X-/EPA United States Environmental Protection Agency Health Effects Research Laboratory Research Triangle Park NC 27711 Research and Development EPA-600/S1-81-067 Feb. 1982 Project Summary Determination of the Effects of Material from Alternate Energy Sources on the Upper Respiratory Tract Clearance Mechanism: Part I and Part II Dorothy Adalis Studies were conducted to measure the toxic effects of a variety of sub- stances from the environment on the clearance mechanism of the upper respiratory tract using an in vitro hamster model system. Studies using hamsters for in vivo exposures to ozone were also con- ducted to determine the effects of ozone on the cilia beat frequency and cytopathology of the trachea! epithe- lium. Organ cultures of hamster trachea! tissue were exposed to graded con- centrations of pollutants to determine effects on the respiratory cilia. Param- eters studied were beat frequency, ciliostasis, cytopathology, and ATP concentration. A preliminary survey to determine the "lethal level" and "no measurable effect level" on ciliary beating for 15 trace metals found in the environment and in the effluents from coal gasifica- tion was conducted. The metals toxic at the 100 Aig/ml level following 24 and 48 hours of in vitro exposure were cadmium, zinc, nickel, manganese, mercury and vanadium. Cobalt was toxic at 10 /ug/ml following 48 hours of in vitro exposure. Copper was lethal at 10 jug/ml and chromium was lethal at 1 /jg/m\ after 24 hours of exposure. Extensive and/or complete data for time and dose levels of all the metals was not determined because of the preliminary nature of this survey. Material from an electrostatic pre- cipitator of a coal-fired power plant was not toxic at levels as high as 1075 jug/ml in suspension following 48 hours of exposure. Exposure of rings for 48 hours to a filtrate of 2477 /ug/ml also showed no observable toxic effect on the epithelial cells and there was no effect on the beat fre- quency due to treatment. In vitro exposures to nickel at 0.63, 6.3 and 63.5/ug/mlfor 24,48, and 72 hours resulted in a dose related pattern of cytopathological changes in the trachea! ring explants. ATP content of nickel treated rings decreased as concentration of nickel increased (P=0.01). Treatment with VO3 in concentra- tions of 0.63 /ug/ml to 12.5 fjg/m\ did not produce statistically significant differences in ATP content of rings. However, dose related histological changes resulted from exposure to vanadium in concentrations of 0.63 fjg/m\ to 12.5 Aig/ml for 24, 48 and 72 hours. Ciliostatis occurred at levels ------- as low as 0.63 //g/ml and the results were statistically significant from the controls at the P < 0.005 confidence level. In vitro exposure studies using zinc, zinc and ammonium and ammonium ion showed dose response changes in ciliostasis and ATP concentration at levels of 50, 75, 100 /ug/ml zinc and 28, 41, 55 /ug/ml NH4. Differences from the controls were statistically significant at P < 0.05. A.combination of zinc plus ammonium ions was significantly more toxic than either ion alone. All treatment levels of zinc or zinc plus ammonium caused a signifi- cant decrease in ATP content as com- pared to the controls (P < 0.001). Results of in vivo exposures to .25 ppm and .5 ppm ozone indicated a dose related reduction in cilia beat frequency that was statistically signif- icant at the P < 0.01 level exposed versus control hamsters. The histological assessment also indicated dose related, adverse patho- logical effects in the tracheas of hamsters exposed to ozone. This Project Summary was devel- oped by EPA's Health Effects Research Laboratory, Research Triangle Park, NC. to announce key findings of the research project that is fully docu- mented in a separate report of the same title (see Project Report ordering information at back). Part I. In Vitro Exposure to Participate Pollutants Introduction Airborne particles emitted into the environment from the utilization of materials from all types of power plants constitute a major source of pollution directly related to the problem of air quality for the human population. A number of new substances from alter- nate and conventional energy sources have not been lexicologically evaluated for their potential adverse health effects in humans Some of these substances, including the ones in this study, are materials derived from shale oil, coal gasification and liquefaction and par- ticulate effluents from both mobile and stationary sources. Once these substartces are inhaled and deposited within the lung, they may exert a toxic effect on the host via one or more of these mechanisms: (1) the substance may be intrinsically toxic due to its inherent chemical or physical properties, (2) the substance may inter- fere with one or more of the clearance mechanisms in the respiratory tract, and (3) the substance may act as a carrier of an absorbed toxic substance. Depending on their site of deposition, these substances come into intimate contact with very specialized host cells, i.e., cilia and alveolar macrophages, whose function is to rid or clear the body of inhaled substances. If these defense systems of the host are reduced m activity then an accumulation of both viable and nonviable substances would occur, which in turn might jeopardize the health of the host. The overall purpose of this project was to screen a variety of substances from the environment for their toxic effect on the clearance mechanism of the upper respiratory tract using an in vitro hamster model system. In testing for cytotoxicity, an in vitro organ culture system was used employing hamster tracheal rings to determine specific effects on normal ciliary activity and ciliated epithelium. This isolated pseudo- stratified ciliated epithelium provided a. controlled means for assessing some direct effects of the pollutants Samples of pollutants tested were supplied by the contractor or the EPA Project Officer. Materials tested and studies completed in this project include the following: a screening study using 15 trace elements from the environment, cytohistological and ATP effects of nickel, a beat frequency study of a sample from an electrostatic precipitator and two in depth studies of the toxicity of vanadium, and zinc/ammonium sul- fate ion. Dose response experiments were designed using various concentrations and lengths of exposures. The various parameters assayed in the hamster tracheal model included histopatholog- ical alterations, ciliary beat frequency, ciliostasis, and ATP determinations. Conclusions The purpose of this project was to evaluate the effects of various materials on the ciliary function of the upper respiratory tract. Experiments com- pleted in this project demonstrate that alterations in ciliary function seem to be indicative of an impairment of the normal clearance mechanism. The results from the data showed statistically significant differences between control and exposed tracheal tissue in an in vitro organ culture model. Data from the screening study of 15 trace metal pollutants indicated toxicity to tracheal ciliary beating activity for mercury, zinc, cadmium, vanadium, nickel, cobalt, copper and chromium. Molydenum, manganese, barium, lead, chromium (in the +3 valence state), sulfite, and magnesium were not toxic after 48 hours of in vitro exposure to concentrations of at least 100 /ug/ml. Histologically, there was evidence of damage to the ciliated epithelium of the tracheal rings following exposure to graded concentrations of nickel and vanadium. Denudation, tufting of cilia, sloughing, blebbing, vascularization of cytoplasm, notched nuclei, eosinophilic inclusions and flattened cells were observed in a dose related pattern tracheal rings exposed to either pol- lutant Treatment with both nickel and zinc sulfate or zinc ammonium sulfate re- sulted in statistically significant (P < 0.05) dose related decreases in the ATP content of tracheal ring explants. In vitro exposure to vanadium did not produce correlating changes in the ATP content of rings. In terms of tracheal clearance, the significant differences in % ciliostasis with both zinc and vanadium indicate impairment of normal ciliary activity. If the effectiveness of the mucociliary activity depends on normal functioning of the ciliated epithelium of the upper respiratory tract, it follows from the data presented that inhaled environmental pollutants can inhibit the normal muco- ciliary function needed to clear possible harmful and infectious agents from the respiratory tract. Recommendations The in vitro hamster tracheal ring model used in this study is excellent for preliminary screening of environmental chemicals. This in vitro system is a convenient and inexpensive means of examining the effect of acute and short term exposure to potentially toxic sub- stances. Such substances, if present in ------- sufficient quantities to allow for testing, should be examined by preliminary screening in such an in vitro system. Previous studies which compare this in vitro method with in vivo exposures support the use of the in vitro model for testing the effects of pollutants on the living animal. In addition, the short term, acute exposures of the materials evaluated in this study should be extended to include multiple exposures and recovery projects. Pollutants should be evaluated in combination with a stress factor such as exercise. Further detailed studies should include in vivo exposures for validation of in vitro results. Our study of ATP concentration was an attempt to elucidate the site of pol- lutant effect on ciliary activity. Further studies might examine oxidative and enzymatic responses to pollutants. Part II. In Vivo Exposure to Ozone Introduction An increase in ozone concentrations of ambient air has become one of the prime concerns of air quality organiza- tions. Ozone, a gas, which has its chemical origin as a product of photo- chemical-oxidant formation, is con- sidered a secondary pollutant since it is formed as a result of chemical reactions in the atmosphere rather than being directly emitted by a pollution source. Primary pollutants (these directly emitted by a pollution source) most responsible for ozone and other oxidant formation in the air are the nitrogen oxides, hydro- carbons, and carbon monoxide. Ozone and other oxidants formation is related to time of day, meteorologic conditions, and the amount of primary pollutants in ambient air. Ozone is used by the U.S. Environmental Protection Agency as an indicator of photochemical smog. The overall purpose of Part II of this study was to determine the effects of in vivo exposure to three concentrations of ozone on the ciliated epithelium of hamster tracheal rings. Specifically, two parameters were studied following in vivo exposures to ozone1 1) a cytopathological assessment was made to determine if ozone alters or interferes with the struc- tural integrity of the ciliated epi- thelium, and 2) quantitative measurements of the cilia beat frequency were deter- mined using a stroboscope and statistically analyzed for signifi- cant differences between control and exposed rings. A variety of times and doses were studied to include 0.25, 0.5, and 1.0 ppm for 1, 3, or 5 days. Conclusions In vivo exposure to 0.25 and 0.5 ppm ozone 3 hrs x 3 days resulted in a significant reduction.in ciliary beat frequency. This reduction in beat fre- quency increased as the concentration of ozone increased. The mean beating frequency in beats per minute for 94 control rings (8 animals, 4 of each sex) was 117.4.6. The mean beating fre- quency for 93 rings from 8 animals exposed to 0.25 ppm ozone 3 hours x 3 days was 1089.2. Mean beat frequency for 8 animals exposed to 0.5 ppm ozone was 1009.0 (89 rings). An analysis of variance indicated a significant decrease (P<0.005) in beat frequency in the ozone treated rings when compared with controls. There was no significant difference in beat frequency between sexes or due to interaction of sex and treatment. The results indicate an altered func- tioning of the tracheal ciliated epithe- lium which would possibly interfere with the host's normal pulmonaryclear- ance mechanism. This in turn would make the host more susceptible to respiratory infection. Effects of in vivo exposure to ozone produced pathological effects which were dose related. Increasing the con- centration of ozone results in increasing disorganization of the pseudostratified ciliated epithelium lining of the tracheal rings. The degree of disorganization was also directly related to the length of time or exposure at any one concentra- tion level. The resulting damage to tissue mor- phology due either to concentration or length of exposure to ozone would alter the normal functioning of the mucociliary escalator. This impairment could in- crease the susceptibility of the host to respiratory infections. Recommendations Because of,the widespread and in- creasing occurrences of ozone pollution, further studies should be conducted. The short term acute exposures to ozone in this study should be extended to include ozone in combination with other pollutants. Also needed are studies to determine the effects of ozone in combination with stress factors such as exercise or in relation to diet. Other studies recommended include the following' 1. Recovery of ciliary activity follow- ing in vivo exposures to ozone. 2. Measurement of ATP production in hamster heart, liver and gastro- cnemius muscle following in vivo exposure to ozone. 3. The effect of ozone on the oxidative capacity of hamster heart, liver and muscle. 4. Effect of ozone on the oxidative capacity of hamster tissues fol- lowing exercise. ------- Dorothy Ada/is is with the Department of Biology, Ball State University, M uncle, IN 47306. Donald E. Gardner is the EPA Project Officer (see below). The complete report, entitled "Determination of the Effects of Material from Alternate Energy Sources on the Upper Respiratory Tract Clearance Mechanism: Part I. In Vitro Exposure to Paniculate Pollutants. Part II. In Vivo Exposure to Ozone." (Order No. PB 82-117 037; Cost: $9.00. subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Health Effects Research Laboratory U.S. Environmental Protection Agency Research Triangle Park. NC 27711 US GOVERNMENT PRINTING OFFICE. 1982 — 559-017/7447 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 ------- |