and of rat tracheal epithelium, as measured by total numbers of necrotic cells in tracheal explants, after ozone exposure was examined in this system. Responses of cultured cells to ozone exposure as measured by bioassay were consistent with the observed low level of variability of ozone concentration between replicate incubation dishes or vials. Responses of cultured cells to ozone were proportional to duration of exposure and inversely proportional to the volume of medium covering the cells. We conclude that this newly developed in vitro exposure system will allow relatively simple and convenient exposure of cultured cells or organs to ozone or other gaseous agents under highly controlled and reproducible conditions.

In Vitro Exposure of Proteins to Ozone

An in vitro system has been developed to expose proteins to ozone. The system is designed to deliver consistent and accurate levels of ozone over a range of concentrations (between 0.1 and ≥10 ppm) with extended exposure times (24 h or longer) in a humidified environment (100%). In the experiment presented in this article, ozone concentrations between 0.1 and 2.0 ppm were used. Ozone was generated by an electrical discharge ozonizer to ensure stability; it was continually monitored by an ultraviolet ozone analyzer and was precisely controlled by mass flow controllers, which gave reproducible results between runs. Humidity was closely regulated in the system to allow small amounts of protein solutions (50 μL or less) to be exposed without significant changes (<0.2%) in sample volume. The degree of surfactant protein-A (SP-A) oxidation by ozone was measured between runs to demonstrate the reproducibility of the system. A detailed description of the system is given, and protein oxidation detection methods and their limitations are discussed. Using these methods, we were able to assess oxidation of SP-A that apparently occurred prior to its isolation from the lung by bronchoalveolar lavage. This in vitro system allowed us to expose small amounts of protein to ozone in a simple, highly controlled, and reproducible manner.

Generatation and characterization of sodium sulfite aerosols for applications in inhalation toxicologic research

A method has been developed for generation of submicrometer aerosols of sodium sulfite suitable for use in inhalation toxicologic research. Concentrations ranging up to about 30 mg/m3 Na2SO3 were achieved in a 0.44 m3 exposure chamber with an air flow rate of 0.20 m3/min for periods up to 16 days. The coefficient of variation of the sulfite aerosol mass concentration was about 4% during a typical exposure period. The measured mass median aerodynamic diameters (MMADar) of the generated aerosols were 1.2 (+/- 0.2SD) microns with a geometric standard deviation (sigma g) of 1.9 (+/- 0.3SD). The chamber was sampled for gas phase SO2 concentration, and aerosol samples were analyzed for particulate sulfite and sulfate. The fraction of sulfur qas sulfite in the aerosol was usually 95% and was always greater than 90%. Gas phase SO2 amounted to less than 2% of the total S(IV) present in the chamber.

Ozone exposure of cultured cells and tissues

Publisher Summary This chapter discusses the effects of oxidant air pollutants, such as ozone (O3) and nitrogen dioxide (NO2) on the respiratory tract using in vivo inhalation exposures, organ culture or explant systems, and cell culture systems. With in vitro systems, a major problem is the design of the exposure system to deliver constant and reproducible concentrations of ozone to replicate culture dishes because ozone is chemically highly reactive. The other challenge is to mimic in vivo exposure conditions where the luminal surfaces of respiratory epithelial cells are exposed almost directly to the inspired air, except for a mucous or surfactant layer of variable thickness. The exposure of organ or cell cultures through a stationary liquid layer is undesirable because relatively insoluble gases have little effect except at high concentrations. Additionally, the mechanism of action may be different if an oxidant first reacts with the components of a relatively thick liquid layer rather than reacting directly with the cell or its surface layer.