OFFICE OF RESEARCH AND DEVELOPMENT National Health and Environmental Effects Research Laboratory CRITERIA AIR POLLUTANTS PARTICULATE MATTER HEALTH EFFECTS RESEARCH PROGRESS REPORT OCTOBER, 1997 ------- CONTENTS Introduction 3 Summary of the Particulate Matter Health Effects Research Program 4 FY96-97 Program Highlights 6 Particulate Matter Problem Characterization Research Program 8 Particulate Matter Dosimetry Research Program 11 Particulate Matter Mechanisms of Toxicity Research Program 14 Particulate Matter Host Susceptibility Factors Research Program 18 Conclusions 21 NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 ------- INTRODUCTION PURPOSE The purpose of this report is to communicate results from the Particulate Matter Research Program of EPA's National Health and Environmental Effects Research Laboratory (NHEERL). CONTENT The report contains a summary of the NHEERL Particulate Matter Research Program, including an explanation of its regulatory and programmatic context, the overall program goal, the rationale for the program, and the research strategy; a section that highlights recent key findings (FY96-97 Program Highlights); and a more detailed description of the NHEERL Particulate Matter Research Program, by program area, including a summary of recent research accomplishments and anticipated progress for the near future. COMMENTS WELCOME The format of this report is still evolving, and we welcome feedback. Readers with comments, questions, or requests for further information are encouraged to contact: John Vandenberg, Assistant Laboratory Director National Health and Environmental Effects Research Laboratory (MD-51A) U.S. EPA Research Triangle Park, N.C. 27711 Phone: (919) 541-4527 or FAX: (919) 541-0642 E-mail: vandenberg.john@epamaii.epa.gov NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 ------- PARTICULATE MATTER RESEARCH PROGRAM SUMMARY REGULATORY AND PROGRAMMATIC CONTEXT The 1970 Clean Air Act (CAA) authorizes EPA to establish National Ambient Air Quality Standards (NAAQS) for criteria air pollutants, including particuiate matter (PM). The Act states that the scientific basis for the NAAQS is to be reviewed periodically and revised as appropriate to reflect new scientific know- ledge. EPA's Office of Research and Development (ORD) provides scientific support for this process. ORD's Particuiate Matter Research Program, which is based upon a peer-reviewed research strategy, is designed to produce scientifically sound data to help guide decision-makers in their considerations of the standard. Four areas of uncertainty have been identified by ORD and are the focus of its research efforts: health effects research, ambient monitoring and exposure research, source characterization and management research, and risk assessment. NHEERL is responsible for health effects research. This document summarizes NHEERL's health effects research on particuiate matter and highlights some of its recent accomplishments in this area. PROGRAM GOAL ,,- To provide credible PM health effects data that reduce the uncertainties in risk assess- ment and thereby support evaluation of the PM NAAQS. RATIONALE Recent epidemiological studies of urban populations, have indicated that current exposures to particuiate matter may lead to increased morbidity from pulmonary disorders and increased mortality from cardiopulmonary diseases. Age and pre-existing cardio- pulmonary disease appear to be important factors in PM susceptibility. PM research is needed (e.g., on toxicological mechanisms) to affirm the biological effects of PM and thereby improve our understanding of these epidemiological findings. There are also uncertainties in the association between particle size/composition, deposition, and adverse effects. Research in these areas will help expand the scientific basis for the assessment-and ultimate reduction-of the public health effects caused by exposure to particuiate matter. RESEARCH STRATEGY To ensure that the Agency is equipped with scientific and technical data relevant to the formulation of sound environmental policy, ORD operates a research program founded on principles of risk assessment. In the area of health effects, the risk assessment paradigm of the National Academy of Sciences (NAS) provides the research context. The risk paradigm consists of 4 fundamental steps that support risk manage- ment decisions: hazard identification, dose- response assessment, exposure assessment, and risk characterization. NHEERL's research programs emphasize two of these steps: hazard identification and dose- response assessment. PM research is being conducted by NHEERL in four critical areas: PROBLEM CHARACTERIZATION, in which we are integrating field and clinical studies to evaluate health effects caused by PM, and we are developing advanced molecular-based methods to provide more definitive assessments of target dose and damage; DOSIMETRY (exposure-dose relationships), in which we are measuring and modeling particle deposition in the lungs while taking into account factors such as age, NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 ------- gender, and disease status; MECHANISMS OF TOXICITY, in which we are determining the role of PM composition, size, and physical properties in provoking health effects; and HOST SUSCEPTIBILITY FACTORS, in which we are examining various host traits, health conditions, and physiological changes responsible for enhancing PM susceptibility in certain population subgroups. NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 ------- NHEERL PARTICULATE MATTER RESEARCH FY96-97 PROGRAM HIGHLIGHTS PROBLEM .CHARACTERIZATION (pg 8) The goal ot this research is to improve our understanding of the epidemiological observations of a relationship between increased mortality/morbidity and PM exposure. In a pilot study of elderly individuals, we found evidence suggesting that cardiovascular- compromised individuals may be at higher risk than the general population to the effects of fine PM. " In collaboration with the Czech Ministry of Health and the Czech Ministry of the Environment, we conducted a study of the impact of fine paniculate matter from coke oven emissions on occupational and urban populations in toe Czech Republic. DOSIMETRY (pg 11) The goal of this research is to develop animal and human dosimetric models to better understand the role of particle size and pre-existing conditions on the health effects of PM and to facilitate animal-to-human extrapolation. A research physicist in our Experimental Toxicology Division has used supercomputers to develop two- and three-dimensional models that simulate the movement of inhaled substances through the human lung. These models were considered so innovative that they won the Smithsonian Institution's 1997 Computerworid Award for Medicine. * We have shown that individuals with pre-existing respiratory disease receive higher doses of fine (PMis) panicles relative to healthy individuals. The dose for patients with chronic obstructive pulmonary disease was as much as 5 times higher than that for healthy individuals; for asthmatics, the dose was 59% higher; and for smokers, 49% higher. > Using our newly developed serial bolus aerosol delivery technique, we conducted the first systematic investigation of regional deposition in human lungs using inert aerosols. We found that the site of peak fine particle deposition is in the distal region (toward the alveoli). With increasing particle size, the peak shifted toward the proximal region (mouth). MECHANISMS OF TOXICITY (pg 14) The goal of this research is to identify and evaluate plausible biological mechanisms that evoke the health effects associated with exposures to PM and its components. » We have found that bioavailable transition metals present on particles play a significant role in inflammation and lung damage. The toxic potencies differ from metal to metal, as do their mechanisms of toxicity. In studies of ambient PM collected in the Utah Valley, our Laboratory has demonstrated for the first time a correlation between a physiological response (production of inflammatory NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 ------- mediators) and epidemiology findings (particle-related mortality), with bioavailable transition metals present on the PM demonstrating a strong correlation with the physiological response. In studies ofneuroreceptors, we have shown for the first time that neurogenic factors are associated with PM-induced inflammation. Neuroreceptors are triggered by PM to stimulate production of inflammatory cytokines. HOST SUSCEPTIBILITY FACTORS (pg 18) The goal of this research is to evaluate various host traits and health conditions that may potentiate the effects associated with PM and to describe the pathophysiology of the susceptibility. When we exposed rodents with pre-existing cardiopulmonary disease to PM, they exhibited an enhanced mortality rate (50%) compared to healthy animals. We showed that death was related to cardiac dysfunction and that specific biochemical changes associated with heart disease may trigger the cardiovascular events. We demonstrated that PM enhances pulmonary infections by altering host defense systems, and we are beginning to explain some of the mechanisms involved in the effects of PM on host defense functions. For example, we found that PM,0 inhibits the antimicrobial defenses of macrophages. NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 ------- PARTICULATE MATTER PROBLEM CHARACTERIZATION RESEARCH PROGRAM NHEERL defines problem characterization research as research to identify and describe the health and environmental risks posed by exposure to environmental contaminants. ISSUE Do current exposures to PM produce increased mortality and morbidity? If so, what are the specific health effects caused by PM and its components, and are there segments of the population who are especially vulnerable to these effects? Recent assessments of epidemiological data have shown significant associations between various measures of ambient paniculate matter and excess mortality and morbidity, raising serious concerns that exposure to PM may impose a heavy burden on human health. However, uncertainties in the data (such as limited characterizations of expo- sure) have clouded our understanding of these observations. There is also the issue of susceptibility. Certain population subgroups, such as children and the elderly, may be more vulnerable than others to the adverse effects ofPM. PROGRAM DESCRIPTION This research program is helping EPA understand the relationship between PM exposures and health effects through more accurate exposure estimates and improved assessments of effect. NHEERL has combined forces with investigators in EPA's National Exposure Research Laboratory (NERL), public institutions, and international governments to implement a two-pronged research strategy. First, we are combining innovative field and clinical approaches to assess physiological changes in small populations exposed to PM in real-world settings. Personal exposure monitors and particle size discrimination are improving our exposure characterizations. Secondly, we are developing molecular and biochemical methods to strengthen epidemiologic assess- ments. By studying changes at the molecular and biochemical level, we can provide more definitive estimates of target dose and contribute to a better understanding of the biological mechanisms that evoke the health effects associated with PM. PROGRAM PROGRESS Field study and clinical approaches. The objective of this research is to couple size- specific PM data with physiological data in our evaluations of health effects relative to PM size. Specifically, we are examining the correlation between PM25 and acute pulmonary and cardiovascular effects in a sensitive subpopulation, the elderly. We are attempting to determine whether daily variations in particulate air quality are associated with daily variations in physio- logical parameters. During FY97, in collaboration with NERL and scientists from the University of North Carolina at Chapel Hill, we completed a pilot study in which we investigated a variety of non-invasive techniques for evaluating the response of elderly individuals to PM exposure. We secured an elderly cohort (individuals >65 years of age) exposed to regional sources of PM and administered a battery of physiological tests measuring lung function, heart rate, blood pressure, blood oxygen saturation, and indicators of immunity (inflammatory response factors). Additionally, we collected environmental monitoring metrics intended to improve exposure assessment; for example, we measured personal particulate exposures and daily indoor and outdoor concentrations of fine and coarse particles. The results of our pilot study showed that elderly individuals are capable of withstanding the demands associated with the NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 8 ------- non-invasive techniques. Preliminary analysis of our health data indicate that cardio- vascular-compromised individuals may be at higher risk than the general population to the effects of fine particulate matter. Future work in this area will focus on more clearly defining methods capable of detecting human health endpoints associated with fine PM and on correlating physiological responses with changing concentrations of PM25 (Contacts: R. Calderon, R. Williams, Human Studies Division) During FY97, we finished constructing and testing an exposure chamber capable of concentrating airborne fine particles approxi- mately 10-fold. We plan to use this particle concentrator in upcoming human studies to assess the degree of lung damage and inflammation associated with ambient urban air particles. Bronchoalveolar lavage samples will be collected, and biochemical and physiological responses will be evaluated. (Contact: R. Devlin, Human Studies Division) Molecular and biochemical epidemiologic methods. In cooperation with scientists in the Czech Republic, NHEERL instituted a multi- disciplinary research program in 1992 to document the relationship between human health effects and PM exposures in a heavily industrialized region of Eastern Europe. Our study is being conducted in the city of Ostrava, an area of exceptionally high levels of PM. During FY96, we assessed the exposure of occupational and urban popula- tions to fine particulate matter (PM25) and carcinogenic polycyclic aromatic hydro- carbons (PAHs) from coke oven pollution sources. We used personal exposure monitors to collect exposure data, and we collected blood samples for the analysis of biomarkers. The biomarker we are studying is the formation of PAH-related DMA adducts, which are structural changes to DNA caused by the binding of a reactive chemical with the DNA helix. Quantitation of these adducts provides a good indication of genetic damage from exposure to carcinogens and mutagens. Our results showed that coke oven workers are exposed to extremely high concentrations (mg/m3) of PM25, while urban populations are exposed to lower levels (ug/m3). In addition, we found that PAH-related DNA adducts increase with increasing exposure to PM at low to moderate concentration levels. At high levels of exposure, however, the formation of adducts becomes nonlinear. This non- linearity in dose-response has been observed not only in humans, but in experimental animals as well. The mechanism for the nonlinearity is uncertain and is under investigation by our Laboratory because this observation has important implications for dose-response extrapolation in risk assessment. (Contacts: R. Williams, J. Lewtas, Human Studies Division) A study in the district of Xuan Wei, China, of a population with high rates of lung cancer (due to exposure to coal combustion emissions) is providing us with a unique opportunity for application and evaluation of biomarkers. Studies are underway in our Human Studies Division to develop methods for measuring protein over-expression of the p53 tumor suppressor gene in sputum- exfoliated airway epithelial cells and lung tissues. It is anticipated that this biomarker can be used for assessing individual lung cancer risk. As a supplement to this study, we are isolating DNA from lung tumors collected from these individuals and attempting to identify the mutations that occur along the p53 gene using PCR (polymerase chain reaction) techniques. This research should help us explain some of the mechanisms involved in the development of PM-related lung cancer. (Contacts: J. Mumford, Human Studies Division; D. DeMarini, Environmental Carcinogenesis Division) During FY97, we devised a comprehensive, multidisciplinary approach to address questions concerning the relationship between ambient PM exposures, internal dose (as measured in lung tissue and NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 ------- surrogate respiratory tract cells and fluids), and particle-induced cell injury. Through scanning electron microscopy analysis, we are determining particle size and chemistry (inorganic and carbonaceous) for a wide variety of air pollution sources. We are then using cell assays to identify the extent to which these parameters are responsible for particle-induced damage. In light of recent evidence suggesting that particle-bound PAHs enhance production of human IgE (a factor that may increase airway allergic disease), we also are examining the role of particle-bound organics in modulating cell loxicity. As a complement to these in vitro studies, we have obtained human lung tissue from autopsy victims and are evaluating possible linkages between chronic PM and ozone exposure, internal particle dose and chemistry, and histopathological changes in the lung. In addition, we are exposing rodents to particles markedly different in size and organic content to assess the differential effects of particle burden, chemistry, and size on inflammation and tumorigenic response. (Contact: J. Gallagher, Human Studies Division) In a collaboration with NERL, we are providing data to the National Human Exposure Assessment Survey (NHEXAS), whose over-arching goal is to assess the every-day exposure of American citizens to potentially hazardous substances. During FY96, we analyzed respirable PM collected as part of the survey and determined that it contained detectable quantities of PAHs, some of which were human carcinogens. Exposure varied with respect to location (urban, suburban, rural), ambient concentration, and chemical specie. Generally speaking, individuals living in urban environments had greater exposures to fine PM and PM-related carcinogenic PAHs. (Contact: J. Lewtas, Human Studies Division) NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 10 ------- PARTICULATE MATTER DOSIMETRY RESEARCH PROGRAM NHEERL defines dosimetry research as research that elucidates the relationship between exposure and dose at the site of toxicity. ISSUE What is the relationship between PM exposure, dose, and effects, especially with respect to sensitive subpopulations? Substantial uncertainties exist regarding the relationship between PM exposure, dose, and observed effects. One reason for this uncertainty is the difference that exists in air flow in the lungs (differences found, for example, in children vs. the elderly or in healthy individuals vs. those with lung disease). Differences in air flow affect the pattern of particle deposition in the lung, which, in turn, affects dose. Understanding deposition patterns and the retention/ clearance of particles is critical for assessing the potential risks of PM. Mathematical or computational models, derived from both human and experimental animal data, help explain dose distribution. Of particular interest are dosimetric models for susceptible subpopulations. PROGRAM DESCRIPTION In an effort to improve our understanding of the exposure-dose-effect relationships associated with PM, we are conducting research on particle deposition in the human lung. Through the combined use of human studies, computer modeling, and physical models, we are relating ambient PM concentrations to delivered dose. Particles differing both in size and in size distribution are being tested. A critical component of our research is the effect of factors such as age, gender, and pulmonary disease on the behavior and fate of particles inhaled and retained by the lung. This research is enabling us to describe the distribution and clearance kinetics of PM in the human respiratory tract, resulting in more realistic estimates of dose distribution and reduced uncertainty in risk assessment. PROGRAM PROGRESS Human studies. During FY96, scientists in our Human Studies Division collaborated with researchers at the University of North Carolina at Chapel Hill to examine the regional deposition of coarse (PM4 5) and fine (PM25) particles in children. Regional deposi- tion is important because particles are not deposited uniformly throughout the lung, and sites receiving higher doses are more likely to become triggering points for adverse biological events. Our results suggest that children have enhanced upper airway deposition of coarse particles relative to adults; the increase is correlated with decreasing height. In light of recent epidemiotogical studies showing increased morbidity in children resulting from airborne particulate pollution, these findings suggest that if morbidity is due to upper respiratory problems, enhanced upper airway deposition may play a role. We also found that the rate of deposition of fine particles-normalized to lung surface area-was greater in children than adults. (Contacts: W. Bennett, K. Zeman, Human Studies Division) During FY96-97, we conducted an investigation of the deposition of particles in the lungs of individuals with pre-existing respiratory disease, including asthmatics, patients with chronic obstructive pulmonary disease (COPD), and smokers. Our objective was to examine differences in particle deposition in diseased vs. healthy lungs. Working with researchers at the University of North Carolina at Chapel Hill, we measured total lung deposition in individuals with varying NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 11 ------- levels of airway obstruction and found a good correlation between lung deposition and spirometric lung function tests. We showed that COPD patients receive a dose of fine particles (PM, ) as much as five times higher than the dose received by healthy subjects. For asthmatics, fine particle deposition was increased by 59% over healthy individuals; for smokers with small airway disease, the increase was 49%. Our data further suggested that the increase in deposition was associated with the obstructed airways. These dosimetric findings are relevant to recent epidemiologic data indicating greater increases in morbidity and mortality in asthmatics and patients with COPD. Enhanced dose likely contributes to the severity of the disease. (Contacts: W. Bennett, C. Kim, Human Studies Division) In order to obtain more detailed quantitative data for inhaled particles, scientists in our Human Studies Division have developed a new method for assessing regional deposition in the human lung. It is called the serial bolus aerosol delivery technique. This method does not require radioactive aerosols, and there are no limitations to the number of lung regions that may be assessed. Initial studies using this technique were conducted during FY95- 96, representing the first systematic investiga- tion ever conducted to determine regional deposition in humans in situ with inert aerosols. Our results showed that particle deposition within the healthy human lung is highly uneven. The site of peak fine particle deposition is in the distal region (toward the alveoli). With increase in particle size (PM30. so), the site of peak deposition shifts from the distal to the proximal region (toward the mouth). These findings suggest that, for coarse particles, early tissue damage may occur in the proximal airways. This technique has the potential for wide application in toxicological studies of aerosols and in the fields of biomedical and health sciences research. (Contact: C. Kim, Human Studies Division) In FY97, we used this novel aerosol delivery technique to study gender differences in particle deposition. We found a significant difference in dose distribution in the lungs of males and females: particle deposition is skewed toward the mouth in females compared to males. This enhancement of deposition becomes more pronounced with coarse particles (PM?0.50). These results support our findings in children, discussed above, which showed that height plays a key role in deposition patterns. They also indicate the importance of dose distribution in interpreting potential gender differences in PM effects. (Contact: C. Kim, Human Studies Division) Computerized models. Using computer modeling that relies on the speed and power of supercomputers, a research physicist in our Experimental Toxicology Division has developed two sophisticated computer simulations of the human lung. These models allow us to visualize the movement of inhaled substances through the lungs in two- and three-dimensional views. One model displays the 20 million airways of the human lung; the other depicts the paths of particles as they flow through and are deposited in the lung. The models are the culmination of many years of research and are so innovative that they won the Smithsonian Institution's 1997 Computerworld Award for Medicine. They are assisting predictions of exposure-dose relationships under a variety of conditions, such as obstructed airways and uneven ventilation. Potential uses for the models include research related to pulmonary diseases (e.g., cancer, asthma, and Cystic fibrosis) and research on the effects of air pollution on children. Other possible applica- tions are in tracking the movement of aerosolized drugs to ensure delivery to diseased areas of the lung. Future research will emphasize the refinement of the models and the development of algorithms for 3-D morphological descriptions and fluid dynamics patterns of respiratory systems. (Contact: T. Martonen, Experimental Toxicology Division) NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 12 ------- Airway models. We have developed branching airway models constructed of glass tubes that permit us to analyze dose distribution under realistic breathing condi- tions. Studies conducted by our Laboratory during FY96 showed that particle deposition in the bronchial airways is highly localized. Surface features, such as rings and ridges, have profound effects on localized PM deposition. We also determined that deposition dose is greater with polydispersed aerosols relative to monodispersed aerosols. (Polydispersed aerosols contain particles of varying sizes and are more representative of real-world exposures than monodispersed aerosols, which are composed of particles of uniform size.) These results are providing new insights into the dynamics of poly- dispersed aerosols and are helping us improve our dose estimates. We are now in the process of using these data to develop an empirical mathematical formula for estimating deposition dose in the bronchial airways. (Contact: C. Kim, Human Studies Division) NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 13 ------- PARTICULATE MATTER MECHANISMS OF TOXICITY RESEARCH PROGRAM NHEERL defines mechanisms of toxicity research as research to identify and characterize the physical, chemical and/or biological mechanisms whereby an environmental agent may induce health or environmental effects. ISSUE What are the causal mechanisms that can provide a biologic explanation for the epidemiologic observations of excess mortality and morbidity due to PM? Despite human evidence linking low-level PM exposures with adverse health effects, there are uncertainties about the biological mechanisms responsible for the observed toxicity. Understanding causal mechanisms would help explain recent findings that relate PM toxicity to differences in particle size and composition. PROGRAM DESCRIPTION -:. . . Our research on the mechanisms of PM toxicity is aimed at determining the effect of particle size and composition on PM toxicity. In vitro tests that utilize cultured lung cells (epithelial cells and alveolar macrophages) are being developed and used to evaluate PM-induced injury at the cellular level. These cell cultures tend to approximate in vivo responses, and they have the added advantage of allowing us to study damage to specific cell types. Our approach is to expose the cells to a wide range of PM in order to test specific hypotheses regarding the relationship between toxicity and particle size/composition. To complement our in vitro work, we are conducting in vivo studies to investigate the role of particles in lung injury and inflam- mation. This involves exposing experimental animals to PM with differing physicochemical properties, and then measuring various physiological and biochemical indicators of damage, such as lung inflammation and oxidant formation. PROGRAM PROGRESS Collectively, our data demonstrate that metal content and bioavailability-as well as particle size, acidity, and sulfate content-are important physicochemical properties that influence PM toxicity. In vitro studies. Scientists in our Human Studies Division and Experimental Toxicology Division have hypothesized that bioavailable transition metals present on particles (e.g., iron, vanadium, and copper) can contribute to inflammation and lung damage. During FY96- 97, we tested this hypothesis at the cellular level. We exposed cultured lung cells to a variety of metals and to residual oil fly ash (ROFA), which is paniculate matter with a high metal content emitted by power plants. When exposed to ROFA, the cells responded by increasing the expression of inflammatory cytokine genes and by producing inflammatory mediators, such as IL-6, IL-8, TNF, and PGE2. When the cells were exposed to pure transition metals, we discovered that different metals have different potencies. For example, vanadium is more reactive than zinc or nickel. We then began to examine some of the molecular mechanisms by which metals exert their effects on lung cells and found that metals are toxic by different means. We found, for example, that vanadium inhibits tyrosine phosphate activity, resulting in elevated levels of phosphorylated proteins. This, in turn, activates nuclear transcription factor NF-KB, which is related to the inflammatory response. Iron, on the other hand, stimulates the cellular production of lactoferritin and ferritin, which internalize iron inside the cell and render it inactive. However, iron complexed with substances such as humic acid, which is NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 14 ------- present on some fine particles, is not internalized and remains active. We will continue to examine the involvement of metals in PM toxicity by studying the molecular mechanisms responsible for PM- induced effects. (Contacts: P. Devlin, S. Becker, A. Ohio, Human Studies Division; J. Dye, K. Dreher, Experimental Toxicology Division) We have used our in vitro methods in several studies of ambient PM toxicity. During FY96 we showed that PM collected from the Salt Lake City airshed stimulates the production of inflammatory cytokines in human lung cells and that copper ts the most active component of these particles. (Contacts: R. Devlin, A. Ohio. C. Kennedy, Human Studies Division) In a collaborative effort with the National Institute of Environmental Health Sciences (NIEHS) and the National Cancer Institute of Mexico, we examined the toxicity of ambient PM collected from various locations in Mexico City. The most toxic samples were from the industrialized northern region. We showed that these particles also contained the highest amount of sulfate and metals. (Contact: K. Dreher, Experimental Toxicology Division) Finally, in a study of ambient PM collected over a three-year period in the Utah Valley, we found that cultured lung cells produced significantly more inflammatory mediators when exposed to PM collected during a time when a major polluting industry was in full operation compared to a time when the industry was on strike. When we compared these results to epidemiology findings reported for the same period, we were the first to demonstrate an association between a physiological response and epidemiology results (reduced particle-related mortality for the year the industry was on strike). We are currently instilling extracts of these particles into the lungs of animals and human volunteers to characterize in vivo response. Preliminary evidence suggests that the in vivo toxicity of the Utah Valley samples is correlated with bioavailable metal content. (Contacts: R. Devlin, A. Ghio, Human Studies Division; D. Costa, Experimental Toxicology Division) In another project, we are studying the role of alveolar macrophages in the clearance of particles and infectious microbes from the lung. While most of our experiments are being performed with alveolar macrophages exposed in vitro, we also plan to obtain macrophages from volunteers experimentally exposed to PM,,, in our chambers on the campus of the University of North Carolina at Chapel Hill. The primary purpose of these studies is to understand how PM interferes with macrophages, which help prevent the development of infections in the lungs. There is interest in this area because it has been shown that high particle pollution levels are associated with increased incidence of pneumonia. The project was initiated in 1995 with the identification of particle-induced cell toxicity and cytokine production in macro- phages. In FY96, we reported that PM10 modulates macrophage surface receptor expression and phagocytosis. During FY97, we found that PM10 can inhibit the anti- microbial defenses of macrophages. These studies are beginning to explain some of the mechanisms involved in the effects of PM1Q on host defense functions. Our future plans are to examine the effects of PM on macrophage antiviral functions, such as virus-induced production of inflammatory mediators. (Contact: S. Becker, Human Studies Division) During FY95, we formulated an artificial lung lining fluid to simulate fluids in the human lung. During FY96-97, we used this in vitro model to investigate a number of mechanisms involved in PM toxicity. We were especially interested in the oxidative effects exerted by PM. We found that particle acidity affects oxidative reactions: high acidity appears to increase the oxidative reactions induced by PM. In tests of the protective effects of Vitamin C and other antioxidants, we showed that Vitamin C inhibits effects caused by low levels of PM, but that the protective effect disappears at high PM exposures. (Contact: NHEERL PARTICIPATE MATTER PROGRESS REPORT, 1997 15 ------- G. Hatch, Experimental Toxicology Division) In a new area of research, we are focusing on defining the role of neuroreceptors in the initial events of PM inflammation. Recent data (FY96-97) produced by our Laboratory suggest that neuroreceptors, which are located on the sensory fibers that innervate the airways, are triggered by PM and stimulate inflammatory cytokines in bronchial epithelial cells. Our data are the first to associate neurogenic factors with PM inflammation. Based on these data, neuro- immunological interactions appear to be a potential mechanism of PM toxicity. We propose that certain types of PM can activate capsaicin receptors, located on sensory fibers and target cells, which in turn causes the release of neuropeptides that trigger the cellular events of inflammation. Currently, we are conducting studies in rodents to determine whether in vivo results can support our findings. In the future, we will examine the role of neuroimmunological interactions in other airway target cells, such as alveolar macrophages. (Contacts: B. Veronesi, Neurotoxicology Division; S. Becker, R. Devlin, Human Studies Division) In vivo studies. During FY97, through a collaboration with ORD's National Risk Management Research Laboratory (NRMRL), we exposed rodents by intratracheal instillation to PM from a variety of sources. Our samples included stationary combustion emissions (ROFA and coal fly ash), a spectrum of inert dusts (such as Mt. St. Helens ash), and ambient air from urban locations (e.g., Washington, DC, and St. Louis. MO). These samples exhibit different metal and sulfate contents, enabling us to obtain information on the association between particle composition and health effects. The health endpoints we are examining include acute pulmonary responses, such as inflammation and airway hyper-reactivity, and chronic responses associated with pulmonary fibrosis. We have found that particle surface chemistry plays a key role in the pulmonary toxicity of PM. The most toxic combustion PM samples are fine (PM^) acidic particles with a high content of sulfate and bioavailable transition metals. Metals are largely, if not entirely, responsible for the initial acute inflammatory response to PM, and specific metal composi- tion is very important in determining the severity of lung injury. For example, we found that a significantly greater degree of lung damage is induced by fly ash containing high levels of zinc, and nickel poses a greater risk of lung injury than vanadium or iron. Although these in vivo findings do not seem to corroborate our in vitro results using cultured cells (discussed above), which showed vanadium to be more reactive than nickel or zinc, the contrast in response is related to differential cell sensitivity. In vivo, nickel causes more of a fibrotic than inflammatory response, which is probably related to effects on alveolar macrophages rather than epithelial cells. Epithelial cells, which measure inflammatory response, indicate vanadium is more reactive than nickel. Thus, vanadium seems to drive the inflammatory component of pulmonary response. While reinforcing the hypothesis that the composition of soluble metals and sulfate is critical to the development of airway hyper- reactivity and lung injury, these results also suggest that differential cell sensitivities may be critical to the overall injury caused by PM. Our findings emphasize that in vitro studies must be conducted using "relevant" cell types in order to approximate in vivo responses. We are currently testing nickel in vitro with alveolar macrophages to see whether we can mimic the in vivo (fibrotic) effects with this cell type. (Contacts: S. Gavett, U. Kodavanti, K. Dreher, Experimental Toxicology Division; A. Ghio, Human Studies Division) We also fractionated ambient air PM from Washington, DC, into a coarse (PM37.20) and two fine (PM<17 and PM17.37) fractions and found that all size fractions induced the same modest increase in airway hyper-reactivity. NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 16 ------- However, PM0 7 induced the greatest degree of injury in terms of pulmonary inflammation, edema, and hemorrhage. Furthermore, we found that the chemistry of ambient air PM changes with particle size. For example, fine ambient particles (PM^.y) are chemically similar to combustion emissions (i.e., they are acidic and have a high content of sulfate and bioavailable metals). (Contact: K. Dreher, Experimental Toxicology Division). NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 ------- PARTICULATE MATTER HOST SUSCEPTIBILITY FACTORS RESEARCH PROGRAM NHEERL defines host susceptibility factors research as research to characterize traits and pre-existing health conditions that may potentiate the health effects caused by an environmental contaminant. ISSUE What are the factors responsible for the enhanced susceptibility to PM exhibited by certain segments of the population? Certain subpopulations, including individuals with pre-existing disease or weakened physical condition, appear to be more vulnerable to the effects of PM and may be predisposed to unusual response, injury, or death when exposed to PM. For example, there is evidence of an association between pre-existing cardiopulmonary disease and excess mortality due to PM. Because risk assessment strategies must consider the spectrum of population responses, it is important to understand variability in susceptibility to PM. PROGRAM DESCRIPTION The issue of PM susceptibility is a recurrent theme throughout this document (for example, the elderly are being studied under PROBLEM CHARACTERIZATION, and DOSIMETRY research is being conducted on children and asthmatics). The objectives of the HOST SUSCEPTIBILITY FACTORS research program are to study various traits or health conditions that make an individual more susceptible to PM and determine how these pre-existing conditions potentiate the toxicity of PM. To accomplish these objectives, we are developing animal models of cardiopulmonary disease, asthma, and infectious disease to represent sensitive human subpopulations. These models then enable us to study the pathophysiology of PM susceptibility. Another facet of our research involves the study of animals and humans to help explain genetic predisposition to the effects of PM. PROGRAM PROGRESS Cardiopulmonary disease. Using a variety of rat models of human cardiopulmonary disease, we have begun to explore susceptibility to PM by examining effects on cardiopulmonary function, pulmonary pathology, and lung damage. In our pulmonary hypertension model, PM exposure resulted in more severe inflammation, greater lung impairment, and higher mortality when responses were compared to normal animals. The mortality rate (50%) was of special interest and prompted us to investigate its cause. We used radiotelemetry to examine cardiac function, and during FY96 we found that both the number and severity of cardiac arrhythmias were significantly increased following ROFA exposure in hypertensive animals compared to controls. These animals also exhibited gas uptake abnormalities, possibly causing a transient hypoxic condition, and fibrinogen levels and leukocyte counts in the blood were elevated, both of which are risk factors for ischemic heart disease. These physiological changes may constitute mechanisms whereby cardiopulmonary events are triggered in susceptible individuals. Collectively, these findings (effects of ROFA on lethality, arrhythmia, and biochemical risk factors) strongly support epidemiology studies that show an association between PM and cardiovascular morbidity and mortality (Contacts: U. Kodavanti, P. Watkinson, Experimental Toxicology Division). Asthma. Our Experimental Toxicology Division has developed two asthma models in rodents. These models are permitting us to examine the effects of co-exposure to allergens and PM. The mouse model, whose development began in 1995, incorporates key NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 18 ------- features of the human asthmatic condition, including elevated levels of serum IgE, pulmonary inflammation highlighted by increased eosinophil numbers, and, most importantly, airway hyper-reactivity. To create the model, we sensitized normal mice with an allergen and then challenged the mice with an aerosol of the foreign protein. During FY96, we used our "asthmatic" mouse to study the impact of PM exposure on physiological and inflammatory responses. A great advantage of our model is that it allows us to investigate mechanisms through widely available immunological and genetic tools. Initial studies were performed using several different particle types, including ROFA and ambient air PM samples from St. Louis, MO, and Washington, DC. Following trachea! instillation of the particles, we studied the time course of responses and assessed possible mechanisms for the observed responses. Mechanisms were examined in detail by measuring levels of allergy-related cytokines in- bronchoalveolar lavage fluid and then relating these levels to inflammatory and physiological responses, such as airway hyper-reactivity. During FY96, we found that ROFA particles increase airway reactivity and that there is a synergistic interaction between allergen and ROFA in elevating eosinophil numbers. Upon further investigation of this phenomenon in FY97, we found that ROFA increases levels of the allergy-related cytokine lnterieukin-4, and that the increase is associated with enhanced pulmonary response. However, IgE antibodies do not increase. In an important discovery in FY97, we found that airway hyper-reactivity following ROFA exposure may be divided into early (1 day) and very late (8 day) phases that are mediated by different mechanisms. In the future, we will study PM-modulated mechanisms of allergy by utilizing various gene knock-out and transgenic mice. Study of these mutant mice will give us the opportunity to examine the role of specific gene products in the pathogenesis of PM effects. (Contact: S. Gavett, Experimental Toxicology Division) In our other allergy model, house dust mite allergen was used to create an asthmatic rat. Using this model in FY96-97, we showed that ROFA treatment enhances the development of immune responses during sensitization, and it augments antigen-induced inflammation during the challenge period. (Contact: I. Gilmour, Experimental Toxicology Division) Infectious disease. Other NHEERL studies are examining the ability of particles to enhance pulmonary infections. These studies demonstrate that particulate matter enhances infectivity by altering host defense systems. For example, studies conducted by our Laboratory prior to FY96 showed that several combustion and ambient air PM samples enhanced susceptibility to pulmonary streptococcal infection in mice. In rats, we showed that dual exposure to ROFA and the influenza virus produces more severe respiratory symptoms and lung damage than either stimulus alone. And in FY97, in collaboration with investigators at the New Jersey Medical School, we demonstrated that ROFA exposure compromises one of the lung's innate immune responses: it inhibits the production of antimicrobial peptides. (Contacts: M. Belgrade, G. Hatch, I. Gilmour, L Ryan, Experimental Toxicology Division) Genetic susceptibility. Another avenue of research is genetic susceptibility. In animal studies conducted during FY96, we demon- strated genetic variability in rats with respect to ROFA-induced pulmonary inflammation, airway hyper-reactivity, and pulmonary fibrosis. Sprague-Dawley rats were shown to be more sensitive than Wistar rats, which were more sensitive than Fischer rats. (Contacts: U. Kodavanti, K. Dreher, D. Costa, Experimental Toxicology Division) Differences in human response to some pollutants have been attributed to genetic NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 19 ------- differences between individuals, including differences in the way in which contaminants are metabolized. For example, humans have multiple enzyme-mediated pathways for the detoxification or activation of DNA-reactive chemicals. The inter-individual variation in metabolic enzyme activities is likely to be associated with variations in susceptibility to pollutants. As part of the China lung cancer project (discussed on page 9), we are evaluating genetic differences in the GSTM1 gene, which is the gene coding for the enzyme important in the detoxification of carcinogens present in PM, Determination of GSTM1 in a lung cancer case-control study is in progress. The results should help us understand the causal mechanisms associated with differences in susceptibility to PM-related lung cancer (Contact: J. Mumford, Human Studies Division). NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 20 ------- CONCLUSIONS As demonstrated clearly in this Progress Report, a variety of methods and models, are being developed by NHEERL scientists in human, animal, and in vitro systems to significantly improve and expand the scientific basis for future evaluation of the PM NAAQS. It is quite unlikely that any research institution in the world is more focused than NHEERL in understanding the biological effects of PM exposures on human health. NHEERL researchers benefit substantially from expertise committed to PM research by NERL and NRMRL, with ORD's National Center for Environmental Assessment poised to incorporate, in the year 2000, the latest research findings in the next PM Criteria Document. We anticipate that critically important results of the research conducted by the laboratories of the Office of Research and Development will be available for inclusion in the next Criteria Document. These results, in combination with extramural studies conducted through the EPA Science to Achieve Results (STAR) grants program and by other institutions, will be used to inform risk management decisions with economic consequences measured in the billions of dollars. NHEERL PARTICULATE MATTER PROGRESS REPORT, 1997 21 ------- |