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Executive Summary In the course of a year, an adult breathes approximately 7 million liters of air. Industrial workers breathe about 20 liters/minute, and runners can create up to 80 liters/minute. As part of the body's autonomic processes, breathing fulfills the vital function of exchanging the gases of oxygen and carbon dioxide. Along with air, respiration can bring in common air pollutants, such as toxic pol- lutants, respirable particulate matter, and the "criteria" air pollutants-nitro- gen dioxide, sulfur dioxide, ozone, and carbon monoxide. Original interest in reducing the intake of these pollutants came directly from episodes of severe air pollution that were clearly linked with immediate in- creases in deaths related to respiratory disorder, such as those in the Meuse Val- ley, Belgium, in 1930 and in Donora, Penn- sylvania, in 1948. By December 1952, the "great fog" of London had killed an esti- mated 4,000 residents, many of whom were elderly persons who already had heart and lung problems. Sobered by such epi- sodes, the United States developed an aggressive program for reducing air pollutants. Still, subtle differences in pulmonary performance can occur and chronic pulmon- ary problems afflict one of every five persons; exposure even of healthy persons to environmental pollutants, even at their current magnitudes, plays a role. Such problems include reductions in the maximal amount of air that can be expelled, increases in chest tightness, wheezing, and possibly even cancer. Young children, elderly persons, and those with chronic diseases are likely to be especially vul- nerable to pollution; respiratory symp- toms can occur in them when concentrations of air pollutants increase even slightly. In light of these longstanding and con- tinuing concerns and corresponding con- cerns for other elements of public health, the Board on Environmental Studies and Toxicology (BEST) of the National Research Council's Commission on Life Sciences undertook a major investigation of the use of biologic markers in health research. At the request of the Office of Health Re- search of the U.S. Environmental Protec- tion Agency (EPA), the National Institute of Environmental Health Sciences, and the Agency for Toxic Substances and Disease Registry, the Committee on Biologic Markers was formed to clarify the concepts and definitions of biologic markers. After completion of its task, the committee or- ganized two subcommittees: the Subcom- mittee on Reproductive and Neurodevelop- mental Toxicology (with individual panels on male reproduction, female reproduc- tion, pregnancy, and neurodevelopment),

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2 and the Subcommittee on Pulmonary Toxicol- ogy, which developed this report. This executive summary encapsulates each of the seven chapters in the body of the report and presents a summary of the major conclusions and recommendations. Preceding the summary of the chapters are introductory sections that present con- cepts, definitions, and selected applica- tions of biologic markers. CONCEPTS AND DEFINITIONS The committee has defined, as described in Chapter 1, the following concepts re- lated to biologic markers. Biologic mark- ers in the context of environmental health are indicators of events in biologic sys- tems or samples. It is useful to classify biologic markers into three types-markers of exposure, of effect, and of suscepti- bility-and to describe the events peculiar to each type. A biologic marker of expo- sure is an exogenous substance or its me- tabolite or the product of an interaction between a xenobiotic agent and some target molecule or cell that is measured in a com- partment within an organism. A biologic marker of effect is a measurable biochemi- cal, physiologic, or other alteration within an organism that, depending on mag- nitude, can be recognized as an established or potential health impairment or disease. A biologic marker of susceptibility is an indicator of an inherent or acquired limit- ation of an organism's ability to respond to the challenge of exposure to a specific xenobiotic substance. Biologic markers of susceptibility are discussed in this report only insofar as they also can serve as markers of exposure or effect. Once exposure has occurred, a continuum of biologic events can be detected. These events may serve as markers of the initial exposure, administered dose (circulating peak or cumulative dose), biologically effective dose (dose at the site of toxic action, dose at the receptor site, or dose to target macromolecules), altered struc- ture or function with no ensuing patho- logic effect, or potential or actual health impairment. Even before exposure occurs, biologic differences among humans might cause some to be more suscep AL4RKERS IN PULMONARY TOXICOLOGY tible to environmentally induced disease. Thus, biologic markers are tools that can be used to clarify the relationship, if any, between exposure to a xenobiotic com- pound and health impairment. Markers of Exposure Exposure is the sum of xenobiotic mater- ial presented to an organism, whereas dose is the amount of the xenobiotic compound that is actually absorbed into the organism. Blood flow, capillary permeability, transport into an organ or tissue, the number of receptor sites, and route of administration (which determines the path of the parent material or its metabolites in the body) all can influence absorbed dose or biologically effective dose. An inhaled carcinogen that is retained in the lung might produce tumors in the lung; if the same material were ingested and eliminated via the kidney, renal tumors might be produced. If the parent material is responsible for the observed toxicity, the amount of metabolite that reaches the target might be of no importance. If metab- olites are responsible, however, metabo- lism in the liver, another target organ, or elsewhere as a result of metabolic coop- eration between several tissues is an im- portant determinant of absorbed dose and biologically effective dose. Markers of Effect For present purposes, the effects of an exposure on an organism (the responses of an organism to an exposure) are consid- ered in the context of the relationship of exposure to health impairment or to the probability of health impairment. An effect is defined as an actual health impairment or recognized disease, an early precursor of a disease process that indicates a potential for health im- pairment, or an event peripheral to any disease process, but correlated with one and thus predictive of development of health impairment. A biologic marker of an effect or re- sponse, then, can be any change that is qualitatively or quantitatively predic

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EXECUTED: sentry live of health impairment or potential impairment associated with exposure. Biologic markers are also useful to iden- tify endogenous components or system functions that are considered to signify normal health, e.g., blood glucose. It is important to recognize, however, that the concentration or presence of such mark- ers represents points on a continuum. Therefore, the boundaries between health and disease can change as knowledge in creases. Markers of Susceptibility Some biologic markers indicate individ- ual or population differences that affect the biologically effective dose of or the response to environmental agents indepen- dently of an exposure under study. An in- trinsic genetic or other characteristic or a pre-existing disease that results in an increase in the absorbed dose, the biologically effective dose, or the tar- get-tissue response can be a marker of increased susceptibility. Such markers include inborn differences in metabolism, variations in immunoglobulin concentra- tions, low organ reserve capacity, or other identifiable genetically deter- mined or environmentally induced varia- tions in absorption, metabolism, and re- sponse to environmental agents. Other factors that can affect individual sus- ceptibilities include nutritional status of the organism, the role of the target site in controlling overall body func- tion, the condition of the target tissue (whether disease is or was present), and compensation by homeostatic mechanisms during and after exposure. The reserve capacity of an organ to recover from an insult at the time of exposure can play an important role in determining the ex- tent of an impairment. EXTRAPOLATION FROM ANIMALS TO HUMANS Extrapolations of data from animals to humans should be based on the most sen- sitive animal species tested, barring clear evidence that that species is toxi- cologically distinct from humans. Recent 3 ly, EPA issued guidelines for evaluating exposure studies and other guidelines concerning various toxic effects (e.g., reproductive mutagenicity). Those guide- lines provide a means to estimate data quality and stipulate the types of data that are necessary to estimate exposure for assessment evaluations. Laboratory animals and humans can dif- fer in structure, physiology, and phar- macokinetics. Thus, data from animals must be used carefully in determining health risks in humans. Important similar- ities and differences in relation to par- ticle deposition and clearance between laboratory animals and humans must be con- sidered when animal data are used to model human diseases. It has been particular- ly difficult to model asthma and emphy- sema. Furthermore, animal models of ef- fects of suspected human carcinogens-such as radon, cigarette smoke, and arsenic- have been difficult to develop. The toxicity of some chemicals is medi- ated by activation or detoxification bio- transformation reactions. Inasmuch as biotransformation differs among species, it is important to establish whether the routes and rates of human and animal meta- bolic pathways are similar. Health risks often are associated with combinations of effects in humans. For example, cardiovascular disease in humans can encompass atherosclerosis and hyper- tension. Although swine provide the most suitable animal model for studying spon- taneous atherosclerosis, young rats might be most appropriate for studying hyper- tension. Estimating human risks neces- sarily would entail some appropriate combination of the relevant animal test systems. A common source of uncertainty in risk assessment is the dose-response relation- ship at low doses or for rare effects. It is often impractical to conduct animal studies of effects at low doses, because large numbers of animals are required to detect a low incidence of an effect. Dem- onstrable health effects in humans, given the limits of epidemiology, often are as- sociated with high doses. Sensitive molec- ular markers being developed will permit study of the relationship between exposure

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4 to chemicals at low ambient concentrations and the formation of a molecular marker predictive of human risk. The development of biologic markers might enable scien- tists to make better use of laboratory- animal data in estimating the effects of chemicals in humans. As a 1986 National Research Council study on drinking water and health ob- served, the timing of exposure and the dose-response patterns in animal studies have important implications for extrapo- lating the resulting data to humans. MARKERS OF EXPOSURE As the portal of entry for airborne pol- lutants, the respiratory tract should be an excellent site for detecting pollu- tant-specific markers. From the fine cilia and mucosa of the nasal passages, through the trachea, to the bronchi, bron- chioles, and moist alveolar membranes, the respiratory anatomy may be considered a system for detecting and identifying a variety of markers. Investigators seek markers of general pulmonary function or toxicity that reflect such deviations from normal conditions as altered breathing patterns and airway constric- tion. Specific exposure markers can be devised by sampling and examining tissue in the nose and lung (e.g., with nasal or bronchoalveolar ravage) and noting con- centrations of pollutants of interest, such as diesel-exhaust particles, chryso- tile asbestos, wood dust, fiberglass, wollastonite, iron, silica, and volcanic ash. This report assesses various respira- tory phenomena as markers of exposure, disease, and in some cases predisposition to disease. The respiratory system can respond to inhaled toxicants in only a few ways. Pollutants, antigens, infec- tion, exercise, cold, and psychologic factors can all alter breathing patterns and lead to airway constriction; and per- sistent alteration of lung structure can occur in persons with chronic obstructive pulmonary disease (such as chronic bronchitis or emphysema, fibrosis, granu- lomatous disease, or neoplasia). None of those responses can be associated with WIRERS IN PULMONARY TOXICOLOGY a specific etiology; each can result from a variety of causative agents or condi- tions. Yet some markers could be chosen as peculiar to a specific disease state and could be quantitatively relatable to the degree or stages of the disease. Chapter 2 describes several new ap- proaches to the use of biologic markers for providing information on respiratory tract dosimetry. The development of bio- logic markers of exposure to xenobiotics offers much promise. New molecular bio- logic techniques permit the measurement of such molecular markers as adducts form- ed with macromolecules in the body, the techniques can be used to detect adducted material in blood, urine, and tissue samples and are sensitive enough for the measurement of adducts formed with DNA or protein in cells washed from the respir- atory tract or collected in sputum. Innovative procedures, such as magneto- pneumography, allow estimation of the lung burdens of some types of particles. Refined histologic techniques have re- vealed the cellular sites of deposition of inhaled particles in the lung and thus created the potential for calculating the dose to critical cells. And techniques for analyzing markers are well advanced; e.g., new techniques allow analysis of exhaled air, sputum, nasal ravage fluid, and bronchoalveolar ravage fluid for chemical evidence of exposure to specific pollutants. The field of mathematical modeling has advanced to the point where models now include physiologic measure- ments, such as blood flow rates, ventila- tion rates, metabolic rates, and both blood-air and blood-tissue partition coefficients. The models have made it much easier to extrapolate data from ani- mal pharmacokinetic studies to predicted deposition and distribution in humans. To make optimal use of the new tech- niques, we must determine the relation- ship between markers of exposure and the characteristics of the exposures that generate them. The markers usually yield only yes-no answers; that is, a particular exposure did or did not occur. But we need to determine the kinetic relationships between formation and breakdown of mark- ers, so that we can use mathematical

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EXECUTM? SPRY models to answer the question, "Given this amount of this marker in this tissue, what exposures could have produced the marker?" In addition, there is a need to explore such readily available respiratory tract flu- ids as nasal fluids and sputum for chem- ical markers of exposure to specific pollutants. Chapter 2 briefly summarizes methods for collecting clinical information. The standardized questionnaire on respir- atory disease can play an important role in identifying markers of potential res- piratory disease. Neither the clinical history of a patient nor information ob- tained from a population with a stand- ardized questionnaire constitutes bio- logic material, but such information is important in identifying the potential for respiratory disease. For instance, severe respiratory illness before the age of 2 years implies a likelihood of res- piratory illness in later childhood; and persistent wheezing in childhood pre- dicts abnormal pulmonary function in adulthood. Since the early 1950s, efforts to develop standardized procedures for gathering clinical and epidemiologic data on pulmonary health status have been in place. One of the most successful ques- tionnaires has been the one developed by the British Medical Research Council and adopted by the American Thoracic Society. This questionnaire is used widely in the United States and throughout the world. Chapter 2 shows the importance of deter- mining the mechanisms by which environ- mental pollutants induce lung disease. What are the sites of toxic actions? How much of a given pollutant is required at a given site to produce a given toxic re- sponse? Knowledge of the mechanisms by which toxicity occurs should provide the most pertinent information on poten- tial early markers of exposure to environ- mental pollutants and initial stages of response to them. MARKERS OF PHYSIOLOGIC EFFECTS IN INTACT ORGANISMS Chapter 3 describes the role of physio- logic measurements of the functional sta- tus of the respiratory tract, which are s applicable for use in intact humans and animals. These measurements range from commonly used clinical tests of respira- tory function to currently developing assays of the integrity of tissue bar- riers. Most of the measurements described in this chapter are relatively noninvas- ive and do not require anesthesia, cath- eterization, or collection of tissue sam- ples. Several are readily adaptable to large-scale epidemiologic studies; how- ever, some that require extensive or high- ly sophisticated equipment are best suit- ed to studies of small groups. The physiologic markers of early biolog- ic effects in intact organisms can be de- scribed in four general categories: Markers of changes in respiratory (gas-exchange) function of the lung. These are derived from measurements of ventilation and its control, lung mechan- ical properties, intrapulmonary gas dis- tribution, and alveolar-capillary gas exchange. A wide variety of such measure- ments have been developed and are in common use. Markers of increased airway reactivi- ty, both to specific environmental agents and to standardized physiologic provoca- tion. These are usually indexes of respir- atory function. They are distinct from markers in the first category, in that the focus is not on gas exchange. Markers of change in clearance of par- ticles. Measurement is used to examine an important set of respiratory defense mechanisms, and alterations of the mech- anisms at times provide an early indica- tion of an adverse effect of inhaled environmental agents. Markers of increased permeability of the air-blood barrier and of increased uptake of potentially injurious materials in the lung. Increase in permeability is sometimes an early feature of lung in- jury due to inhaled particles. MARKERS OF ALTERED STRUCTURE OR FUNCTION Chapter 4 describes two of the simplest methods of assessing alterations in lung structure: examination with the naked

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6 eye and microscopic examination. Of course, access to relevant materials in vivo is problematic, requiring in most cases direct access to lung tissue during surgical procedures. Lung tissue at autopsy is very helpful in establishing the burden of inhaled particles. Separate autonomic regions can be sampled, and quan- titative data obtained. Apart from gross examination of lung tissue, both light and electron microscopic examination can be useful. Light microscopy has the ad- vantages of speed, accuracy, and low cost. It also has some limitations, in that the types of measurements of tissue compart- ments are restricted. Such measurements are more easily made with electron micros- copy. However, electron microscopy re- quires expensive equipment and more highly trained personnel. Newer techniques using electron and x-ray microscopy can also identify elements such as silica, cobalt, and nickel in human lung. The proliferation of epithelial cells, fibroblasts, and macrophages can be meas- ured microscopically. Proliferation of those cells can serve as markers of lung damage. For example, oxidant gases and chrysotile asbestos fibers cause rapid incorporation of tritiated thymidine into bronchiolar and alveolar cells. Detec- tion and analysis of inhaled particles are possible with microscopic techniques. The size and distribution of particles in pulmonary tissue can be assessed with current morphometric techniques. MARKERS OF INFLAMMATORY AND IMMUNE RESPONSE Chapter 5 discusses markers associated with the inflammatory and immune responses of the respiratory tract. Inflammation in the lungs starts with the macrophage, the resident cell in the lower respiratory tract. The macrophage response is usually followed rapidly by an influx of neutro- phils, which can damage tissue directly. Neutrophils are, however, most effective for destroying bacteria, and their pres- ence constitutes a primary response to injury in any part of the body. The presence and timing of the neutrophil influx are MARKERS IN PULMONARY TOPOLOGY good markers of inflammatory response in the respiratory tract. Neutrophils are seen in nasal washings and in other biolog- ic samples from the respiratory system after exposure. Another inflammatory cell, which is less commonly seen, is the eosinophil. It is attracted to the lung by chemotactic factors released during an inflammatory response. Like neutrophils, eosinophils can release chemotactic factors for other cells. They can also release several potential irritants, including oxygen radicals and leukotrienes. The influx of neutrophils and eosino- phils is usually seen in the first 3-7 days of an inflammatory response. If the in- flammatory response is sustained, it is usually accompanied by a specific immune response. The immune response is general- ly mediated by lymphocytes, which migrate into the inflamed area within days of the original insult. Apart from cells in the inflamed area that serve as markers, proteins and other products of cells can be detected. Those products often appear in predictable pat- terns that allow one to estimate the dura- tion and intensity of the inflammatory response. Increases in proteins and other products in biologic fluids, such as bron- choalveolar ravage fluid, can serve as sensitive markers of the inflammatory response. The human respiratory tract contains a complex array of host defenses-anatom- ic barriers, mucociliary clearance, phag- ocytic cells, and various components of cellular and humoral immunity-that col- lectively cleanse inhaled air and inac- tivate infectious and other injurious agents that are inhaled. In particular, the mucosal lining of the small airways and alveolar airspaces contains many com- ponents of the immune system that are im- portant in protecting the normal lung. However, some of the components also play an important role in immunologic lung disease. Antigen-antibody complexes are the basis of immune response. The initial phase of the immune response usually be- gins with antigen processing by phago- cytes, such as macrophages. That includes

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EXECVTM: SPRY degradation of foreign substances and exposure of lymphocytes to antigens, which stimulate the production of antibody, sensitized cells, or both. Chapter 5 describes the mechanisms of the immune response and the different cells and fac- tors involved in the immune response that can be used as markers. MARKERS OF CELLULAR AND BIOCHEMICAL RESPONSE Chapter 6 discusses current and devel- oping cellular and biochemical techniques that provide a possible source of markers peculiar to the lung and upper respiratory tract. The most important of the new tech- niques are bronchoalveolar ravage and nasal ravage. The predominant cell in bronchoalveolar ravage fluids from healthy people is the macrophage. Howev- er, in an inflammatory state, neutrophils, eosinophils, and mast cells might be pres- ent. Macrophages play an important role in the regulation of the immune cellular response, acting as both promoters and suppressors of events during inflamma- tion. Thus, bronchoalveolar ravage fluid is ideally suited to the study of cells from respiratory tract and their response to chemical insult in both laboratory ani- mals and humans. For example, alveolar macrophages secrete interleukin- 1; the role of interleukin- 1 as a mediator of inflammation is uncertain, and broncho- alveolar ravage is providing important mechanistic information on it. Biochemical markers of pulmonary dis- ease processes are also present in ravage fluid. Lactate dehydrogenase, a cytoplas- mic enzyme released from damaged or lysed cells, can be used as a marker of cytotox- icity. An increase in this enzyme ac- tivity can be used to distinguish between toxic events and physiologic responses. An increase in serum proteins in the ravage fluid serves as an indicator of increased permeability of the alveolar-capillary barrier. The activity of hydrolytic and proteolytic enzymes released into the epi- thelial lining fluid has been shown to be correlated with the toxicity of inhaled particles. Biochemical and cellular mark- ers of inflammation are useful in animal 7 toxicity studies to rank the toxicity of a series of compounds and to study the mech- anisms of development of late-occurring lung diseases, such as fibrosis and emphysema. Testing for immune cellular response has often used whole animals or humans. The interaction of several components of the immune system can be tested, and body responses can be measured, so one need not rely on extrapolation from results obtained in vitro. The examination of individual cellular components is de- scribed in detail in Chapter 6. Exposure to environmental toxicants can cause damage in individual cells at the level of DNA and other cellular com- ponents. New techniques are now available to measure molecular markers of damage to cell components. Molecular markers have proved useful in the detection and diagnosis of some infectious diseases and in sickle-cell anemia and alpha- and beta-thalassemia, and they are being de- veloped for other diseases, such as Duchenne muscular dystrophy, cystic fi- brosis, Lesch-Nyhan syndrome, phenylke- tonuria, antithrombin III deficiency, and alpha,-antitrypsin deficiency. The development and use of molecular markers to identify cellular responses to en- vironmental toxicants will be important in increasing understanding of the mech- anisms involved in pulmonary disease. RECOMMENDATIONS The recommendations of the Subcommittee have been divided into four major groups: exposure dosimetry, physiologic measure- ments, structural and functional meas- urements, and cellular and biochemical measurements. Exposure Dosimetry More information is needed on factors that affect the dosimetry of inhaled toxi- cants at specific sites along the respira- tory tract. Specifically needed is infor- mation on the following: Regional deposition of inhaled pol- lutants at various sites along the respir

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8 atory tract. Considerable information is available on regional deposition of inhaled particles larger than 0.1 ,um in aerodynamic diameter, but relatively little is known about factors that govern the deposition of inhaled gases, vapors, and ultrafine particles (smaller than 0.1 Em in aerodynamic diameter). Specific factors that affect deposition, particu- larly airway structure, need to be assess- ed in both laboratory animals and humans. Pollutant effects on clearance of deposited material. Dosimetry at speci- fic sites in the respiratory tract depends both on how much is deposited at the site and on how quickly it is removed. Inter- species studies of regional clearance are required. The capacity of tissues at the site of deposition to metabolize a pollutant to a more toxic or a less toxic form. The toxicity of an inhaled organic compound might be increased by metabolic activity at some sites of deposition. Effects of chemical and physical char- acteristics of pollutants on the site of sequestration and on the induction of injury in the respiratory tract. Physiologic modeling of the pharmacoki- netics of inhaled materials in animals and humans shows promise for allowing ex- trapolation of dosimetry data between species, sexes, and regimens. Extension of that approach to the active metabolites of inhaled compounds would greatly in- crease our understanding of the toxicity of inhaled materials. Physiologic model- ing also requires information on deposi- tion, clearance, and metabolism. One region of the respiratory tract that has received little attention, but is read- ily accessible for sampling, is the nose. The analysis of nasal rinses or sputum to detect exposures to specific pollutants could be useful. It must be applied with appropriate knowledge of dosimetry dif- ferences between the nose and the rest of the respiratory tract when different toxi- cants are inhaled. Macromolecular adduct formation offers a promising new method of measuring expo- sure to organic chemicals that are reac- tive or can be metabolized to reactive MARKERS IN PULMONARY TOXICOLOGY forms. Further research on the kinetics of adduct formation and clearance is required to determine the relationship between exposure history and the concen- tration of adducts in blood or tissue samp- les. Most research has been on formation of adducts with DNA and hemoglobin. Ad- ducts with other macromolecules, particu- larly those with site specificity, should be explored as markers of dose. Physiologic Measurements Existing physiologic tools need to be extended and new tests need to be developed and evaluated to focus on specific sites of action of environmental pollutants and specific effects of given pollutants. That requires evaluation of pathophysio- logic correlates assessed initially in animals and later in humans, both in con- trolled exposure settings and in popula- tion-based samples. Further research is required on the role ~ ~. ~ OI lIlCreaSeS In llOIlSpeC11 1C airway reac- tivity in identifying persons susceptible to environmental agents and on the role of increases in airway reactivity in the natural history of chronic obstructive pulmonary disease (COPD). The role of transient changes in airway reactivity in response to specific environmental agents needs to be assessed in regard to risk of development of COPD. Links among alterations in particle clearance, environmental exposures, and development of lung disease need to be studied further to determine the useful- ness of clearance as a marker of suscep- tibility and response. Markers of early endothelial changes that would identify persons likely to de- velop acute or chronic vascular injury are needed. Markers of endothelial dys- function that demonstrate toxicant speci- ficity should be sought. More information is needed on how endothelial barrier func- tion is correlated with nonbarrier functions. Refinements in techniques are needed to render them applicable to the screening of large numbers of people for vascular function. Immunologic, biochemical, cytologic, and structural markers identified as re

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EXECUTED? SPRY lated to specific lung injury need to be correlated with physiologic measures of respiratory function, airway reactivi- ty, particle clearance, and indexes of air-blood barriers. Understanding of those relationships could be important in developing methods for assessing risks associated with environmental exposures. Structural and Functional Measurements Animal studies are needed for increasing understanding of the pathologic sequelae of particle disposition at specific sites in the lung. Examples of some analytic methods that can be made highly site-spe- cific and cell-specific are morphometry, immunocytoche mistry , his to c hem is try , and in situ hybridization (i.e., formation of RNA-DNA hybrids). Research is required on the specific cell kinetics of response to injury. Labeling indexes determined by autoradi- ography are not adequate for describing cell kinetics. New techniques, such as a combination of autoradiography with morphometry to measure cell pool sizes before and after injury, can make it pos- sible to determine changes in the entire cell cycle during lung injury. Three-dimensional reconstruction of cells and tissues could establish changes in intracellular organelles and cell-cell relationships that result from exposure and injury. Such techniques as computer- assisted tissue reconstructions, time- lapse photography, and high-voltage elec- tron microscopy can be applied to obtain data on cell function and cell regulation. Lavage fluids should be analyzed to de- termine whether exposure to particles or gases changes chemotactic activity. Alterations could be due to depletion or activation of pulmonary C5, oxidants, arachidonic acid metabolites, growth factors, and other chemotactic factors that might be important markers of response. Cell- and organ-culture models should be developed for extrapolating animal data on histologic changes to humans. 9 Findings on early histologic markers of exposure and injury in animals are diffi- cult to apply to humans, because they re- quire invasive techniques. New ways to maintain and use human cells obtained by bronchoalveolar ravage, transbronchi- al lung biopsy, and tracheal exploration need to be developed. Cellular and Biochemical Measurements Bronchoalveolar ravage has proved use- ful for evaluating lung inflammation, but further research is required to deter- mine its utility for assessing pollutant exposure. Interspecies studies are needed to determine relationships between changes in bronchoalveolar-lavage fluid constituents and site-specific and pol- lutant-specific injury. Where applic- able, clinical studies should be used for confirmation of results. Cell and mediator changes found in bron- choalveolar and nasal-ravage fluid need to be related to physiologic and patho- logic changes to assess their utility as biologic markers. Nasal ravage needs to be investigated as a means of evaluating pollutant expo- sure and as an epidemiologic tool. The characteristics of bronchoalveolar- lavage fluid, nasal-ravage fluid, and whole-lung specimens need to be correlated in humans and animal models. Monoclonal antibodies and molecular genetic techniques need to be applied to characterize types and functions of cells of the respiratory tract. As those tech- niques are introduced, relationships between phenotypic changes, pollutant exposure, and cell function should be established. It would be of value to identify markers of susceptibility. Changes in cells and mediators in ravage fluid should be examined as possible markers of susceptibility. Markers of the early events leading to late-stage disease should be developed to serve as molecular probes of mechanisms of disease.

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