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Markers of Physiologic Effects in Intact Organisms This section discusses markers of pul- monary response that can be applied in studies of intact human subjects (all can also be applied to animals). The focus is on physiologic tests of respiratory system function that reflect early biologic ef- fects or underlying changes in lung struc- ture, function, or sensitivity to inhaled materials. Most of the techniques reviewed here can be applied in a relatively noninvasive manner and do not require anesthesia, cath- eterization, or collection of tissue sam- ples. Many are suitable for epidemiologic studies of large populations and have the characteristics of mobility of equipment, short subject interaction time, minor requirement for subject training, ability to be performed by technicians, and auto- mated data processing; an example of such a technique is spirometry, the most common of those discussed in this section. The equipment, personnel, and sample- collection requirements of others imply that they are likely to be used only in sta- tionary facilities. Many centers are suit- able for these tests, but they are most appropriate for evaluation of small popu- lations. Techniques that use gamma-camera imaging are examples of this class because gamma-camera equipment is large. Other 43 approaches require equipment and exper- tise that are likely to be present only in a few specialized laboratories, for ex- ample, measurements of the dispersion or clearance of inhaled boluses of particles. The markers of early biologic effects or altered structure/function discussed here are in four general categories. The first includes markers of respiratory or gas-exchange function of the lung. These are derived from tests of ventilation and its control, lung mechanical properties, intrapulmonary gas distribution, and alveolar-capillary gas exchange. A wide variety of such tests have been developed and are in common use to evaluate lung func- tional competence. Although all these tests yield markers of lung response, only a few thought most likely to have potential for detecting responses of populations to environmental exposures are discussed in detail. The second category includes markers of increased airway reactivity, both to specific environmental agents and to standardized physical or pharmacologic bronchial provocation. Although these are usually indexes of respiratory func- tion, they are distinct from the first category, in that the focus is not on gas exchange. The third category includes markers derived from measurement of the .. . . ~
44 clearance of particles. These measure- ments examine an important set of defense mechanisms of the respiratory system, and alterations of the mechanisms sometimes give an early indication of an adverse impact of inhaled environmental agents. The fourth category includes markers of increased permeability of the air-blood barrier, which is sometimes an early feature of lung injury due to inhaled materials. The usefulness of assays of physiologic function is generally limited to their ability to demonstrate responses to in- haled environmental agents. They reveal the functional manifestations of struc- tural changes in the respiratory system, whether the changes are transient (e.g., bronchoconstriction) or lasting (e.g., fibrosis). As a group, the assays have little specificity for specific environ- mental agents. The lung responds to injury in a limited number of ways, and the func- tional manifestation of a given type of injury is often the same, regardless of the causative agent. An exception to both those generalizations might be the useful- ness of an increase in airway sensitivity to specific agents as a marker of exposure and sensitization. The sensitivity of physiologic tests varies, but many demonstrate substantial intersubject variability. The sensitivi- ty is often improved when control popula- tions are studied or when individual base- line values are obtained; however, there is often a reliance on predicted normal values. The results of physiologic assays generally depend on technique, and both the degree of standardization of technique and the magnitude of the data base for pre- dicting normal values vary widely from assay to assay. Some of the physiologic tests offer the advantage that a response, once detected, can be placed in a context that can be interpreted in terms of its practical impact on the subject. That is based on knowledge of correlations among measured values related to function, sub- jective perception of ill health, clinical lung disease, and impairment of physical performance. MARKERS IN PULMONARY TOXICOLOGY RESPIRATORY FUNCTION The respiratory functions of the lung include mechanisms involved in ventila- tion, gas distribution,alveolar-capil- lary gas exchange, and perfusion. Although developments in the understanding and evaluation of respiratory function are continuing, most of these physiologic phenomena are well-studied, and many as- says of function are long established. Table 3-1 lists several common assays of respiratory function. Note that each list- ed category contains numerous individual tests or measured characteristics. Only a few of the many assays of respira- tory function are discussed in detail in this section, but all the assays listed are useful in a clinical setting and yield potential markers of response to environ- mental exposures. As there is considerable literature on the performance and inter- pretation of the assays; it would be inap- propriate to review it all here. The intent of this review is to comment on the function tests that the Committee thought were most likely to have potential for studies of the effects of environmental exposures in occupational groups or more general populations. Considerable attention has been focused on the development of respiratory function tests that are sensitive to alterations in the region of terminal bronchioles and respiratory bronchioles-i.e., small- airway disease. That anatomic region is at particular risk from several types of inhaled toxicants. Therefore, respira- tory function tests that have been proved (or proposed) to have particular sensi- tivity to small-airway disease are dis- cussed here. Current knowledge suggests that a given test has little usefulness for producing markers of effects of speci- fic environmental exposures. Spirometry The forced expiratory maneuver, which records the time taken to expel as quickly as possible as much gas as possible from a full deep inspiration, is the mainstay of both clinical pulmonary physiology and epidemiologic field studies designed to
PHYSIOLOGYIN INTACT ORGANISMS 45 TABLE 3-1 Assays of Respiratory Function Breathing pattern Respiratory frequency, tidal volume, and minute volume Inspiratory-expiratory times and flow rates Alveolar ventilation Physiologic subdivisions of lung volume Total lung capacity Vital capacity Functional residual capacity Residual volume Inspirato~y and expiratory resents volumes Spirometry (forced exhalations Forced vital capacity (FVC) Forced expired volume in 1 second (FEV') and %FVC in 1 second Peak aspiratory flow Mean midexpiratory flow Flows at selected absolute lung volumes or portions of FVC Breathing mechanics Dynamic lung mechanics Dynamic lung compliancea Total pulmonary resistance Airway resistance and conductance, and specific airway resistance and conductance Oscillation mechanics (respiratory system impedance, composed of compliance, resistance, and inertanceJa Static-quasistatic lung compliancea Intrapulmona~y gas distribution Single-breath gas washouts Multiple-breath gas washout Imaging of radiolabeled gas and particlesa Particle bolus distributions Alveolar-capillary gas transfer Blood gases, pH, and alveolar-arterial gas tension differences Oxygen and carbon dioxide exchange at rest Diffusing capacity for carbon monoxides Gas exchange during exercises Multiple-gas evaluation of ventilation-perfusion relationships Evaluation of respiratory control . aDiscussed In this report. explore the development of chronic ob- structive pulmonary disease. The volume of air expelled in the first second is termed the forced expiratory volume in 1 second (FEVER. The total amount expelled is called the forced vital capacity (FVC). The flow-volume curve is a plot of expira- tory flow rate against expired volume, and it is also analyzed to evaluate flow limitation. Patients with chronic obstructive res- piratory diseases are those whose airflow limitation prevents them from continuing activities that they would otherwise be able to perform (Speizer and Tager, 1979~. There is a continuum between the normal state and the diseased state. Most people would consider themselves substantially disabled when their FEV, approached 40- 50% of the predicted value. Most physi- cians would take an FEV1 below 65% of the predicted value as indicative of obstruc- tive disease. LEVI tends to decline smoothly in adult life with modest acceleration with in- creased age (Figure 3-1 ) (Speizer and Tager, 1979~. Several studies have shown that airflow function is a strong predictor of morbidity (Fletcher et al., 1976~. Thus, FEVER is a good biologic marker of risk of developing obstructive pulmonary disease.
46 FIGURE 3-1 Decline of FEVER at normal rate (solid line) and at accelerated rate (dashed line). "A" represents a person who has attained a "normal" maximal F~V~ during lung growth and development. "B" represents a person whose mammal FEVER has been reduced by childhood respiratory infection (CAO). Repnnted with permission from Samet et al., 1983. Additional measures of ventilatory function that are general biologic markers of risk of chronic respiratory disease are based on a forced expiration. To use them, one must have recording devices that capture the essential components of the maneuver or the entire curve. The total volume of an expiration, the FVC, has been recognized since the mid-nineteenth cen- tury as a predictor of "vital status" (Hut- chinson, 1846~. In the 1960s, the Framing- ham heart program found FVC to be a predic- tor of total mortality (Kennel et al., 1980~. More recently, FEW and FVC measure- ments obtained some 20 years previously were used to predict specific respiratory disease mortality (Peso et al., 1983~. Other components that are believed to rep- resent the flow characteristics of the airways include flow after 50 or 75% of the volume has been forcibly expelled (forced expiratory flow of 50 or 75%, FEF50 or FEF75) and the maximal midexpiratory flow (MMEF, the slope of the line drawn between points at 25 and 75% of the FVC). The same measures have all been used (with other measures discussed below) to test the effects of exposure to an array of environmental agents, generally at concentrations that exceed only slightly those occurring in the environment. In each case, pre-exposure, postexposure, and recovery measurements were compared. The interpretation of those measures as biologic markers of risk is only partly MARKERS IN PULMONARY TOXICOLOGY 5 4 2 1 o B ~ Normal Normal ~ CAO 25 35 45 55 YEARS 65 75 understood. When asthmatic subjects are exposed at rest to SO2 at 0.25-0.5 ppm, some undergo significant reductions in FIVE (Sheppard et al., 1980~. Whether the re- sults predict which asthmatics are more susceptible to naturally occurring en- vironmental insults is as yet unknown. However, we do know that children who re- port wheezing or asthma generally have more respiratory symptoms than those who do not when exposed to ambient environments with particulate pollution (Ware et al., 1984~. No relation between pollutant con- centration and magnitude of FEV, FVC, or MMEF change has been discerned, but chil- dren with a history of wheezing clearly have lower MMEF. In most chronic respiratory diseases other than asthma, lost ventilatory func- tion does not return. By the time impair- ment is judged to be significant, there is little need for subtle biologic markers of risk of disease (it might still be ap- propriate to use markers to study mechan- isms). Therefore, it is important to con- sider how the sensitivities of the other markers compare with the sensitivity of FEY,. For example, subtle decreases in flow at 50 or 75% of vital capacity (FEF50 or FEF75) might be associated with cigar- ette-smoking without necessarily being simultaneously associated with reduced FEY,. Some have argued that those findings represent changes in small airways (Gelb and Zamel, 1973~. Typically, reduced FEF50
PHYSIOLOGYIN INTACT ORGANISMS or FEF75 without a reduced FEVER has not been used to define obstructive airway disease (Speizer and Tager, 1979~. MMEF similarly might be reduced in association with ex- posure to respiratory irritants, but this has not typically been used to define ob- structive disease without a reduced FEW. Those more subtle measures are useful as biologic markers, because they might indi- cate earlier or more subtle damage to small airways that, if not reversed, could lead to more severe and irreversible damage reflected in reduction in LEVI and even- tually in FVC. Mechanical Properties of the Lung Dynamic Lung Mechanics Measurement of dynamic lung mechanics is a means of assessing the work of breath- ing. The work of breathing is incurred in the need to overcome elastic, resistive, and inertial forces of the lung tissue and air column (Mead and Agostoni, 1964; Rodarte and Rehder, 1986~. Dynamic lung mechanics-lung mechanical properties during breathing-are usually expressed in terms of dynamic lung compliance (indicative of work required to stretch the lung) and airway resistance (indic- ative of work required to overcome resis- tance to airflow). Classical measures of dynamic lung mechanics are often useful in evaluating clinical lung disease, but by themselves have low potential as sensi- tive markers of lung response to environ- mental exposures. Tests commonly applied in epidemiologic studies to detect abnormalities of respir- atorY function typically evaluate lung volume and resistance to airflow, but do not examine the compliance. Lung compli- ance is reduced in disorders such as in- flammation and fibrosis, which increase lung elastic recoil, and is increased in diseases like emphysema, which decrease elastic recoil. Measurement of static or quasistatic compliance demonstrates those changes more sensitively than does measurement of dynamic compliance. Aside from forced-oscillation methods, measure- ment of dynamic compliance requires place- ment of an esophageal balloon catheter. 47 Although that is not difficult or hazar- dous, it is sufficiently time-consuming and unpleasant for its use to be limited usually to selected clinical subjects and to the physiology laboratory. Oscillation techniques now constitute the most likely use of lung compliance as a marker of response. Dynamic lung compliance depends on breathing frequency. Compliance de- creases with increasing breathing fre- quency, because of regional inhomogen- eities among lung units (Otis et al., 1956~. Measurement of the frequency de- pendence of compliance was introduced as one of the first tests "specific" for small-airway disease (Woolcock et al., 1969~. Compliance is measured as the sub- ject breathes over a range of frequencies, and the magnitude of the reduction is noted. Although the dependence of compli- ance (or resistance) on frequency is often mentioned as a potential marker of small- airway disease, these changes have actual- ly been correlated with structural changes in the lung in only a few studies (Berend, 1982~. The dependence on frequency has been shown to be abnormal in a large portion of asymptomatic young smokers (Martin et al., 1975~. The compliance test is not broadly used, and there are few data on which to base either an estimate of its usefulness in population studies or es- timates of normal values. The most common method of assessing re- sistance to airflow is spirometry during forced exhalation. Resistance to airflow during either forced exhalation or tidal breathing is commonly used to indicate response in studies of airway sensitivity and in evaluating experimental exposures of humans to inhaled toxicants. Although resistance can be measured during tidal breathing with esophageal catheters or oscillation methods, it is most commonly measured with plethysmography (Leith and Mead, 1974; Zarins and Clausen, 1982~. The subject is seated within a body pleth- ysmograph (a box with transducers that sense changes in pressure) and breathes with a panting pattern while flow at the mouth and pressure changes within the plethysmograph are measured. The airway is then occluded, and mouth pressure is
48 measured as representative of alveolar pressure. The resulting data are used to calculate resistance and thoracic gas volume. Resistance can depend on volume, so it is often divided by volume and ex- pressed as specific airway resistance (resistance per unit volume) or its recip- rocal, specific airway conductance. Current measurements of dynamic lung mechanics are useful clinical tools, but are unlikely to gain substantially broader use in population studies. The information obtained represents the integrated re- sponse of the entire lung; the lack of re- gional specificity and the lack of sensi- tivity due to intersubject variability reduce its utility as a marker of subtle effects. The greatest potential for devel- opment as a marker appears to lie in the use of oscillation methods because they are noninvasive and provide considerable information without requiring difficult procedures as described below. Respiratory System Impedance (Oscillation MechanicsJ Measurement of "oscillation mechanics" is a means of evaluating the mechanical properties of the respiratory system. The technique provides a marker of response in the form of information on changes in the mechanical properties of the lung. The technique is rapid and and requires little cooperation from the subject-char- acteristics that make it suitable for epi- demiologic studies. It provides indexes of lung compliance, as well as resistance; thus, it has potential for adding to the spectrum of information obtained in popu- lation studies. Tests of oscillation me- chanics are in use for measuring the in- tegrated compliance and resistance of an entire lung, but its potential as a marker is primarily in describing mechanical properties of specific regions of the res- piratory system. The extent of the poten- tial is uncertain and is the focus of cur- rent developmental work. Oscillation mechanics was recently reviewed by Peslin and Fredberg (1986~. The general approach is to superimpose an oscillating pressure signal on the air- way during normal tidal breathing with MARKERS IN PULMONARY TOXICOLOGY a loudspeaker or pump. The frequency of oscillation is higher than the respiratory frequency of the subject, and the oscillat- ing pressure and flow changes are small. The resulting pressure, volume, or flow perturbations in the air column are meas- ured and used to calculate values of com- oonents of the mechanical impedance (re- s~stance, compliance, and inertance) of the respiratory system. The response of the respiratory system to an oscillating signal is determined by its impedance, which in turn is deter- mined by its anatomic and mechanical prop- erties. The overall response of the system represents an integration of the elastic, resistive, and inertial charac- teristics of each component of the system. In the simplest form of the assay, oscilla- tion at a single frequency is used to meas- ure dynamic compliance and airway resis- tance of the entire lung, without the need for a body plethysmograph or esophageal catheter. By manipulating the oscillating signal and analyzing the resulting re- sponse, one can theoretically extract information specific for different mech- anical properties and for different ana- tomic structures. Two approaches have been used for inter- preting respiratory system impedance. One is the empirical association of changes in impedance with lung abnormalities (Kejeldgaard et al., 1976~. The second is the estimation of specific impedance parameters by fitting impedance data to mathematical models of the respiratory system, which are based on mechanical or electric analogues (Jackson et al., 1984; Peslin et al., 1986~. The latter approach should provide more descriptive informa- tion, if model parameters can be correlated with physiologic elements of the respira- tory system. Much of the effort in this field is di- rected toward development of improved models of respiratory system impedance. Previous work focused primarily on oscil- lating frequencies of 2-32 Hz, and it now appears that such data allow reliable ex- traction of only the integrated compli- ance, resistance, and inertance of the total respiratory system (Jackson et al., 1984~. By extending the range of oscillat
PHYSIOLOGYIN INTACT ORGANISMS ing frequency, one can obtain statistical- ly reliable estimates of additional param- eters. For example, Jackson and Watson ( 1982) differentiated between central and peripheral resistance, compliance, and inertance by fitting oscillation data from rats to a six-parameter model. Their group has obtained similar results with other animal species, but has encountered difficulties in applying such models to data from humans. They hypothesize that models for humans need to account for shunting of flow in upper airways and for acoustic phenomena that occur in the rela- tively long airways of humans. No models have yet been shown to be satisfactory for clearly discriminating between mechanical properties of central and peripheral air- ways of human lungs. It is not clear whether oscillation meas- urements will constitute improved tools for detecting and describing abnormali- ties of respiratory system mechanics due to environmental exposures. Considerable work remains to be done to develop appro- priate models and to confirm associations among impedance changes, physiologic correlates, and alterations in respira- tory system structure. That will require both application of the method to patients with known abnormalities of representa- tive types and the study of animals with specific, experimentally induced abnor- malities. Those lines of research are just now being pursued, and the utility of the approach is not likely to be fully known for a few years. Measurements of oscillation mechanics with substantially improved descriptive value beyond that of tests currently in use would require specialized equipment. The oscillating system would consist of computer-generated signals fed to care- fully calibrated loudspeakers or pumps. The frequency-response characteristics of the measurement system would have to be optimized. The data-reduction and mod- el-fitting systems would be computer- based. Although the equipment would be specialized, it could probably be packaged into a mobile unit that could be operated by people with only modest training. Pro- fessional input would be required for cali 49 bration, supervision of maintenance, and interpretation of results. In summary, oscillation mechanics has potential for development into a useful marker of response. Its advantage lies in its ability to distinguish mechanical abnormalities on a site-specific anatomic basis. Its primary disadvantages are its dependence on an appropriate model for fitting data and the likelihood of substan- tial variation among individuals in re- gional mechanical properties of the res- piratory system. General acceptance and widespread use will require substantial effort to demonstrate physiologic and clinical correlates, standardization of procedures and analysis, and packaging into measurement systems that are readily used. Static-Quasistatic Lung Pressure-Volume Analysis Lung compliance measured during breath- ing is usually lower than the actual com- pliance of lung tissue, because of the lack of time for tissue relaxation and because of differences in compliance among lung units. Measurement of static or quasi- static compliance avoids such frequency dependence by plotting transpulmonary pressure against lung volume during a single, slow exhalation. The current tests are assays that examine the elastic properties of lung tissue most directly and are the procedures of choice, if a spe- cific index of lung elastic recoil is de- sired as a marker of response. Standardized procedures for measuring lung compliance were recommended in a re- port from NIH (Macklem, 1974~. Transpul- monary pressure is measured with an esopha- geal balloon catheter (Dawson, 1982~. The subject inhales to total lung capacity and then exhales slowly while the exhaled volume and transpulmonary pressure are recorded. The test is termed quasistatic if exhalation is continuous, and static if exhalation is interrupted periodically to allow flow to cease and elastic forces to come to equilibrium. The elastic char- acteristics of the lung are expressed either by calculating compliance as the
so slope of some portion of the pressure- volume curve or by simply displaying the entire curve. The lung pressure-volume curve shifts to the left (compliance increases) when lung elastic recoil is reduced (e.~.. in emphysema) and shifts to the right (compli- ance decreases) when elastic recoil is increased (e.g., in fibrosis) (Macklem and Becklake, 1963~. The curve represents the integrated elastic characteristics of the entire lung. It is not specific for the anatomic site of the change in recoil. Nor is it specific for the cause of the change in recoil. For example, fibrosis, inflammation, edema, and proliferative disorders could all cause similar shifts of the curve to the right (showing reduced compliance). Regardless, the test could be a useful marker of response in popula- tions in which abnormal elastic recoil is a likely response. Intrapulmonary Gas and Particle Distribution Gas Distribution Properties- Single-Breath Gas Washout Patterns of the washout of gases inhaled in a single breath have received consider- able attention as indexes of small airway disease. Although the test can be perform- ed by having the subject inhale a bolus of inert gas (bolus technique), the most com- mon approach is to evaluate the washout of nitrogen from the lung after an inhala- tion of oxygen (resident-gas technique). The single-breath nitrogen washout (SBNW) test was introduced in 1969 as a test of small-airway function (Anthonisen et al., 1969~. The air-breathing subject exhales to residual volume, inhales a sing- le breath of oxygen, and exhales again to residual volume. The nitrogen concentra- tion of the expirate is plotted against its volume. The normal curve has a charac- teristic shape, first noted in 1949 (Fowl- er, 1949), in which the nitrogen is ini- tially low (washout of dead-space oxygen), rises to a plateau that represents the nitrogen-oxygen distribution in the ma- jority of the lung, and then increases again near the end of the exhalation. The M4RKERS IN PULMONARY TOMCOLOGY slope of the curve depends on the uniform- ity of gas distribution among ventilating units and is affected both by asymmetry of airway path lengths and by nonuniformity of compliance among ventilating units (Engel and Macklem, 1977~. The slope in- creases as gas distribution becomes less uniform. The onset of the terminal nitro- gen rise has been termed "closing volume" and is thought to indicate the lung volume at which airway closure begins (Engel et al., 1975~. The phenomena responsible for determining the closing volume remain incompletely defined, but it is generally agreed that an increase in, if not onset of, airway closure is primarily respon- sible (Forkert et al., 1979~. It is interesting that Ernst et al. (1986) examined the relationship of clos- ing volume and fluoride air pollution in children living near an aluminum smelter. In both sexes, there was a significant linear relationship between increased closing volume and the amount of fluoride found in urine samples from the children. Since 1969, the SBNW test has been the focus of numerous physiologic studies and has been applied in several population studies. Standardized measurement proce- dures were disseminated by NIH in 1973 (Martin and Macklem, 1973), partly to fa- cilitate multi-institutional collabora- tive studies funded by the National Heart and Lung Institute (NHLI). Methods for computerizing analysis of the curves have been published (Craven et al., 1976; Cramer and Miller, 1977~. Equipment for perform- ing the test is available commercially, and several equations have been developed for predicting normal values of SBNW para- meters (Gold, 1982~. Although the SBNW test continues to be included in lists of tests sensitive to small-airway disease, its usefulness as a marker of responses to environmental exposures has not been clearly demonstrat- ed. In 1973, a workshop on screening pro- grams for early diagnosis of airway ob- struction (NHLI, 1973) concluded that, "although closing volume and closing capa- city are sensitive tests, they are probably of low specificity and moderate precision, and their validity as an early diagnostic test is unknown." The presumed usefulness
PHYSIOLOGYININTACT ORGANISMS of the test is founded largely on the find- ing in numerous studies that it can detect abnormalities in asymptomatic smokers, often in the absence of abnormalities in "conventional" lung function tests (McCarthy et al., 1972; Buist and Ross, 1973; Nemery et al., 1981; Teculescu et al., 1986). Recent work has more directly demon- strated associations between SBNW abnor- malities and small-airway pathologic conditions. Cosio et al. (1978) and Berend et al. ~ 1981 a,b) found significant cor- relations between abnormal values of wash- out slope and closing volume in human sub- jects and small-airway disease in excised lung tissue. The latter study demonstrated that closing volume was related more close- ly to small-airway inflammation than to lung elastic recoil. Petty et al. (1980) performed SBNW tests on excised human lungs and found that increased closing volume was associated with inflammation and squa- mous metaplasia in small airways. Those results further confirm and define the morphologic basis for SBNW abnormalities. Incalzi et al. (1985) recently published regression equations of SBNW parameters with age, height, and lung volume for 234 normal subjects 20-80 years old with no history of smoking, occupational exposure to known pulmonary toxicants, or chronic respiratory illness. The authors conclud- ed that the variability was too great for detection of subtle changes in population studies. In summary, the utility of the SBNW test as a marker of responses to environmental exposures remains uncertain. The test reflects small-airway abnormalities, but its sensitivity and specificity are questionable. Inhaled-Particle Distribution The deposition of inhaled particles is a function of particle characteristics, airway geometry, and ventilation. The latter two can be altered by exposure to pollutants or by disease, so it follows that aerosols can be used as markers of exposure or response to environmental agents. Although the specific equipment required for tests of aerosol distribution 51 is not generally available in the standard pulmonary function setting, the technol- ogy is neither new nor very complicated. Establishment of guidelines for their use, which do not now exist, could result in uniform application in the future. Aerosol particles can be used to assess pulmonary structure and function, because they can trace the convective motion of air in the lungs and their deposition is related to the dimensions of the airways through which they pass. Three techniques can be used to obtain information from inhaled particles; they allow assessment of airway sizes and inhomogeneities of ventilation and gas mixing. The use of aerosols to assess mechanical clearance from the respiratory tract is discussed later. Assessmentof gasmixing. Intrapulmonary mechanical mixing of gases can be assessed by injecting an aerosol during the entire tidal-volume inhalation or in a pulse dur- ing a portion of this inhalation and then examining the particle concentration recovered in exhaled air. The procedure requires the use of particles with a mini- mal probability of deposition-approxi- mately 0.3-0.5 ,um-so that their loss from the inhaled air occurs largely because of nondiffusive gas mixing in the lungs, i.e., bulk transfer from tidal to reserve air. By separating mixing due to molecular diffusion from mechanical mixing due to airflow, one can estimate the role of molecular diffusion in ventilation (Alt- shuler et al., 1959~. Although the proced- ure provides some assessment of bulk transfer, conclusions as to the sites at which this occurs await the further devel- opment and use of models of aerosol dynam- ics and gas transport in the lungs that take into account the effects of geometric com- plexities on lung ventilation (e.g., Engel,1983~. The distribution of exhaled aerosol in normal people shows a fair degree of inter- subject variability; nevertheless, gener- al profiles are reproducible within groups of subjects, and exhaled-aerosol measure- ments have been used to assess airway ab- normalities, in which case the shape of the aerosol exhalation curve is different from that in normal subjects. In airflow
52 obstruction, for example, the shape of the curve is different because the recovery of particles is decreased, owing to an increased rate of particle deposition and a change in mixing characteristics. A correlation has been found between the percentage of aerosol recovered and the predicted percentage change in FEVER over a wide range of degrees of airway obstruc- tion (Muir, 1970~. A difference in aerosol recovery (i.e., a decrease) has also been demonstrated in coal miners with various forms of pneumoconiosis (Hankinson et al., 1979~. Aerosol probe procedures. The aerosol probe technique allows inferences con- cerning small-airway (< 1 mm) and al- veolar dimensions; it is of particular use for assessing changes in diameter, such as those associated with obstructive lung disease. Conducting-airway obstruc- tion can also be detected with conventional pulmonary function tests, but the latter might be less sensitive than the probe procedures in detecting early changes. However, alveolar size, which is important · · - in assessing emp ~ysema progression, can be estimated in viva only with particle probes. The aerosol probe procedure is a modifi . · ~. ~ cation of the aerosol mixing technique discussed above, in that a period of breath-holding is generally imposed be- tween aerosol inhalation and exhalation. It is based on the concept that the amount of deposition of inert, nonhydroscopic monodisperse particles with a known, but low, rate of sedimentation during the breath-holding period depends on the set- tling distance required for the particles before they come into contact with an air- way wall; this distance is a reflection of the overall dimensions of the airspaces in which the aerosol is found. Particles that are deposited and are removed from the air will not be recovered during ex- halation. In reality, and as should be evident from the discussion of the mixing technique above, not all the inhaled aero- sol will be recovered even if there is no breath-holding period between inhalation and exhalation. In practice, the aerosol probe procedure requires determination of the extra loss of particles due to gravi AL9RKERS IN PULMONARY TOXICOLOGY rational settlement. The ideal particle size range used in the procedure is 1-1.5 Am MMAD (Gebhart et al., 1981~; however, the increased impaction deposition in people with obstructed airways might re · · - Cure some size adjustment. The work of Palmes et al. (1967) estab- lished a basic method for estimating the effective dimensions of the respiratory airspaces with monodisperse aerosols. An aerosol is inhaled; the breath is then held at close to total lung capacity (TLC), with different breaths held for periods of 0-30 seconds; and a volume equal to twice the inhaled volume is then exhaled, to ensure recovery of all remaining airborne particles. The persistence of the aerosol-i.e., the probability of its remaining suspended in air and thus being exhaled-decreases exponentially as a function of breath-holding time. Aerosol inhalation was completed near TLC, so most of the aerosol mass and most of its deposi- tion were assumed to be in the region of the respiratory bronchioles and alveolar ducts; the contribution of anatomic dead- space volume was considered to be small, and its influence on the shape of the aero- sol recovery curve could be ignored. The logarithm of percent of aerosol recovery is plotted against breath-holding time; this results in a curve whose slope (or slopes) is related to the average size of the airways within which residual aerosol remained before exhalation. Results are usually expressed in terms of the half- time of aerosol persistence in the lung. Subjects differ substantially, but the aerosol probe procedure is sensitive to changes in airway dimensions and does yield reproducible results in normal subjects repeatedly tested (Lapp et al., 1975~; in addition, the variability in airway size measured in healthy people was found to be quite similar to that measured in fixed lungs obtained from accident vic- tims. However, the wide intersubject vari- ability in normal people is a negative feature of the test if applied to people with lung abnormalities; e.g., in obstruc- tive disease, the results can be equivocal (Palmes et al., 1971, 1973~. In some cases, increased half-time of aerosol persis- tence indicates enlarged airspaces; but
PHYSIOLOGYIN INTACT ORGANISMS results in patients with diagnosed emphy- sema can be comparable with those in healthy people. Inasmuch as the aerosol has access only to airspaces to which it is delivered by convective airflow, it can penetrate to either predominantly normal or diseased lung tissue, depending on the site of airflow obstruction (if any). Thus, half-times observed in pa- tients are more variable than those observ- ed in healthy subjects. In addition, the amount of aerosol at zero time of breath- holding was generally lower in patients than in normal persons; that indicates increased deposition during the dynamic phase of the breath-holding maneuver, probably due to obstruction and the result- ing narrowing of airways. Another group examined with the aerosol probe were coal miners with pneumoconiosis (Hankinson et al., 1979~. There was some correlation between disease type, aerosol persistence, and calculated airway dimen- sions, but a lack of correlation between persistence and recovery suggested either that the mechanisms that cause changes in those two phenomena are differ- ent or that the changes occur at different sites in the respiratory tract. The latter possibility is of concern, because the results of the tests are a reflection of airway dimensions at various depths In the lungs. A slight modification of the aerosol probe technique might be used to examine particular regions (depths) in the lungs. The modification, known as the bolus probe technique, involves either inhalation of an aerosol bolus followed by a preset (but variable) volume of particle-free air (Palmes et al., 1973) or inhalation of the bolus at various stages of inhala- tion rather than at a particular fixed stage (Heyder, 1983~. When this procedure is used, inhalation of equivalent tidal volumes leads to decreases in measured effective airway diameter as lung volumes decrease. In addition, the measured per- sistence and thus the actual average airway diameter that is measured depend heavily on the inhalation volume containing the aerosol, in that, the more deeply a bolus is inhaled, the greater is the dispersion of the bolus later exhaled and the smaller 53 are the airways being "probed." The airway dimensions calculated from aerosol recov- ery curves for different depths of inhala- tion have been found to agree well with what would be expected, on the basis of comparisons with both morphometric models of the human lung and measured airways in fixed lungs (Gebhart et al., 1981; Heyder 1983; Nikiforov and Schlesinger, 1985~. In an aerosol rebreathing procedure described by Kim et al. (1983), subjects breathe 1-,um particles 30 times/minute from a volume held at 500 ml. That results in magnification of the differences in particle recovery after a single breath. The test is able to screen reproducibly for airway constriction and might also indicate the extent of such concentration; variability in both normal subjects and those with chronic obstructive airway disease was 5-10%. The ability of the test to detect changes early in pathogenesis is not yet known (C. S. Kim et al., 1985~. A variation of the single-breath aerosol bolus technique that is sensitive in de- tecting early airway changes has been de- scribed (McCawley and Lippmann, 1984; McCawley, 1987~. It involves precise con- trol of volume and flow during introduction of a bolus of monodisperse (0.5-pm MMAD) particles. With a dispersion index, it was possible to differentiate between healthy smokers and nonsmokers; thus, the procedure seems capable of detecting the early changes known to occur in the small airways of smokers. There were found to be no differences in tests of forced ex- piration (e.g., FIVE andFVC) betweensmok- ers and nonsmokers, and the coefficient of variation of the dispersion parameter (approximately 17% in nonsmokers and 36% in smokers) was less than that of the pul- monary mechanics tests. The procedure optimizes the protocol for rapid screening and is useful in epidemiologic studies that attempt to assess airway obstruction. Regardless of the specific probe proced- ure used, there are always differences between the simple theoretical assump- tions of the models of aerosol deposition and actual complex situations. Direct anatomic interpretations should be made with caution, and one must bear in mind effects of a number of factors, e.g., axial
54 dispersion of the aerosol bolos, nonuni- formity of the aerosol concentrations in cross-section at the end of inhalation, the relative shortness of the airways (which become increasingly short with greater depth in the lung), the irregulari- ty of branching, and the regional distribu- tion of airflow in the lungs. Radioimaging Techniques. Particles with MMADof 1 ~mormorearesubjecttodeposi- tion by impaction and thus might provide information on obstruction in small air- ways before it is measurable with conven- tional pulmonary mechanics tests. In all cases, the pattern of deposition depends on the distribution of ventilation in the lungs; in obstructive disease, deposition is also influenced by the sites of obstruc- tion, and there is no deposition in regions where there is no ventilation. Radioimag- ing procedures involve the external detec- tion of deposited particles labeled with a radioactive tag. The types of detectors used are the same as those used in measuring particle clearance and are discussed in MARKERS IN PULMONARY TOXICOLOGY might alter amounts present and lead to false conclusions as to regional deposi tion. In addition, 24-hour retention var ies widely in normal people and even more in smokers and people with lung disease. Another technique makes use of gamma camera imaging to differentiate deposi tion in central (hilar) and peripheral regions of the lungs (Dolovich et al., 1976; Emmett et al., 1984~. In the simplest procedure, which uses a calibration with an 81-mKr ventilation scan, it is possible to obtain the ratio of outer to inner zone radioactivity of the tracer particles and to describe a "penetration" index; the index relates the penetration of radio labeled aerosol into peripheral airways to the degree of ventilation of these air ways. Such procedures do show differences between healthy, nonsmoking subjects and asymptomatic smokers; thus, they can de tect changes often not seen with conven tional pulmonary function tests. Penetra tion index has also been found to correlate fairly well with pulmonary function meas Chapter2.ures, such as FEY', in people with diag In normal people, the radiolabeled-nosed chronic obstructive disease (Agnew aerosol image appears to indicate a fairlyet al., 1981; Agnew, 1984~. Unfortunately, even distribution of radioactivity, espe-the tests appear to yield a high number of cially if particles 1-4 Em in diameter arefalse-positives, even though they are very inhaled at flow rates that occur at rest.sensitive. However, obstructive disease or obstruc-Theaboveproceduresprovidenoinforma tion is indicated by the presence of areaslion on the precise anatomic location of deposited particles. An approach to as sessing gamma-camera images to allow bet ter definition of sites of deposition in volves development of concentric zones around the hilar region, each of which constitutes a different percentage (e.g., 25%, 50%, and 75%) of the entire lung field (Foster et al., 1985~. The innermost 50% is considered as the central fraction. Differences in aerosol penetration sometimes observed between smokers and nonsmokers have resulted in the suggestion that radioimaging procedures can be used to produce markers of developing or early disease. They also seem better as sensi tive indicators of obstruction in large airways than in small airways (Chopra et al., 1979~. However, there is an inherent problem in the techniques: they require the use of radioactive aerosols on a rou tine screening basis. Thus, such proced ot concentrated Deposition ano areas ot minimal or no deposition. Much of the form- er occurs in the central bronchi, and the latter in peripheral areas. Early studies (Ramanna et al., 1975; Taplin et al., 1977) indicated qualitative differences in the pattern of deposition between normal people and people with clin- ical indications of airway obstruction; the differences were ascribed to differen- ces in regional ventilation. The quantita- tion of particle deposition to assess ob- struction is generally performed in two ways. One procedure is to use a measure of retention at an appropriate time after exposure to the tracer particles as an index of regional deposition, i.e., tra- cheobronchial versus alveolar. General- ly, retention at 24 hours is the measure used. That is not necessarily accurate, in that changes in particle clearance rates
PHYSIOLo~YIN INTACT ORGANISMS ores should not be used if other, more conventional tests are available for screening. Alveolar-Capillary Gas Transfer Diffusing Capacity for Carbon Monoxide The diffusing capacity for carbon monox- ide (DCCO), sometimes called "transfer factor," is considered the most sensitive measure of alveolar-capillary gas-ex- change efficiency performed with the sub- ject at rest and is recommended by the Amer- ican Thoracic Society (1986) as one of the two primary measures for evaluating impairment of respiratory function (spir- ometry is the other). DLCO is the rate of uptake of a low concentration of inhaled CO, normalized by the alveolar-capillary difference in the partial pressure of CO. CO is used as the indicator gas, because of its high affinity for hemoglobin, which, at low concentrations, obviates the sam- pling of blood to measure the pressure difference. There are several methods for measuring DLCO and multiple variations of each meth- od; however, the single-breath technique (Ogilvie et al., 1957) is preferred. The measurement technique and methodologic factors that influence the results were described in detail in a recommendation for standardization published by the Amer- ican Thoracic Society (1987~. The subject inhales a large breath of a mixture of CO and an inert gas in air, holds the breath for a few seconds. and exhales. A sample of "alveolar" gas is taken late in exhala- tion, and concentrations of CO and the inert gas are measured. The inert gas al- lows the lung volume at end inspiration (termed "alveolar volume," although air- way volume is also included) to be calcu- lated. DLCO is volume-dependent, so the DLCO:alveolar volume ratio helps in deter- mining the contribution of reduction in lung volume to reduction in DLCO. As with most measures of respiratory function, DLCO is influenced by several factors; thus, the test has little specifi- city. DLCO is influenced by membrane fac- tors, such as thickness and surface area; by capillary blood volume; and by the rate 7 55 of combination of CO with hemoglobin. The latter factor makes the test somewhat sen- sitive to hemoglobin concentration and to altitude. Methods for correcting for hemoglobin concentration were included in the recommendations of the American Thoracic Society ~ 1987~. Ayers et al. (1975) reviewed some of the physiologic factors that influence DLCO and a scheme for differential diagnosis. Of the tests performed at rest, measure- ment of DLCO has been shown to be the most sensitive to some abnormalities, such as radiation-induced pneumonitis in humans and animals (Mauderly et al., 1980~. It was the first measure shown to change in a progressive disease. Its inherent sen- sitivity is limited, however, by the pres- ence of considerable variation among in- dividuals. Coefficients of variation among individuals are typically about 4% for normal persons and 7% for subjects with severe obstructive disease (American Thoracic Society, 1987~. It has been recommended that, for a subject to be con- sidered "mildly impaired," the measured value be below 79% of predicted (American Thoracic Society, 1986~. Generally ac- cepted formulas for predicting normal values are available (Crapo and Morris, 1981~. Automated equipment for measuring DLCO is available commercially and is in general clinical use. The test requires sufficient subject cooperation to perform the single-breath maneuver, but is rapid and readily adapted to mobile facilities. The trace amount of CO used in the test is innocuous. The test could be used in large population studies; however, because of variability, it would have questionable utility in such a setting. It would be more useful in studies of more selected popula- tions, such as occupational cohorts. The test is well developed; recent progress has been largely in standardization of technique and development of commercially available equipment for reproducible performance. Gas Exchange During Exercise A major problem in using respiratory function tests at rest to detect subtle
56 abnormalities is the large reserve func- tional capacity of the lung. Exercise testing provides an examination of the performance of both the respiratory and cardiovascular systems under metabolic conditions that increase the demand on them. Although exercise testing requires cooperation from the subject and takes longer than the functional assays describ- ed above, it need not be invasive (i.e., blood samples are not necessarily requir- ed), and it can be performed in the field. If blood samples are obtained, the approach should provide greater sensitiv- ity to subtle gas-exchange abnormalities than any of the tests performed at rest. Excellent reviews of exercise testing have recently been published. Wasserman et al. ( 1987) presented a thorough review of theoretical and practical aspects, including case reports; and Hansen (1982) presented a concise review, including procedures, equations, and sources of error. Only selected facets of the ap- proach are discussed here. Gas exchange between the cells and the environment requires the effective inter- action of the lung and chest "bellows" (thoracic wall and diaphragm), a heart capable of pumping sufficient blood, a vascular system that can selectively dis- tribute flow to match requirements, and respiratory control mechanisms capable of regulating blood-gas tensions and pH. In a healthy person, the responses of those systems to exercise are predictable and depend only on the rate of work perform- ed and the fitness of the subject (Wasser- man et al., 1981~. The usefulness of exer- cise testing is based on the ability to measure deviations from predicted physio- logic behavior. There are several variant protocols for exercise testing, but all have the goal of using work by large muscle groups to increase metabolic demand. Both treadmill exercise and cycle ergometer exercise are used, but the cycle is usually preferred. The work expended on a cycle is more readily calibrated than that on a treadmill. Vari- ability during treadmill exercise associ- ated with body build, pattern of leg move- ment, and holding onto side rails is mini- mized with a cycle, and there is less risk AL9R~RS IN PULMONARY TOMCOLOGY of slipping. Both prolonged, steady-state exercise and non-steady-state incremental exercise are used. The latter might be preferable, because it can be matched to the subject's ability, and the various factors that affect performance can be distinguished. The basic measurements include electrocardiography, determina- tion of ventilatory flows and volumes, and measurement of oxygen and carbon diox- ide concentrations at the mouth. Manual collection of exhaled gas is usually ob- viated by computer integration of breath- by-breath volumes and gas concentrations; however, manual collection is possible. Measurements of arterial pressure, blood gases, alveolar-arterial gas tension differences, and acid-base status con- tribute substantially to the informa- tion gained from the test, but require arterial puncture. Much information can be gained, however, from numerous other measurements that are. Characteristics that can be measured during exercise and their interpretive usefulness have been listed and discussed at length (Wasserman et al., 1987~. The anaerobic threshold and relationships between heart rate and oxygen uptake appear to have good overall sensitivity to gas exchange and cardiovascular inefficien- cies. The anaerobic threshold is the high- est oxygen uptake that can be sustained without metabolic acidosis, and it is low- ered by any factor that limits oxygen flow to exercising muscles. Although blood samples are required for precise defini- tion of the anaerobic threshold, it can be satisfactorily estimated noninvasively from a plot of carbon dioxide output versus oxygen uptake over a range of work magni- tudes. The slope of the ratio of heart rate to oxygen uptake, plotted over a range of work magnitudes, is sensitive to both air- way and heart abnormalities and can some- times be used to distinguish between the two. The "oxygen pulse," oxygen uptake per heartbeat, equals the product of stroke volume and the arteriovenous oxygen dif- ference and is also sensitive to anemia and carboxyhemoglobin. Several additional characteristics can be measured noninvasively, including maximal exercise ventilation, ratio of
PHYSIOLOGY IN INTACT ORGANISMS dead space to tidal volume, ventilatory equivalents for oxygen and carbon dioxide, respiratory-exchange ratio (ratio of CO2 output to O2 input), expiratory flow pat- terns (examined as are those obtained dur- ing forced exhalation), and ratio of tidal volume to inspiratory capacity (a reflec- tion of lung stiffness). Each of those has a relatively specific sensitivity for particular physiologic abnormalities. The addition of arterial-blood sampling extends the usefulness of exercise testing greatly. Not only are blood-gas tensions and acid-base status of interest, but the alveolar-arterial differences in oxygen and carbon dioxide tensions during exer- cise are probably the most sensitive of all tests of pulmonary gas exchange at the alveolar level. Exercise testing is being applied to clinical subjects in numerous laborator- ies. Application in population studies (discussed by Wasserman, 1985) has been limited. Sue et al. (1987) compared the sensitivities of exercise testing and resting single-breath CO diffusing capa- city (DLCO) to detect abnormalities among 276 current or former shipyard workers. The criterion for abnormal DLCO was set at 70% of predicted, and several prediction equations were evaluated. Only 16 subjects had an abnormal DLCO, and exercise gas exchange was abnormal in all but two of them. In contrast, resting DLCO was abnor- mal in only 14 of 96 men with abnormal exer- cise gas exchange. Thn.ce results suggest that exercise testing can greatly improve the detection of subtle gas-exchange ab- normalities, in addition to providing considerable discrimination among the factors that cause the abnormalities. In summary, exercise testing appears to have one of the strongest potentials among respiratory function assays as a sensitive marker of pulmonary injury as- sociated with environmental exposures. The degree of interaction required from the subject and the extended usefulness of the assay if blood samples are included tend to make it more suitable for studies of limited populations in stationary fa- cilities under the direct supervision of experienced researchers. Equipment for performing exercise tests in a standard 57 ized manner is now becoming available com- mercially. It is practical to consider use of at least some aspects of exercise testing in mobile facilities in the field. Exercise testing would have particular usefulness in studies in which gas-ex- change or cardiovascular abnormalities were of primary interest. AIRWAY HYPERREACTIVITY Although airway provocation testing dates to the late 1940s, it did not emerge as a clinical diagnostic and research tool until the early 1970s. Nonspecific airway hyperreactivity is defined as an exagger- ated bronchoconstrictor response to a variety of chemical, physical, and phar- macologic stimuli. There is now nearly a consensus that nonspecific airway hyper- reactivity is a characteristic shared by virtually all asthmatics (Boushey et al., 1980~; that is, the asthmatic develops bronchoconstriction after inhaling a lower concentration of a provoking agent than is needed to cause a similar degree of change in airway tone in a healthy sub- ject. Thus, in one sense, airway hyper- reactivity serves as a marker of asthma. Its mere presence alone does not define asthma, however, inasmuch as increased bronchial reactivity can occur in other- wise healthy people. Furthermore, airway hyperreactivity has been observed in healthy people after viral upper respira- tory infections (Little et al., 1978), after acute inhalation of ozone (Golden et al., 1978) or sulfuric acid aerosols (Utell et al., 1984), or after inhalation of irritants in the workplace (Brooks et al., 1985~. Whether such transient in- creases in airway hyperreactivity serve as markers of long-term respiratory se- quelae is unknown, but they might well offer a clue to the pathogenesis of chronic respiratory diseases. Nonspecific airway hyperreactivity presumably reflects or mimics events in naturally occurring asthma. Thus, it should not be surprising that many of the proposed mechanisms of asthma have also been linked with the exaggerated contrac- tion of smooth muscle that characterizes airway hyperreactivity. However, it
58 should be noted that airway hyperreactivi- ty does not correlate with histamine reac- tivity of bronchial smooth muscle (Roberts et al., 1985~. Possible mechanisms include alterations in airway geometry, disorders of autonomic regulation of smooth muscle, structural alteration in airway smooth muscle, increased accessibility of stimu- li to the muscle, and release of locally acting mediators of inflammation (Boushey et al., 1980; Sheppard, 1 986b). Present evidence suggests that both neural and nonneural mechanisms contribute to airway hyperreactivity. For example, abnormally small baseline airway caliber increases reactivity, but cannot explain the hyper- reactivity seen in asthmatics in remission or in persons with respiratory viral infec- tions. Autonomic regulation of the airways seems an attractive explanation in studies in which responses can be abolished by directly interfering with nerve transmis- sion or inhibited by pretreating with blocking agents (Nader and Barnes, 1984~. Epithelial injury from viral infections or inhalation of pollutants might result in more direct exposure of nerve endings to an agent that provokes bronchial re- sponse, but evidence of such injury is often not present. Recently, the develop- ment of acute inflammatory response has been linked to the pathogenesis of airway hyperreactivity, especially after ozone inhalation (O'Byrne, 1986~. Several prod- ucts of arachidonic acid metabolism re- leased during the inflammatory response (Sheppard, 1986a) or by immunologic ac- tivation of human lung mast cells (Schul- man, 1986) have been targeted as possible mediators. If inflammation and mediator release are linked with the acute develop- ment of airway hyperreactivity, then re- current episodes would have a greater like- lihood of producing chronic airway effects. Methods of Assessment In the laboratory, airway reactivity testing is divided into two general cate- gories, depending on the choice of nonspe- cific versus specific agents. In both, the increased bronchoconstrictor response is assessed with pulmonary function tests. M'4R=RS IN PULMONARY TOXICOLOGY Nonspecific stimuli include pharmacologic agents, such as methacholine, carbachol, and histamine; exercise; hyperpnea with cold or dry air; and inhalation of hyper- tonic or hypotonic aerosols. Although pharmacologic challenge is used most often in the clinical laboratory, it is less suitable for population studies, espe- cially those involving children. Cold- air challenge with hyperventilation has been used effectively, and response is generally correlated closely with metha- choline responses. Challenges with speci- fic agents, such as common antigens, such chemicals as isocyanates, and such organic materials as plicatic acid (from western red cedar) attempt to identify specific sensitizing agents. Those approaches can be particularly powerful in incriminating occupational chemicals and confirming the diagnosis of occupation-related air- way disease, but might provoke immediate and/or late pulmonary responses that do not resolve spontaneously. Even with spe- cific agents, the interpretation of re- sponses can be difficult and confounded by a variety of factors, such as dose and irritant effects (McKay, 1986~. Inhalation challenge testing with chol- inergic agonists and histamine has varied considerably among laboratories, and efforts to standardize it have been advo- cated (Hargreave and Woolcock, 1985~. Standardized approaches could minimize variability in aerosol generation and inhalation, in methods of measuring re- sponse and expressing results, in prepara- tion and handling of test solutions, and in concomitant use of medications that affect bronchoconstrictor response. Aerosols are usually generated and inhaled with two techniques: continuous genera- tion of an aerosol inhaled during tidal breathing and generation of a puff of aero- sol with a dose-measuring device or hand- held nebulizer and inhalation of the puff during a single deep breath. Aerosol par- ticles range from submicrometer size to several micrometers and from nearly mono- disperse to widely heterodisperse. Such variables affect lung deposition and in- fluence airway reactivity (Dolovich, 1985~. The bronchoconstrictor response is
PHYSIOLOGYININTACT ORGANISMS commonly measured with two methods-on the basis of airway resistance and maximal expiratory flow (FEVER. Both have short- comings. The plethysmographic measure- ment of airway resistance includes resis- tance of the larynx, so an increase in re- sistance could reflect laryngeal narrow- ing or smooth muscle contraction. One potentially confounding problem with expiratory maneuvers is that the test it- self might alter the characteristics being tested. The deep inhalation that precedes measurements of expiratory flow causes transient bronchodilation and lessens the bronchoconstriction in a normal sub- ject, whereas it might increase broncho- constriction in an asthmatic. Never- theless, the bronchoconstrictor response is usually measured as the change in LEVI. Increasing doses of an aerosol are inhaled to construct a dose-response curve, and the results are expressed as the provoca- tion concentration (pc) necessary to pro- duce a decrease in FEW of 20% (PC20~. The PC20 is obtained from the log-dose-re- sponse curve by linear interpolation of the last two points; the lower the PC20, the greater the reactivity. PC20 has been found to correlate closely with methacho- line and histamine concentrations in the aerosol (Hargreave et al., 1983~. An alternative approach is provocation testing with physical stimuli, such as exercise or isocapnic hyperventilation with cold air. Those are naturally occur- ring stimuli and their use obviates the inhalation of pharmacologic agents. They can cause bronchoconstriction by airway cooling or airway drying, as well as through the endogenous release of media- tors in the airway (Barnes and Brown, 1981~. The degree of airway reactivity to methacholine or histamine tends to be correlated with reactivity to exercise or isocapnic hyperventilation. That sug- gests that the reactivity to chemical medi- ators is an important determinant of the response to exercise and hyperventila- tion. When assessing airway reactivity with exercise or isocapnic hyperventila- tion, one must control such variables as magnitude of ventilation, duration of exercise or hyperventilation, workload, and inhaled air temperature and water con 59 tent (Hargreave and Woolcock, 1985~. A1- though the provocation tests effectively identify hyperresponders, they provide less quantitative dose-response informa- tion. Otherwise there is little justifica- tion for concluding that one method is superior to another for assessing airway hyperreactivity; the choice depends on the experience of the investigator and the laboratory, although pharmacologic challenge might have the advantages of requiring less expensive equipment and being technically simpler. Nonspecific airway hyperreactivity testing has proved to be highly useful for assessing airway responses to low concen- trations of environmental air pollutants. Even after the return to baseline lung function on removal from acute nitrogen dioxide (Bauer et al., 1986) or sulfuric acid aerosol (Utell et al., 1984) exposure, asthmatics demonstrated increased airway reactivity to cold air and hyperventila- tion or to carbachol aerosols, respec- tively. Likewise, after removal of healthy volunteers from ozone environments, in- halation of methacholine or histamine increased bronchoconstriction (Golden etal., 1978~. In the assessment of asthma induced by occupational agents, airway reactivity testing with nonspecific and specific agents serves a diagnostic function. Chan- Yeung and Lam (1986) recently published a comprehensive review on the subject of occupational asthma and the role of airway reactivity testing. Nonspecific airway hyperreactivity occurs in most workers with occupationally induced asthma, de- spite the absence of predisposing factors, such as atopy. Furthermore, Lam and co- workers ( 1983) found a good correlation between the degree of nonspecific bron- chial hyperreactivity and severity of response to the provoking agent plicatic acid in workers with red cedar asthma. Measurements of hyperreactivity also assist in providing objective evidence of sensitization (Chan-Yeung and Lam, 1986~. The demonstration of an increase in bronchial reactivity on returning to the workplace and a decrease away from work, with appropriate changes in lung function, establishes the causal rela
60 tionship between symptoms and the work environment. To pinpoint the etiologic agent in the workplace responsible for asthma, specific challenge is necessary. Chan-YeungandLam~ 1 986)emphasizedthat such testing can be dangerous and should be performed only by experienced persons in a hospital setting for the following conditions: studying previously unrecog- nized occupational asthma, determining the precise etiologic agent in a complex industrial environment, and confirming a diagnosis for medicolegal purposes. Detailed guidelines and testing proced- ures have been developed and published (Pepys end Hutchcroft, 1975;McKay, 1986~. In summary, increased airway hyperreac- tivity is a hallmark of clinical asthma and occupationally induced asthma. In patients with asthma, the greater the reac- tivity, the greater the likelihood of symp- toms. Despite variability in testing meth- ods, the tests have proved reproducible, relatively simple to perform, and closely correlated with each other. Distribution of Airway Hyperreactivity in the Population There is a broad distribution of airway reactivity to cholinergic agents and his- tamine in the general population, with asthmatics among the most responsive group. However, increased airway hyper- reactivity occurs in other pulmonary con- ditions, including cystic fibrosis, chronic bronchitis, and sarcoidosis. It is found in about 4% of people who have never had symptoms, in about 200/0 of patients with isolated chronic cough, and in about 10% of patients with rhinitis without chest symptoms (Hargreave et al., 1985~. Several population studies have concluded that bronchial hyperreactivity occurs in a unimodal, rather than bimodal, distribu- tion. For instance, in a distribution of bronchial reactivity, nonasthmatics could be placed at one end of the curve and asthmatics at the other, most reactive end. Subjects with hay fever or allergic rhinitis are often intermediate in non- specific airway reactivity. Few epidemiologic studies have examined the distribution of reactivity in unse MARKERS IN PULMONARY TOMCOLOGY lected populations. In a study of 300 rela- tively young, randomly selected college students, Cockroft and colleagues (1983) studied nonspecific reactivity to inhaled histamine. Asthmatics responded to a con- centration of histamine of 4 mg/ml or less. When responders were defined by such cri- teria, 20% of the nonasthmatic group demon- strated equivalent reactivity in the ab- sence of symptoms. In a larger, popula- tion-based study, Weiss and colleagues ( 1984) assessed nonspecific bronchial reactivity with eucapnic hyperpnea in response to subfreezing air in 134 adults and 213 children. Children and young adults were significantly more likely than older subjects to be responders. Nearly 20% of asymptomatic children had increased airway hyperreactivity. Of current asth- matics, 92% responded to the cold-air chal- lenge. One can conclude that the preva- lence of increased reactivity in the popu- lation-based studies exceeds the preva- lence of asthma in the general population. The observation that asymptomatic airway hyperreactivity occurs in many children and young people presents an opportunity to examine hyperreactivity as a marker of susceptibility to disease. Followup of such populations should determine whether bronchial hyperreactivity plays a role in the development of progressive lung disease; if it does, it would serve as a key risk factor (i.e., a marker of sus- ceptibility to) for lung disease. Airway Hyperreactivity as a Marker Population-based studies that examine the effect of bronchial hyperreactivity on the rate of decline of pulmonary func- tion in asymptomatic subjects are pre- requisites to understanding airway reac- tivity as a risk factor. Several lines of evidence suggest that airway hyperreac- tivity is important in the development of chronic lung disease. Barter and Camp- bell ( 1976) demonstrated that subjects who were hyperreactive to methacholine had a more rapid decline in FEV1 than did nonreactive subjects. Despite concerns that the two groups were not ideally match- ed and the need for further study, the find- ings are impressive.
PHYSIOLOGYININTACT ORGANISMS Britt and colleagues (1980) reported that almost half a group of subjects who were first-degree relatives of patients with chronic obstructive lung disease showed increased reactivity to metha- choline. In those with hyperreactive air- ways, a markedly accelerated loss of lung function was observed (150 ml/year), com- pared with normal loss of lung function in the nonreactors (30 ml/year). Thus, airway hyperreactivity might constitute a marker useful for detecting risk of ac- celerated loss of lung function. Efforts are needed to determine whether transient but recurrent episodes of airway hyper- reactivity, such as follow pollutant exposure or viral respiratory tract infec- tion, also serve as a risk factor for pro- gressive lung disease. Similar issues emerge in examining the relationship between airway hyperreac- tivity and occupational asthma. Although most patients with occupational asthma demonstrate nonspecific bronchial reac- tivity, it is unclear whether it is the result of exposure or a predisposing fac- tor. However, most evidence points to airway hyperreactivity in this setting as a marker of exposure, in that reactivity often wanes with removal from the environ- ment, it can recur with re-exposure, and it can persist for long periods in previ- ously asymptomatic persons exposed to high concentrations of irritating gases (Brooks et al., 1985~. Studies incorporat- ing measurements of airway hyperreac- tivity in pre-employment examinations and after workplace exposure should help to resolve the issue of whether increased hyperreactivity is a marker of exposure · . Or Injury. A large, multicenter study sponsored by the National Heart, Lung and Blood In- stitute is examining whether airway hyper- reactivity affects (or predicts the rate of) loss of lung function in those at risk of chronic obstructive lung disease. If that is the case, airway hyperreactivity will prove to be a powerful marker for as- sessing susceptibility to environmental agents. 61 CLEARANCE OF PARTICLES FROM THE RESPIRATORY TRACT Components of clearance systems are commonly the initial points of contact with inhaled pollutants; clearance func- tion might therefore be useful as a marker of response or a predictor of respiratory tract disease. This discussion focuses on techniques that can be used to assess clearance in viva; various elements of clearance systems can be examined with methods that use isolated organs, tissues, or cells. The latter could be important in attempts to evaluate individual com- ponents of clearance systems to explain mechanisms of impairment, but separation of mechanisms has generally not been pos- sible, and the cause of clearance impair- ment cannot always be determined. The broad mechanisms of clearance in conducting and respiratory airways are similar in humans and most other mammals. Interspecies differences do occur. for example, in secretory cell structure and distribution, macrophage function, relative roles of clearance mechanisms, and rates and efficiencies of clearance from various regions. Although such dif- ferences might be reflected in quantita- tive differences in response to inhaled agents, responses to numerous toxicants are qualitatively similar between humans and many experimental animals Phalen et al., 1984; Lippmann and Schlesinger, 1984~. Thus, although there are anatomic and physiologic differences, they do not preclude the use of clearance as a marker of exposure or response to inhaled toxicants. A valid marker should meet appropriate basic criteria. It should be reproducible (with minimal variability), it should be fairly sensitive to change after exposure to appropriate agents, and it should have a relationship to respiratory tract disease. Clearance rates from conducting and respiratory airways are well-defined functional characteristics of a person. However, interindividual variability is high. It is difficult to develop a li- brary of values that are considered normal, for comparison with those obtained after pollutant exposure or in disease states.
62 Nevertheless, the current database does allow characterization of a person's clearance as within or outside a normal range (Agnew et al., 1986~. Measured clearance rates depend heavily on the tech- nique used to assess them, so some stand- ardization of clearance measurement pro- cedures is needed, if this function is to be used as a reproducible marker. In both humans and experimental animals, bronchial mucociliary clearance altera- tion has been shown to be a sensitive physi- ologic indicator of response to exposure to many pollutants-such as nitrogen dioxide, ozone, and sulfuric acid-and occur after acute exposure to low concen- trations of pollutants and early in chronic exposures (Wolff, 1986; Schlesinger and Driscoll, 1988~. Studies with experimen- tal animals have shown that alteration in rate of clearance from the respiratory region can be a sensitive indicator of exposure. Although transient alterations in clearance after acute exposure to in- haled irritants might be adaptive (helping to maintain organ homeostasis), such changes are more likely to be pathophysio- logic responses of the airways and, al- though temporary, might foreshadow per- manent alterations or progressive changes that would follow continued exposures (Schlesinger et al., 1983; Gearhart and Schlesinger, 1988~. Interpretation of mucociliary clearance alterations in terms of potential health problems is speculative. Dysfunction of mucus transport is probably involved in the pathogenesis of some acute and chronic respiratory diseases. An absence of muco- ciliary function in some people is directly responsible for the early development of recurrent respiratory tract infections and, eventually, chronic bronchitis and bronchiectasis (Wanner, 1980; Rossman et al., 1984~. Partial impairment of the mucociliary system can also increase the risk of lung disease. In this regard, the rate of mucociliary clearance might affect the development of infectious disease. The upper respiratory tract is continuous- ly exposed to potentially pathogenic or- ganisms, and both these and organisms that reach the lower conducting airways are confronted with the mucociliary barrier. MARKERS IN PULMONARY TOXICOLOGY The rate of penetration of mucus by disease vectors to the underlying cells relative to the rate of mucociliary transport out of the respiratory tract could determine the capacity of inhaled pathogens to in- itiate disease (Proctor, 1979; Niederman et al., 1983~. There is a predisposition to respiratory infection in conditions (e.g., chronic bronchitis) characterized by retarded clearance from conducting airways (summing and Semple, 1980~. In addition, destruction of the functional integrity of the ciliated epithelium re- sults in impaired defense against bacteria (Laurenzi and Guarneri, 1966), and impair- ed transport has been observed in associa- tion with viral respiratory infections (Bang and Foard, 1964; Lourenco et al., 1971a). Retardation of mucociliary clearance might also be a factor in the genesis of bronchial cancer, by increasing the resi- dence time of carcinogens at initial depo- sition sites or those being carried through the lungs on the mucociliary escalator. For example, the selective distribution of lesions at bifurcations in the upper bronchial tree could result from both se- lective deposition and slowed clearance and in turn result in prolonged retention of high local concentrations of inhaled carcinogens. Although there is no direct evidence that ineffectual clearance con- tributes to the development of broncho- genic carcinoma, a causal relation has been suggested between adenocarcinoma, sites of local particle retention, and inadequate clearance in the nasal passages of furniture workers (Hadfield and Mac- beth, 1971; Morgan et al., 1973~. Evidence is accumulating that dysfunc- tion of bronchial clearance plays a role in the pathogenesis of chronic bronchitis; mucus transport is impaired in people who have the disease (Wanner, 1977~. Cigar- ette-smokers and persons with chronic obstructive pulmonary disease show a wider variation in clearance rates than do non- smoking healthy people (Albert et al., 1973; Gongora et al., 1981~. In the former groups, the within-subject variation is also great. That suggests that loss of control of mucociliary transport could either cause or result from chronic ob
PHYSIOLOGYININTACT ORGANISMS structive lung disease. It has also been shown in experimental animals that modest changes in mucociliary clearance rates are associated with secretory epithelial changes in small bronchi and bronchi- oles-changes that, if continued, could lead to clinical manifestations of bron- chitis (Schlesinger et al., 1983; Gearhart and Schlesinger, 1987~. Other studies with humans have suggested that mucocili- ary dysfunction is an early indication of pathogenic changes in the lungs. For example, retarded mucociliary clearance has been demonstrated in bronchitic people who showed no sign of airway obstruction (MossbergandCamner,1980),whereasyoung smokers with various degrees of impairment of tracheal mucus transport had no overt bronchitic symptoms and had normal results for pulmonary function tests of airway obstruction (Goodman et al., 1978~. The pathogenetic implications of al- terations in clearance from the alveolar region have not been examined to the same extent as changes in mucociliary clear- ance. Alveolar clearance rates appear to be reduced in people with chronic ob- structive lung disease and in cigarette- smokers (Cohen et al., 1979; Bohning et al., 1982~; that suggests some relation between altered defense and disease devel- opment. Clearance dysfunction has also been shown in animals that have viral in- fection (Cresia et al., 1973~. The adequate performance of alveolar macrophages is critical to the effective- ness of lung defense in minimizing the residence time of deposited toxicants. For example, phagocytosis plays an import- ant role in the prevention of particle entry into fixed tissues of the lung, from which clearance is very slow; accumula- tions of several types of dust have been directly linked to development of lung disease. Damage to macrophages has been implicated in the pathogenesis of chronic lung diseases involving proteolysis- e.g., emphysema, fibrogenesis, silicosis, and asbestosis (Brain, 1980; Warheit et al., 1984-as well as in an increased risk of viral and bacterial infections (Hocking and Golde,1979~. In vivo clearance studies are generally performed by examining the rate of removal 63 or transport of tracer particles from the lungs as a whole or from specific individu- al airways. Serial sacrifice or fecal analysis techniques can be used with ex- perimental animals. Measurements of rates or times are, however, strongly influenced by specific methods. Local Mucus Velocity Mucus transport velocities in the nasal passages, trachea, and main bronchi can be measured directly. The techniques in- volve monitoring of tracers on the epithel- ium, measurement of the movement of boluses of particles selectively deposited in these airways, or moving through them from more distal areas. Rates of movement are measured by determining the time needed for a bolus or tracer to traverse a cali- brated distance or to move between two anatomically defined areas. The tracers can be cellular debris or material speci- fically introduced into the airways, e.g., India ink droplets, Teflon disks, radio- labeled resin beads, powders, colored solutions or dyes, or pollen grains. They are generally introduced into the trachea by an airstream or by instillation (both via bronchoscopy) or into the nasal pas- sages by an airstream or simply by place- ment at the desired site. Various measurement techniques have been used. For example, saccharin parti- cles have been placed in the nose, and the time until the subject reported the first taste of sweetness measured (Proctor et al., 1977~. Some nasal markers have been viewed directly in the nasopharynx after placement on the anterior nasal mucosa (Van Ree and Van Dishoeck, 1962; Bang et al., 1967~. The most objective and sensi- tive procedures involve placement or in- halation of radioactive or radiopaque tracers into the upper respiratory tract or central airways and then viewing with external monitoring methods, including fluoroscopy (Friedman et al., 1977; Good- man et al., 1978; Mezey et al., 1978), cine- bronchofiberoscopy (Sackner et al., 1973; Santa Cruz et al., 1974; Toomes et al., 1981), scintillation detection (Proctor et al., 1977; Man et al., 1980; R. K. Wolff et al., 1982), and gamma-camera imaging
64 (Quinlan et al., 1969; Chopra et al., 1977; R. K. Wolff et al., 1982~. A modification involves measurement of transport veloci- ties in the trachea or main bronchi by moni- toring of the movement of boluses of radio- labeled particles, either inhaled in a manner that maximizes deposition in the central airways or coming from more distal bronchi. Movement along the trachea can be monitored with a gamma camera or scin- tillation detectors (Yeates et al., 1975, Foster et al., 1978, 1982; Schlesinger et al., 1978; R. K. Wolff et al., l 982~. The advantage of local-velocity tech- niques is that they allow measurement in anatomically defined airways. In addi- tion, because a specific site is used, there is no question as to whether altered clearance rates due to toxicant exposure resulted from alterations in the mucus system or from a change in tracer-particle deposition pattern; the latter is a pos- sibility when whole-lung clearance assays are used. However, there are a number of disadvantages. Many of the techniques are invasive, in that tracer particles are introduced into the airway of interest, the use of anesthetics might affect trans- port rates, and the procedure of introduc- ing particles might result in trauma to the airways. Alterations in nasal or tracheal trans- port rates have been used as markers of disease or of response to inhaled pollut- ants, because they are easier to measure than is whole-lung clearance (Sackner et al., 1978; Wolff et al., 1981; Majima et al., 1983; Stanley et al., 1985~. However, findings in the upper respiratory tract or trachea cannot be extrapolated to the lower lung and might not be adequate in- dexes of overall respiratory tract effect. Although nasal and tracheal mucus clear- ance could be affected in any impairment of overall clearance, altered bronchial clearance due to pollutant exposure is often associated with no change in nasal or tracheal transport rates (Albert et al., 1974; Schlesinger et al., 1978, 1979; Leikauf et al., 1981). Thus, the use of alterations in clearance in specific air- ways as a marker of mucociliary function is valid only if the agent of interest or a disease affects the region being meas LL4RKERS IN PULMONARY TOXICOLOGY urea. In any case, neither nasal nor tra- cheal transport tests can provide direct assessment of mucociliary function in the small bronchi and bronchioles; however, it is important to note that mucociliary dysfunction is probably of greater conse- quence for pathogenesis than many other changes. To avoid problems in the respira- tory system associated with regional meas- urements, whole-lung clearance can be used as a marker. Whole-Lung Clearance The technique most commonly used to meas- ure whole-lung clearance involves in- halation of a radiolabeled tracer aerosol. Tracer materials used include Teflon, polystyrene latex, hematite, magnetite, and clay. The total amount of radioactivi- ty remaining in the lungs at selected times is measured with external detector sys- tems. The decline in rate of emission of radioactivity, corrected for radioactive decay, represents clearance. Unlike the aforementioned methods, this technique does not measure the movement of individual particles or boluses, so actual rates of transport are not obtained. Various types and configurations of scintillation - detectors have been used to measure lung clearance; they may be divided into scanning and stationary de- tector systems. In the former arrangement, either the detectors move relative to the thorax of the subject, who has inhaled the tracer aerosol (LaBelle et al., 1964; Holma, 1967a,b; Camner and Philipson, 1971, 1978; Camner et al., 1971), or the subject is moved relative to stationary detectors (Albert et al., 1968~. Measure- ments are obtained when the subject is in preselected positions or during continu- ous scanning, i.e., during movement at a constant rate. Scanning systems are relatively independent of the apex-to- base distribution of deposited tracer particles in the lungs, in that they pro- vide a longitudinal activity distribution map. They also reduce counting variations due to slight differences in positioning of the subject or due to subject movements, in that they depend less on the exact equiv- alent geometry of the subject's location
PHYSIOLOGYIN INTACT ORGANISMS in relation to the detectors than do sta- tionary systems. In stationary or fixed systems, collim- ated detectors are placed in positions relative to the subject's thorax. Config- urations used include anterior placement of a single, central detector (Albert and Arnett, 1955; Toigo et al., 1963; Thomson and Short, 1969~; placement of two detec- tors, one centrally and one laterally (Luchsinger et al., 1968~; anteroposteri- or or bilateral placement of twin, axially opposed detectors (Booker et al., 1967; Thomson and Paria, 1974; Leikauf et al., 1981; Schlesinger et al., 1982, 1986~; and use of multiple detectors (Albert et al., 1969; Stahlhofen et al., 1981; Bailey et al., 1982~. Two other systems have also been used: the gamma camera (Lourenco et al., 1971 b; Sanchis et al., 1972; Puchelle et al., 1982) and the whole-body counter (Cresia et al., 1973; Bohning et al., 1982; Snipes etal., 1983~. Unilateral detector systems require exact positioning for reproducible re- sults, whereas dual or multidetector sys- tems (in which the signal output is com- bined) are less sensitive to changes in position of the subject in the measurement plane or to effects of redistribution of particles in the lungs. The gamma camera permits assessment of total clearance and allows visualization of the distribution of retained particles at various times after exposure. The airways within which the test aerosol is deposited are in a three-dimensional array in the lungs and are therefore at various depths relative to the detectors. Thus, the efficiency with which retained activity in each airway is measured varies. It follows that detector configuration affects the shape of the clearance curve. In addition, because of sensitivity dif- ferences, the amount of activity needed for successful analysis varies with the type of detector used. The gamma camera offers the greatest advantage in spatial resolution, but has low sensitivity and requires large amounts of radioactivity. Scanning systems have poorer spatial reso- lution, but better sensitivity. Multiple stationary detectors offer little infor- mation on intrathoracic particle distri 65 button, but are the most sensitive. The most responsive fixed system is the whole- body counter; however, it is not suitable for use during the first few days after tracer exposure, because, not being col- limated, it cannot effectively dis- tinguish between activity in the lungs and that cleared into the stomach during the initial, rapid tracheobronchial clearance phase. But it can be used to moni- tor long-term clearance, once the activity in the rest of the body is lower than that in the lungs. Thus, some other technique must be used to monitor the mucociliary clearance phase and allow determination of the time when the lung is the only organ with appreciable remaining activity. One of the major problems associated with external monitoring techniques is the dependence of the observed mucociliary clearance pattern on the pattern of initial deposition of the tracer aerosol. That dependence exists because the techniques are indirect and clearance characteris- tics are influenced by mucociliary transit rates. For example, an apparent increase in clearance rate after pollutant exposure could be due to a proximal shift in deposi- tion of the tracer aerosol, rather than to an effect on the clearance system it- self. That could be a special problem in the comparison of different groups; e.g., subjects with chronic obstructive lung disease tend to have greater central airway deposition of a given tracer aerosol than healthy subjects (Lippmann et al., 1980~. Such differences must be borne in mind in the assessment of clearance changes as markers of exposure or disease. The shapes of mucociliary clearance curves depend heavily on tracer-particle deposition. In studies of respiratory- region clearance, however, different clearance rates can also be associated with particles of different sizes (Bailey et al., 1982), because there can be size- dependent differences in macrophage phag- ocytosis, in viva solubility, etc. Dif- ferences in long-term clearance of par- ticles of equivalent size but consisting of different materials have been noted in both humans and experimental animals (Stahlhofen et al., 1981, Schlesinger et al., 1982~.
66 The experimental assessment of clear- ance from the respiratory region requires that measurements be performed over, per- haps, several months. If radioactively tagged tracer aerosols are used, a nuclide having a relatively long half-life is re- quired. In addition, because the total dose to a subject should be minimized, long counting times might be required for data to be statistically reliable. Thus, very long-term clearance studies that use hu- mans could preclude use of radioisotopic tracers, because of potential health risks. A technique that avoids the difficulties associated with the assessment of clear- ance is magnetopneumography (MPG). As discussed in Chapter 3, MPG allows direct assessment of lung burdens of magnetic materials in suitably exposed popula- tions. In addition, humans and experimen- tal animals have been exposed to inert magnetic dusts to assess clearance (Valberg and Brain, 1979; Cohen et al., 1979; Freedmanand Robinson, 1981; Halpern et al., 1981~. Measurement of the rema- nent field over time provides an index of clearance. MPG techniques have some advantages over the radioaerosol techniques, with respect to both temporal resolution and spatial resolution in the measurement plane. In addition, magnetic techniques have poten- tial advantages, in that they yield some information that cannot be obtained with other clearance techniques. The time de- pendence of decay of magnetic field after the external field is applied is affected by the viscosity of the medium in which the deposited particles reside (Williamson and Kaufman, 1981~. Therefore, as free particles are translocated from alveolar surfaces or engulfed by macrophages, the response to an externally applied field can change. When magnetic measurements are used to assess clearance over a long period, some or all of any observed decline in remanent moment could be due to immobil- ization of particles. In addition, the hysteresis curves of magnetization and relaxation (loss of magnetic alignment) can provide information on the amount of fibrosis in lung tissue (Cohen, 1975~. One technique used for whole-lung clear A[4RKERS IN PULMONARY TOXICOLOGY ance in both humans and experimental ani- mals allows visualization of tracer par- ticle distribution without the need for radiolabeled aerosols. It involves insuf- flation of radiopaque tantalum powder through a tracheal catheter. Serial chest x-ray pictures are then taken over a period of months, to provide visualization of clearance, which is measured on the basis of visual scoring of film intensity (Gamsu et al., 1973; Wood et al., 1973~. The tech- nique provides some measure of whole-lung and regional clearance patterns and allows measurement in anatomically defined air- ways, but it is invasive, is only semiquan- titative, and requires high radiation doses, so it is unsuitable for routine use in humans. It also requires several grams of tantalum, which might overload clear- ance systems. The technique therefore has not found widespread use. INJURY TO AIR-BLOOD BARRIER Conducting-Airway Permeability The epithelium of the conducting airways is normally fairly impermeable, so materi- al deposited on airway surfaces is absorbed slowly. This protective barrier depends on the integrity of the tight junctions between epithelial cells. Exposure to ozone (Kehrl et al., 1987) and other at- mospheric agents can alter barrier func- tion, increasing penetration of inhaled materials into the blood. Hyperpermeabil- ity has been demonstrated in the absence of morphologic alteration after exposure of experimental animals to a number of noxious agents, including cigarette smoke, nitrogen dioxide, and zinc oxide (Simani et al., 1974; Boucher et al., 1980; Ranga et al., 1980; Hulbert et al., 1981~. Thus, assays of epithelial permeability can be used to screen for, and act as markers of, epithelial damage. However, the damage assessed is nonspecific. Hyperpermeability has pathophysiologic implications (Boucher, 1980~. A decrease in the ability of the epithelium to act as a barrier might result in an increase in the translocation of inhaled, deposited materials through the airway wall. In- creased loading of antigens, for example,
PHYSIOLOGYIN INTACT ORGANISMS could increase immunologic responsivity. Hyperpermeability could also result in an increased flow of bronchoactive agents to effective sites, and thus produce hyperreactivity (Boucher et al., 1977a,b, 1979~. Hyperpermeability might alter the efficiency of mucociliary transport by changing ionic or macromolecular trans- port across the epithelium; permeability changes could alter mucus hydration and viscosity. Epithelial permeability might be assessed by examining ion transport or by measuring the passage of specific molecules across the epithelium. Epithelial Ion Transport Airway epithelial cells exhibit active ion transport. In concert with submucosal gland secretions, this capability con- tributes to regulation of the volume and composition of the liquid lining of airway surfaces. Fluid absorption, driven by active sodium transport, is the dominate basal solute flow across the surface of proximal human airway epithelia (Knowles et al., 1984~. It eliminates the necessity for a large reduction in the volume of air- way surface liquid as it is moved by ciliary activity from distal sites of large aggre- gate surface area to proximal sites of smaller surface area. Whereas fluid is absorbed under basal conditions, fluid secretion driven by active chloride trans- port can be induced in the presence of a favorable electrochemical gradient and an apical cell membrane that is permeable to chloride. The flux of ions across the airway epi- thelium generates a transepithelial po- tential difference (PD), which reflects the magnitude and direction of active ion transport and the passive ion permeabili- ties of cellular and paracellular path- ways; PD differs by site in the respiratory tract (Knowles et al., 1981~. Some sites might be better than others for assessing effects of pollutant exposure. The nasal epithelium is a particularly attractive model for studying toxic effects, because of its accessibility; the ability to per- form parallel in vitro studies in freshly excised or cultured epithelial cells; the availability of techniques, such as the 67 use of nasal filter paper or nasal wash, to obtain cell markers, mediators, or a measurement of the concentration of in- jurious agents; and the ability to obtain nasal epithelial cells for more direct assessment of changes in cell structure or biochemical function. The measurement of epithelial PD in viva has been used to explore normal epithelial function and dysfunction in acquired and genetic disorders (Knowles et al., 1983~. In addition, some information regarding use of in vivo PD as a marker of disease due to pollutant exposure has been obtained. For example, the tracheal PD of a group of young asymptomatic smokers was reduced (Knowles et al., 1982), thus, the tracheal PD could indicate epithelial damage before symptoms are noted. Some of the individual values lie within the range of normal bio- logic variability. Nasal PD can be reduced in the presence of inflammatory processes. A number of other agents have been shown to alter airway epithelial permeability and inhibit PD in viva (Boucher, 1981; Stutts and Bromberg, 1987~. They include ozone, nitrogen dioxide, and sulfur diox- ide. Some agents tested in vitro-e."., zinc, mercury, and formaldehyde-have induced similar responses (Slutts et al., 1981, 1982, 1986~. Therefore, changes in PD are unlikely to serve as markers of specific exposures, or to provide accurate information on exposure concentration. Although measurement of PD might serve as an index of exposure or response to a pollutant, the mechanism of the effect on epithelial barrier function cannot be ascertained from this measurement alone. It is difficult to differentiate between a change in ion current and a change in tis- sue resistance. If there is no change in PD, one cannot be sure that several barrier properties, such as ion transport and tis- sue resistance, were not altered in an equal and opposite fashion. The ability to estimate epithelial resistance in vivo would be useful; such measurements appear feasible and might improve the ability to characterize the nature of any insult (Knowles et al., 1986~. The ability to perform more carefully controlled experiments with fresh tissues in vitro might also allow assignment of
68 an observed effect to a change in ion cur- rent or to a change in cellular or paracel- lular resistance. Potential limitations to parallel in viva and in vitro studies are related to dosimetry, blood flow, and mucus barriers. The preservation of normal ion transport in primary cultures of mam- malian (including human) pulmonary epi- thelia and the expression of genetic dys- function in epithelial cells (as in cystic fibrosis) suggest that further assessment of toxic exposures could be pursued in parallel in epithelial cell culture sys- tems and in vivo. Thus, ion transport char- acteristics of the airway epithelial bar- rier might represent useful biologic mark- ers to monitor the effects of acute and chronic exposures to pollutants. Molecular Tracer Procedures A change in permeability can be assessed by examining the transepithelial trans- port of molecules into the blood. A good molecular tracer should have several prop- erties: · It should be nontoxic. · Its movement across the epithelium should be the rate-limiting process in its clearance from the lungs. The marker should be cleared rapidly from the lungs by the pulmonary, bronchial, or lymphatic circulations, and its clearance should not be limited by its movement through the interstitial matrix. Otherwise, a change in blood flow due to edema or lung disease could affect its clearance independently of changes in epithelial permeability. · Its route of clearance should be known. An accurate interpretation of the clear- ance data with respect to lung injury is difficult, unless it is known whether in- creased clearance is due to opening of the gaps between epithelial cells, an increase in vesicular transport, or simply an in- crease in the epithelial surface area for diffusion. · Its clearance should increase only in the presence of lung injury. The marker must be able to distinguish lung injury from the normal changes in a noninjured lung, such as an increase in lung volume, an increase in blood flow, or an increase AL4RKERS IN PULMONARY TOXICOLOGY in interstitial fluid volume due entirely to high lung vascular pressures. · It must not be transformed in the lung. The marker must not be denatured by the delivery process or degraded in the lung airspaces or interstitium. In addition, if a radioactive tag is used, the binding between the isotope and the marker must be tight enough to prevent its separation. · Its kinetics must facilitate measure- ment. The marker must clear rapidly enough to allow accurate measurement of its clear- ance within approximately 1 hour for clini- cal studies, but it must not clear so rapid- ly that the time required for deposition interferes with analysis. · It should be easily defected. A marker that requires numerous blood samples or labor-intensive assays would make meas- urement too cumbersome, invasive, or costly. Those criteria apply to tracers used for assessing conducting-airway permea- bility, as well as alveolar epithelial permeability. Two basic techniques have been used to assess conducting-airway permeability with macromolecular markers. The first involves use of horseradish peroxidase (HRP), a glycoprotein with a molecular weight of approximately 40,000 daltons. HRP is instilled into the trachea, and epithelial permeability is assessed by measuring plasma concentration of HRP at various times after instillation with radioimmunoassayorenzyme - linked immune - assay; electron microscopy of tissues allows localization of HRP for assessing routes of movement (Conner et al., 1982; Simani et al., 1974; Hulbert et al., 1981, Ranga et al., 1980; Boucher et al., 1978, 1980~. The procedure is used in experimen- tal animals, and the effects of inspired pollutants can be assessed by comparing test with control animals; in normal ani- mals, HRP transfer rates are very low (Hog" et al., 1979~. The finding of HRP in inter- cellular spaces when concentrations in the blood are increased indicates that the blood assays actually measure a loss of barrier function. Studies with cigar- ette smoke in animals have suggested that the exudative phase of the inflammatory
PHYSIOLOClYININTACT ORGANISMS response is associated with hyperpermea- bility and that function returns to normal when the repair phase begins (Hulbert et al., 1981~. In addition, changes in tra- cheal permeability are related in a dose- dependent fashion to dose of cigarettes; effects are seen in guinea pigs after as few as 20 puffs (Boucher et al., 1980~. It must be borne in mind, however, that correct interpretation of plasma HRP requires knowledge of the HRP clearance rate from plasma; any toxic effect on this rate could affect interpretation of the permeability assay (Conner et al., 1985~. Another procedure to assess tracheo- bronchial airway permeability (and one that is used in humans) involves inhalation of radioactively tagged (99mTc) aerosols of diethylenetriaminepentaacetate (DTPA), which has a molecular weight of 492 daltons, and its analysis in the blood or with thoracic scanning to obtain a clearance half- time. With proper adjustment of particle size and breathing characteristics, the inhaled aerosol can be preferentially deposited on the tra- cheobronchial tree with minimal respira- tory-region (alveolar) deposition (Oberdorster et al., 1986~. Clinical studies have yielded a wide range of re- ported half-times of DTPA (Jones et al., 1980; Rinderknecht et al., 1980; Mason et al., 1983; Kennedy et al., 1984; O'Byrne et al., 1984~; some of the variation could be due to differences in deposition sites of the aerosol in different studies and differences in absorption at these sites. Cheerma et al. (1988) examined the dif- fusion and binding characteristics of DTPA in measuring permeability across alveolar epithelium and bronchial mucosa. They found that, because DTPA has a high affini- ty for the mucosal layer of the bronchial epithelium, it might not be suitable for measuring tracheal and bronchial clearance, but is more useful for measuring alveolar clearance. Thus, there is a need to standardize the assay for particle size and breathing char- acteristics, if it is to be a reliable mark- er of response. In addition, the sensitiv- ity of the procedure depends on the site of action of the pollutant being assessed. For example, rats exposed to ozone showed 69 a greater and more persistent response in the bronchoalveolar zone than in the trachea, because the site of major ozone deposition was the bronchoalveolar zone (Bhalla et al., 1986~. Alveolar Epithelial Barrier The layer of epithelial cells lining the airspaces of the lungs forms a tight barrier that greatly restricts the move- ment of most solutes. It is not clear what happens when lung injury causes an increase in the permeability of the bar- rier, but two responses are possible: en- zymes gain access to the lung tissue and cause increased tissue damage; and solutes and fluid pass more easily from the inter- stitial spaces and vascular spaces result- ing in either an alteration in the composi- tion of the liquid lining of the airspaces or alveolar edema. The specific response to increased permeability undoubtedly depends on the location and on the size of the solute to which the epithelium has become more permeable. The study of the clearance of tracers from the alveoli is complicated by the diverse and numerous obstacles between the airspaces and the blood. Tracers can move by diffusion or vesicular transport from the alveoli, across the epithelium and endothelium, and into the blood (Path 1, Figure 3-2~. However, in regions where the basement membranes of the epi- thelium and endothelium are not closely apposed or in conditions where blood ves- sels are not perfused, a tracer can diffuse or flow through the interstitium into a perfused blood vessel or a lymphatic vessel (Paths 2a and 2b, Figure 3-2) (Peterson and Gray, 1987~. It can also diffuse or flow into a fluid reservoir in the inter- stitium, such as those which develop around airways in the presence of interstitial edema (Path 2c, Figure 3-2) (Havill and Gee, 1984~. The formation of those reser- voirs can complicate the measurement of epithelial permeability, because they can prevent the tracer from leaving the interstitial spaces. Macklin ~1955) pro- posed the existence of a mechanism for clearance of particulate matter and large solutes via "sumps" found at bronchioles;
70 Fluid reservoir , (with interstitial edema) ~Lymphatic Lymphatic <2C I Alveolus 2 =~ FIGURE 3-2 Clearance pathways for markers In the air~paces. the sumps allow the solutes to flow by con- vection from the airspaces directly into the lymphatic vessels (Path 3, Figure 3-2~. The existence of a variety of pathways and mechanisms for the movement of a tracer from the airspaces suggests that clearance of a tracer can be complicated by simple changes in the activity of a normal trans- port mechanism. The most commonly used marker of changes in alveolar epithelial permeability is DTPA. Its clearance rate has been found to increase in response to a variety of conditions and insults in humans, includ- ing interstitial lung disease (Rinder- knecht et al., 1980), ozone exposure (Kehrl et al., 1987), sarcoidosis (Dusser et al., 1986a), and cigarette-smoke exposure (Dusser et al., 1986b). Animal studies have also shown that DTPA clearance in- creases in response to inhaled cigarette smoke (Minty and Royston, 1985), ozone (Bhalla and Crocker, 1986), and irradia- tion (Ahmed et al., 1986~. Some problems are evident in the use of 99mTc-DTPA as a marker of alveolar epithe- lial injury. Its clearance rate increases with lung inflation (Rinderknecht et al., 1980; Peterson et al., 1986; Rizk et al., 1984~. The 99mTc label can dissociate from DTPA in the presence of oxidants (Nolop et al., 1986~; this could be a problem in assessing response to some pollutants. Because free technetium clears more rapid- ly than does 99mTc-DTPA, dissociation of the label would yield an increased clear- ance rate (Egan, 1980~; that could explain AL4RKERS IN PULMONARY TOXICOLOGY the increased clearance rate measured in cigarette-smokers (Nolop et al., 1986~. The use of ~3In-DTPA has been proposed, because indium is more tightly chelated by DTPA than is technetium (Nolop et al., 1986~. Finally, although the relatively rapid clearance of DTPA from the lungs of healthy subjects and those with lung injury allows accurate measurements of clearance within 30 minutes, it might also cause a measurable background concentration to appear, because of recirculation of the tracer. Other molecular tracers have been inves- tigated. Clearance of instilled tracers with an Einstein-Stokes radius greater than 2 nm from the airspaces might not be affected by lung inflation (Egan, 1980), so the use of a tracer larger than DTPA (ra- dius, 0.6 nm) might overcome one potential problem with DTPA. Bovine serum albumin (radius, 3.6 nm) has been labeled with 99mTc (Hnatowich et al., 1982; Peterson et al., 1989~. In anesthetized sheep, the albumin clearance rate measured with nuc- lear imaging increases in the presence of lung injury, but is unaffected by changes in lung volume or by lung edema due to increased lung vascular pressures (Fig- ures 3-3, 3-4, 3-5~. However, the process of labeling and aerosolizing the albumin could cause the formation of macroaggre- gates, which complicate interpretation of the data. Furthermore, albumin might leave the airspaces by specialized trans- port mechanisms (K. J. Kim et al., 1985~. Mannitol has also been proposed as a marker of airway clearance (Taylor et al., 1983), but its use might suffer from some of the problems associated with using DTPA, in that mannitol is small (radius, 0.3 nary). In summary, the search for an accurate and useful technique for the in vivo meas- urement of changes in epithelial permea- bility is worthwhile, but has not been totally successful. It might be necessary to use simpler experimental models of lung epithelium-e.g., isolated perfused lungs or monolayers of cultured epithelial cells-to identify markers before that can be used in vivo.
PHYSIOLOGYIN INTACT ORGANISMS 100 ~ . oh Z 80 Z z Z 60 IL '_ 40 C) z ~ 20 CL i_ ~T O _ \1 I I ~1 1~ Control Lung Inflation 0 20 40 60 80 100 MINUTES FIGURE 3-3 10 cm H2O PEEP increases DTPA clearance rate. Source: Re~nnted with permission from Peterson et al., 1988. Vascular Injury Endothelial cells found in pulmonary blood vessels function to retard passage of fluid, protein, and some other blood components from the vessel lumen into the interstitium and the airspaces of the lung. In addition to that barrier function, pul- monary vascular endothelium performs such functions as the removal or metabolism of endogenous and exogenous circulating agents and the synthesis of biologically active substances (e.g., prostacyclin and factor VIII antigen) that help to main- tain vascular homeostasis. In health and disease, the endothelium interacts with blood cells (such as platelets and leuko- cytes) and with cells that form the vas- culature (such as fibroblasts, pericytes, and other cells of the interstitium), as well as with smooth muscle cells in precap- illary portions of the vascular tree. Those qualities change when endothelial cells are injured. In theory, all those and perhaps other characteristics of en- dothelial cells can be exploited as mark- ers of injury. The purpose of this section is to review and to comment briefly on cur- rently used indicators of endothelial injury and to provide an example of a speci- fic functional characteristic of endothe 71 lium that might constitute a useful bio- logic marker of injury. Chemically induced Injury to Endothelium Structural alterations in pulmonary endothelium have been demonstrated ex- perimentally after toxic insult with a number of chemicals, including such di- verse agents as a-naphthylthiourea, pa- pain, ethchlorvynol, bleomycin, bromo- carbamide, iprindole, some gases (e.g., oxygen), epinephrine, and the pyrolizi- dine alkaloid plant toxins (Witschi and Cote, 1977~. Blebbing and swelling of endothelium occur with each of those toxicants. The changes in endothelium are usually associated with changes in other lung cell types. However, the exact temporal relationship between injury to endothelium and changes in other cell types varies from one toxicant to another. For example, after administration of bleomycin, iprindole, or oxygen, changes in endothelium are seen before changes in other cell types; after intratracheal administration of papain, changes in alveolar Type II cells and fibroblasts are seen before changes in endothelium; after administration of other toxicants,
72 MARKERS IN PULMONARY TOXICOLOGY Zo~ Z ~ IS ~ I,V. Oleic Acid (4) ~ ~ '~~~ ~ ~ PEEP (8) 6 Z ~ PEEP (8) L ~ 20 ~ I k1 = 7 9 + 2.9 %/min \\ 50 I 1 1 1 10 ~I I 0 1 2 0 1 2 HOURS FIGI~E 3-4 Albumin clearance discnm~nates between lung injury and lung inflation. such as epinephrine, changes in endotheli- um and changes in other cell types seem to occur simultaneously. The location of the earliest damage to pulmonary endothelium might also very from one toxicant to another. For example, in oxygen toxicity, capillary endothelium is the first to show changes; after bleomy- cin administration, the earliest damage is to arterial and venous endothelium. Thus, markers that could distinguish in- jury in various parts of the vascular bed might provide important clues to mechan- isms and risks associated with various pulmonary diseases. Loss of Endothelial Barrier Function A major role of endothelium is to prevent loss of fluid from vessel lumina. Endo- thelial cell injury is usually manifested clinically as evidence of pulmonary edema. Pulmonary edema is classified into two types. In hydrostatic or hemodynamic edema, abnormally high intravascular pressures in small parenchymal vessels lead to flux of fluid from them. In permea HOURS FIGURE ~5 Compartment analysis of DTPA clearance. bility edema, intravascular pressures can be normal, but leaks in alveolar capil- laries allow increased flux of water and protein into the extravascular compart- ment. The former type does not always en- tail injury to the vascular endothelial barrier, but can arise, for example, as a result of constriction of pulmonary ven- ules. The most common examples of hemody- namic pulmonary edema result from chronic left-sided heart failure or mitral valve disease (Fishman, 1980~. Permeability edema is associated with formation of a protein-rich lymph that arises from an injured endothelial barrier that allows increased passage of plasma proteins into the interstitium of the pulmonary paren- chyma. Permeability edema results from exposure to some noxious airborne agents, such as nitrogen dioxide, and also occurs in acute respiratory diseases. In animal studies, it also results from a number of chemical insults to pulmonary capillary endothelium, e.g., after exposure of the pulmonary vasculature to oleic acid, al- loxan, a-naphthylthiourea, or phorbol ester.
PHYSIOLOGYININTACT ORGANISMS In animals, permeability edema has been measured on the basis of protein leakage from the pulmonary vasculature. Lung lymph flow, lymph fluid protein concentration, and accumulation in excised lungs of radio- labeled protein introduced into the blood all can be measured. They have provided much important information on vascular leak in laboratory animal studies, but not directly in humans. Loss of barrier functions is also re- flected in an increase in extravascular lung water. Regardless of the cause, ex- cess extravascular water can be detected with a variety of methods. In general terms, those methods can be divided into invasive and destructive methods, inva- sive and nondestructive methods, and non- invasive and nondestructive methods (Table 3-2). The destructive techniques 73 dyspnea and tachypnea) and physical find- ings of cyanosis and rates during chest auscultation (Staub, 1974, 1986~. How- ever, other conditions can cause similar findings. In any case, the approach is nonquantitative and relatively insensi- tive to smaller accumulations of extravas- cular water. Nonetheless, because of its simplicity and lack of expense, the clinical examination remains an Important means for detecting the presence of acute pulmonary edema. Of the other available techniques, four deserve special consideration: chest roentgenography, the indicator-dilution method, and the newer techniques of posit ron-emission tomography and nuclear mag netic resonance. Chest roentgenography has many favor able features for use as a marker of lung have the mayor advantage ot accuracy and injury. It is practical, widely available thus are often reported as the putative in a variety of useful settings, and rela "gold standard" by which other techniques lively inexpensive. Its accuracy and sen are judged (Staub, 1974; 1986~. Clearly, sitivity in detecting pulmonary edema are disputed. When strict attention is paid to technical factors, some have argued that the chest roentgenogram is quite sen sitive to changes in lung water content, and accurate inferences can be made about the magnitude of such changes (Pistolesi and Guintini, 1978; Milne et al., 1985). However, several other groups have tested it against presumably more accurate tech however, they are unsuitable for clinical studies. The nondestructive techniques, although useful in a clinical setting, suffer to various degrees from inaccuracy, non- specificity, impracticality, and expense. For instance, pulmonary edema can be diag- nosed clinically in a patient with a char- acteristic history (e.g., acute onset of TABLE 3-2 Methods for Detecting Excess Extravascular Lung Water Accumulation Category Methods Invasive and destructive Invasive and nondestructive Noninvasive and nondestructive Gravimetrics (Staub, 1974,1986) Histology (Staub, 1974, 1986) Indicator dilution (Baudendistel et al., 1982; Grover et al., 1983; Sibbald et al. 1983; Eisenberget al., 1987;Sivak and Wiedemann, 1986;Effros, 1985;Lewis et al., 1982) Clinical examination (Staub, 1974, 1986) Pulmonary mechanics (Staub, 1974, 1986) Chest roentgeno~aphy Mane et aL, 1985; Pistolesi and Guintini, 1978; Baudendistel et al., 1982; Grover et al., 1983; Seybold et al., 1983; Eisenberg et al., 1987; Sivak and Wiedemann, 1986) Soluble-gas uptake (Overland et al., 1981) Microwave transmission (Iskander et al., 1979) Compton scatter (Loo et al., 1986) X-ray computed tomography (Hedlund et al., 1984; 1985) Positron emissiontomography(Schusteretal., 1985;Rhodeset al., 1981;Wollmer et al., 1984; Schober et al., 1983; Schuster et al., 1986; Cutillo et al., 1984) Nuclear magnetic resonance (Cutillo et al., 1984; Morris et al., 1985; Wexter et al., 1985)
74 MARKERS IN PULMONARY TOXICOLOGY niques and have not been able to demon- called the thermal-green dye double-in strate an acceptable degree of accuracy dicator dilution technique, has been veri (Baudendistel et al., 1982; Grover et al., fled by numerous groups as accurate (in 1983; Sibbald et al., 1983; Sivak and most. although not all. instances in which Wiedemann, 1986; Eisenberg et al., 1987~. Two techniques, indicator dilution and positron-emission tomography, measure the intravascular components of lung water. Thus, they can measure extravas cular lung water accumulation, which is in fact the entity of interest, inasmuch as the abnormal accumulation of extravas cular water represents breakdown in en dothelial cell barrier function. In addi tion, gas rebreathing techniques have been used to estimate lung tissue volume and pulmonary capillary blood volume. From those two volumes and estimates of ratios of wet to dry weight of tissue and blood, intravascular and extravascular water can be estimated. Indicator-dilution methods of measuring extravascular lung water are based on the concept that the mean transit time of an indicator through a fluid depends on in dicator flow rate and the volume of the fluid (Lewis et al., 1982; Hedlund et al., 1984; Sivak and Wiedemann, 1986~. For a given flow rate, if volume is small, the mean transit time will be small, and vice versa. To measure extravascular lung water, two indicators are used: one that can diffuse through the entire lung water volume and one that is limited to the in travascular, nondiffusible volume. A1 though a number of indicators have been used, the two that have achieved the great est acceptance are heat (actually, temper ature change) as the diffusible indicator and dye (e.g., indocyanine green) as the nondiffusible indicator. The green dye binds immediately in viva to albumin and thus remains intravascular during the period of lung water measurement. Extra vascular lung water (EVLW) can be calcu lated as EVLW = CO (MTT~ - MTTg&), where CO is the cardiac output (i.e., a measure of vascular flow) and MTT is the mean transit time of the thermal (t) or green dye (ad) indicator. This method, EVLW is increased), reproducible, and reasonably sensitive to changes in EVLW (i.e. it will reliably detect approximate- ly a 20% change) (Sivak and Wiedemann, 1986~. Nonetheless, it is moderately in- vasive (catheters in the pulmonary and femoral arteries are required) and thus is not suitable for general population screening studies. Positron-emission tomography (PET) is a nuclear-medicine technique that pro- duces quantitative tomographic images of the tissue distribution of a previously administered positron - emitting radio - nuclide. It uses image reconstruction algorithms identical with those used dur- ing routine x-ray computed tomography (CT). However, unlike x-ray CT, which cannot distinguish between intravascular and extravascular water (Hedlund et al., 1984, 1985), PET measurement of EVLW is feasible because it subtracts the intra- vascular water content (IVW) of a region from the total lung water content (TLW) of the same region (Schuster et al., 1985~. Unlike the indicator-dilution method previously described, PET is less sensi- tive to errors caused by the underestima- tion of EVLW in poorly perfused areas of lung. The intravascular component of EVLW is measured during PET by scanning the subject at least 2 min after inhalation of i50- labeled carbon monoxide, a gas that avidly binds to hemoglobin. IVW is calculated by comparing the radioactivity in a given lung region with activity in blood samples taken during the scan. A similar procedure is used to measure TLW, except that the scan is obtained during equilibrium of the bolus infusion of )50-labeled water. The cal- culation of extravascular water content of a region involves the subtraction of IVW from TLW. Alternatively, a constant infusion of )50-labeled water, or iiC- in- stead of )50-labeled carbon monoxide or density measurements instead of TLW meas- urements may be used (Rhodes et al., 1981; Schober et al., 1983; Wollmer et al.,
PHYSIOLOGYIN INTACT ORGANISMS 1984~. Recent studies in whole animals have suggested that PET provides measure- ments of EVLW in both normal and edematous lungs with acceptable accuracy and is sen- sitive to small changes in EVLW after phys- iologic intervention (Schober et al., 1983, Schuster et al., 1986~. Values ob- tained in humans have been comparable with those obtained in experimental animals. PET appears to be ideal for measuring regionalEVLW content. Because of technical problems associated with radioactivity counting in heterogenous tissues, whole- lung values for EVLW are more difficult to obtain. More important, however, are the cost and impracticality of PET as a clinical tool in that a scanner, a com- puter, a cyclotron, and several highly trained personnel are required for obtain- ing the measurements. Proton nuclear magnetic resonance (NMR) imaging is a new, complex, and expensive technique for evaluating lung water con- tent (Cutillo et al., 1984; Wexter et al., 1985~. NMR depends on the electromagnetic properties of nuclei of some atoms that cause them to act like small, spinning bar magnets when placed in a strong magnetic field. The most abundant of those atoms is hydrogen, which contains one proton. The proton is the principal nucleus used in current magnetic resonance Imaging experiments. When it is placed in a strong magnetic field, there is a slight net or- ientation of the protons along the magnetic field direction. The introduction of a ,= radiofrequency (RF) excitation at a fre- quency specific for both the magnetic field strength and the protons under considera- tion causes the reorientation of the pro- tons; when the RF excitation is removed, the protons return to their original orien- tat~on. ~ hat process (i.e., return, or relaxation, of the proton) emits RF energy, which is detected by a sensitive antenna or coil, amplified, and processed by a computer. The computer processing of space- and time-dependent RF emission creates an image of the concentration (i.e., density) and environment of protons in fat and water of soft tissue. Desirable features of proton NMR imaging are that no ionizing radiation is necessary and 75 there are no bone artifacts in the image. Although Now imaging will probably yield the most accurate in viva measurement of lung-water distribution, subtraction of the vascular component remains diffi- cult. That problem, signal-to-noise ratio characteristics in the imaging of lung tissue, and the complexity of the technol- ogy as a whole make NMR imaging, like PET imaging, unlikely candidates for screen- ing general populations for evidence of endothelial lung injury. It is probably unwise to use lung-water measurements obtained with any technique to evaluate early lung injury. The abnor- mal accumulation of excess lung water rep- resents not only a failure of endothelial barrier function, but also a failure of various other mechanisms (the most impor- tant of which is lymphatic function) that the lung can use to maintain normal water homeostasis. More useful as a marker of early injury would be a technique that detected breakdown of endothelial barrier function itself. Several groups have measured the flux of radiolabeled proteins across the pulmonary endothelium with external radiation detectors of various sorts (Gorin et al., 1978; Mintun et al., 1987~. Although those techniques indeed seem to be more sensitive markers of lung injury than is the measurement of EVLW, they are still too new for prediction of how accurate, reproducible, and practical they will be in detecting lung injury in groups of humans. In summary, no ideal means exists, or is likely to exist in the near future, for the detection of lung endothelial injury on the basis of either lung-water or capil- lary protein-flux measurements. The tech- niques that are simple, inexpensive, and practical to apply to large groups of hu- mans are generally nonspecific and insen- sitive. The techniques that improve on specificity and sensitivity suffer in being expensive, impractical, and com- plex. The choice of method will depend largely on the specific goals of the pro- gram involved.
76 Nonbarrier Properties of Endothelium As noted above, the nonspecificity, insensitivity, invasiveness, require- ments for sophisticated equipment, and expense of currently available measures of endothelial barrier function limit their usefulness in diagnosing early per- meability defects or subtle endothelial cell injury that can be associated in some people with a predisposition to serious pulmonary vascular disease. Obviously, markers associated with subtle, early defects in pulmonary endothelium that are sensitive, specific, and minimally in- vasive could be useful in identifying people at risk. Similarly, predisposition of people to diseases associated with de- fects in the endothelium might be predicted and such diseases prevented more effec- tively. For example, diverse types of trauma result in adult respiratory dis- tress syndrome (ARDS) in some patients. Markers to identify subtle changes in en- dothelium might aid in identifying pa- tients at risk of developing ARDS and in understanding its pathogenesis. Research during the last several years has led to the identification of several non-barrier functions of pulmonary en- dothelium. From the standpoint of increas- ing our knowledge of mechanisms of lung injury, there is a need to understand bet- ter both barrier and nonbarrier functions of endothelium, to attain the capacity to assess them, and to determine how non- barrier functions of endothelium are cor- related with barrier properties. It should be recognized that changes in nonbarrier functions of endothelial cells might be useful predictors of deficits in the bar- rier function of the endothelium. Metabolic Activity of Endothelium The pulmonary vasculature performs a number of potentially important nonbar- rier functions, some of which involve the modification of circulating concentra- tions of naturally occurring, biological- ly active substances, as well as drugs. Because the lung has a large vascular sur- face area and receives all of the cardiac MARKERS IN PULMONARY TOXICOLOGY output, it is uniquely situated to alter rapidly the circulating concentrations of vasoactive agents before they reach the arterial circulation. The capacity of the lung to clear the circulation of chemical agents and the potential impor- tance of this function have been the sub- ject of several reviews (Gillis and Pitt, 1982; Roth, 1985). The ability to remove and metabolize substances reflects properties of endo- thelial cells of small vessels and capil- laries in lung. For example, carrier- mediated transport of biogenic amines, such as 5-hydroxytryptamine (SHT) and norepinephrine (NE), into pulmonary vascular endothelium occurs. Available evidence indicates that SHT and NE are taken up at different sites at the endothe- lial surface. After removal by the lung vasculature, those amines are metabolized by enzymes like monoamine oxidase and catechol-O-methyltransferase. However, the rate-limiting step in their initial removal from the circulation is transport from the vascular space, rather than intrapulmonary metabolism. Circulating adenine nucleotides (adeno- sine monophosphate, adenosine diphos- phate, adenosine triphosphate) are also altered on passage through the lung. Aden- osine triphosphate, for example, does not survive passage through the pulmonary circulation. Biochemical and cytochemi- cal studies have shown that, when those nucleotides are perfused through isolated lungs, all the radioactivity entering the pulmonary circulation is recovered in the effluent, but none remains in the form of the adenine nucleotide. The mean transit time and volume of distribution of those nucleotides are the same as those of in- travascular markers. This indicates that the adenine nucleotides are metabolized in the pulmonary circulation without leav- ing the vascular space. Cytochemical data confirm that, although several cell types and organelles have phosphate esterases that hydrolyze nucleotides, only the en- zymes that face the vascular lumen are exposed to and metabolize them. The loca- tion of the enzymes along the vascular lumen accounts for the fact that the meta
PHYSIOLOClYININTACT ORGANISMS boric products of adenine nucleotides appear in the venous circulation with no delay or tissue uptake. The lung is also capable of hydrolyzing circulating peptide hormones, such as bradykin in and angiote ns in I. B radykinin is nearly quantitatively converted to shorter peptides in a single pass through the pulmonary circulation. The peptide is not taken up by lung, and its mean transit time and volume of distribution in perfused lung preparations are identical with those of intravascular markers, such as indocya- nine green or blue dextran. Similarly, angiotensin I is extensively converted to angiotensin II on passage through the pulmonary circulation. Angiotensin- converting enzyme is located on the luminal surface of pulmonary endothelium; indeed, immunohistochemical studies have confirm- ed pulmonary endothelium as the only site of angiotensin-converting enzyme in the lung. Studies in animals have revealed that impaired pulmonary metabolic function results from exposure to numerous toxi- cants. However, structural injury to pul- monary endothelium is not always asso- ciated with deficits in each type of metabolic function. For example, the pyrolizidine alkaloid, monocrotaline, produces pulmonary endothelial injury experimentally that is associated with reduced intrapulmonary clearance of SHT by isolated lungs from treated animals (Roth, 1985~. However, 5'-nucleotidase and angiotensin-converting enzyme ac- tivities in isolated lung preparations are apparently unaffected by treatment of rats with monocrotaline. Thus, chemi- cally induced damage to lung might affect some functions of endothelium without altering others. This suggests some specificity in the endothelium-damaging action of some toxicants. A number of studies have suggested that pulmonary metabolic functions may provide sensitive markers of endothelial injury. For example, exposure to the herbicide paraquat results in pulmonary lesions in humans and experimental animals. In rats, marked structural changes in alveolar epithelium have been commonly observed 77 after paraquat administration, but al- terations in vascular endothelium are much more subtle and infrequent. A modest but reproducible decrease in the ability of isolated lungs from paraquat-treated animals to remove perfused 5HT has been reported (Roth, 1985~. The demonstration of impairment in SHT clearance resulting from a treatment that produces little, if any, structural alteration in endothel- ium suggests that the pulmonary metabolic function could be a sensitive index of damage to pulmonary endothelium under some circumstances. That view is supported by studies of oxygen toxicity. Structural alterations in pulmonary capillary en- dothelial cells are an early manifestation of exposure to oxygen at 1 atmosphere. Block and Fisher ( 1977) reported that, although ultrastructurally demonstrable endothelial damage is not apparent until 48 hours of exposure to 100% oxygen, ex- posure for as little as 18 hours produces a significant decrease in SHT clearance by lungs of exposed animals. Those studies of pulmonary metabolic function in animals were performed in iso- lated lung preparations. The functions have also been studied in vivo both in ani- mals and in humans with the multiple-in- dicator-dilution techniques described previously. For example, angiotensin- converting enzyme activity in the pulmon- ary vasculature has been studied with the synthetic substrate 3H-benzoyl-phe-ala- pro (BPAP). BPAP and an intravascular marker are injected intravenously as a bolus, and the concentrations in the arter- ial (i.e., postlung) blood are compared over time. With this technique, the frac- tion of BPAP metabolized in a single pas- sage through the pulmonary vasculature can be calculated. There are potential pitfalls in using that and related methods to assess pulmon- ary microvascular injury (Stalcup et al., 1982~. For example, pulmonary metabolic function can be influenced by changes in transit time and by inhomogeneity of perfusion, edema, and other factors that affect vascular surface area. When exogen- ously administered, radiolabeled sub- strates (e.g., 3H-BPAP) are used, it is
78 possible for endogenous substrates (e.g., angiotensin I and bradykinin) to compete with the tracer for metabolism and thereby confound interpretation of results. Fur- thermore, the lung might simultaneously synthesize and release the same test sub- stance being removed or metabolized, and that would make interpretation of pulmon- ary extraction data difficult. In addi- tion, questions have been raised about how to normalize metabolism data (e.g., whole lung vs. per unit lung weight, pro- tein, DNA, etc.~. Careful monitoring of perfusion, in- travascular pressures, and ventilation aid somewhat in ensuring reliability of data but do not resolve many of the poten- tial problems. As mentioned above, the choice of a specific metabolic function and substrate can be of critical importance with regard to usefulness of a metabolic process in assessing lung microvascular function. For example, substrates that are removed entirely in a single pass through the pulmonary vasculature might not provide needed sensitivity. In this case, reductions in enzymatic capacity might have to be quite large for effects on intrapulmonary metabolism to be detectable. Some of the potential pitfalls can be addressed through refinements in tech- niques. Indeed, if BPAP doses that provide both saturating and nonsaturating con- centrations of substrate at enzyme sites in the pulmonary vasculature are measured serially, enzyme kinetics can be deter- mined from the resulting indicator-dilu- tion curves. Thus, the Michaelis constant (Km) for the enzyme can be calculated, as can Amp which is the product of the maxi- mal velocity (Vm:,x) of the reaction and the microvascular plasma volume. From those estimates, information can be ob- tained on changes in enzyme quality (as measured by affinity) and amount (as meas- ured by Vmax) in toxicoses or other dis- ease states. With angiotensin-converting enzyme (ACE), for example, a reduction in Ama,, could reflect specific inhibition or destruction of the enzyme or a decrease in capillary surface area. Changes in Km, however, reflect alterations in endothel M'4R=RS IN PULMONARY TOXICOLOGY . ial metabolic function that are indepen- dent of effects on capillary surface area. This technique has been used to study effects of pneumotoxicants and ACE inhibi- tors on pulmonary endothelium. Indeed, alterations in metabolic function of en- dothelium have been described for such toxicants as PMA and nitrofurantoin and for radiation-induced injury. Studies in rabbits, for example, revealed an in- crease in Km for BPAP soon after administra- tion of PMA when no histologic evidence of lung injury was observed (McCormick and Catravas, 1986~. The data suggest that, under some circumstances, pulmonary metabolic function can provide a sensitive index of injury to pulmonary endothelium. As with several other potential markers of lung injury, the use of pulmonary meta- bolic function to assess endothelial injury in the lung requires further devel- opment and validation before it can be con- sidered useful. The equipment and techni- cal sophistication required to perform such assessments are considerable, so modifications would clearly be needed if the method were to be used in routine clini- cal or screening situations. Thus, it is clear that measurements of pulmonary meta- bolic functions or other nonbarrier func- tions of endothelium have not reached the status of clinically useful, diagnostic tests. However, with recent and forthcom- ing advances in technology, it is not out- side the realm of possibility that such techniques will be useful both in the clin- ic and in the field. Some of the needs for future research and development are increased basic knowl- edge of how nonbarrier endothelial func- tions, such as transporters and enzymes, work in vivo; investigation in animal mod- els of how acute and chronic lung injury changes several endothelial metabolic functions, especially in the absence of surface area phenomena; determination of the specificity and sensitivity of vari- ous probes in various injury models with the goal of matching the cause of injury with the probe; and simplification of tech- niques to make them more useful in human applications.
PHYSIOLOGYININTACT ORGANISMS 79 TABLE 3-3 Summary of Charactenstics of Physiologic Assays Characteristicsa and Ratings A B C D E F Measure Respiratory function Spirometry Lung mechanics Dynamic compliance, resistance, + + and conductance Oscillation impedance Static pressure-volume Intrapulmonary distribution Single-breath gas washout + Particle distribution Exhaled particles Particle deposition Alveolar-capillary gas transfer CO diffusing capacity Exercise gas exchange Airway reactivity ++ + ++ + ++ + + + ++ ++ + + + ++ ++ + ++ + + + + + + + + + ++ + + + ++ O + O + + + + + + + ++ + ++ + + + + + Nonspecific reactivity + + +- + + + + + + Specific reactivity + + + +- + + + + Particle clearance Radiolabeled aerosol Magnetopneumography Air-blood ba'Tier function Conductingairway permeability Clearance of inhaled DTPA + Transepithelial potential + Alveolar permeability by + radiolabeled aerosol Vascular permeability Radiolabeled protein leakage Chest x ray for edema Extravascular lung water by indicator dilution, PET, or NMR + + + + + + + ++ ++ ++ + + + + + O + o + + O + + + + - Rebreathing soluble gases + + + +- + + + Endothelial metabolic function + + + - +- + aCharacteristics: A. Current State of Development. Considerations in this category included the number of groups using the tech- nique, the availability of the required equipment, the magnitude of the present data base, and the degree of standardiza- tion of procedures. B. Estimated Potential for Development. This category reflected the current estimate of the potential for substantial development of the assay beyond its present state. Although it was recognized that advancements are possible for any assay, this category was intended to reflect potential for substantial technical refinements, adaptation for use in large populations, or advancements in ability to interpret results. C. Current Applicability of Assay to Humans. Primary considerations were the invasiveness of the technique and the requirement for radionuclides. All the assays can be applied to animals, but some are less suitable than others for evaluating humans. D. Suitability for Measuring Large Numbers of Subjects. The focus of this category was the suitability of the assay for use in studies of large populations of people, as might be required for evaluating effects of some environmental expo- sures. Considerations included adaptability of equipment for mobile use, length and nature of subject interaction (i.e., degree of cooperation required), resources required to analyze samples and data, and subject safety. For example, a low rating might suggest a low suitability for field use in evaluating hundreds of subjects of various ages and both sexes, whereas the assay might be quite suitable for studies of dozens of selected subjects brought to a stationary facility.
80 AilARKERS IN PULMONARY TOMCOLOGY E. Reproducibility. This category focuses on the variability of results within and between subjects. F. Interpretability. This category reflects the current understanding of (and degree of consensus as to) pathophysio- logic correlates, anatomic sites of effect, and causative agents. For many of the assays, there is little disagreement on the physiologic function affected, but the specific mechanism or site of change is uncertain. For example, it is agreed that reduced carbon monoxide diffusing capacity reflects reduced efficiency of alveolar-capillary gas transfer, but the test does not distinguish among the effects of a thickened membrane, reduced surface area, and reduced capillary blood volume. bRatings: 0 = Unknown, or information is insufficient. - = Current information suggests inadequate development, little potential for development, little applicability to humans, poor suitability for large populations, poor reproducibility, or poor interpretability. +- = Current information suggests some development, some potential for development, limited applicability to humans, limited suitability for large populations, questionable reproducibility, or questionable interpretability. + = Current information suggests adequate development, potential for further development applicable to humans, suitability for large populations, reproducibility, and interpretability. + + = Current information suggests high development or good potential for substantial development, great applicability to humans, great suitability for large populations, reproducibility, or very good interpretability. SUMMARY Assays of physiologic function in intact subjects are largely markers of response. Few have potential as indicators of expo- sure or susceptibility. Measured charac- teristics often reflect the integrated impact of multiple pathologic altera- tions; they are seldom indicators of speci- fic, single lesions. The respiratory sys- tem responds to injurious agents in only a few ways, so changes in physiologic char- acteristics are seldom specific to causa- tive agents. Many assays are well established and have been used extensively for evaluating patients in the clinic and for studying basic physiologic phenomena. In many cases, therefore, there is information on the relationships among changes in pul- monary function values, subjective sense of illness, and performance disability. Although there is much less information on these relationships for some assays, physiologic assays generally provide a key means of estimating the practical mean- ing of alterations reflected by other types of markers and of estimating the human health impact of environmental exposures. A primary role of the assays, therefore, is to help to determine the extent to which environmental exposures have an impact on health. We have summarized the current clinical assessment of injury to pulmonary endothe- lium and described an example of a biologic .. . . marker of endothelial cell injury that might become useful in either clinical · - or screening programs 1n 1umans. Metabolic lung function was chosen as an example of a biologic property under development as a potential marker of lung injury. That choice was intended not to imply that it is expected to be more useful than other potential markers, but rather to illustrate the challenges that must be met in assessing injury to the pulmonary circulation. Indeed, the techniques re- quired to assess this and other biologic markers of endothelial cell injury are cumbersome and require considerable equipment and technical expertise; those are the limitations to their potential application. The need for further valida- tion is also clear. However, the rapid advances in technology that we have wit- nessed in the recent past and others that are probably imminent might, with commit- ment and effort, render some of the tech- niques useful and bring others to light. Intravascular serotonin is transported into endothelium, where it is either se- questered or metabolized by intracellular monoamine oxidase. The resulting metabo- lite appears in the pulmonary venous blood. Angiotensin I is hydrolyzed to angiotensin II by angiotensin-converting enzyme on the luminal cell surface. Exposure to endothelium-damaging toxicants might alter these processes of carrier-mediated uptake, metabolism, and sequestration. Numerous diverse assays are described
PHYSIOLOGYIN INTACT ORGANISMS in this section, and a tabular summary of their characteristics was thought to be a useful adjunct to the more detailed in- formation in the text. Such tabulation is difficult, because no system of charac- teristics or rating codes fits all the measurements well. If the difficulty and the resulting cautions are appreciated, however, the information in Table 3-3 can provide a useful overview. The definitions 81 of characteristics and codes follow the table. The definitions of characteristics and codes vary from assay to assay and a plus-minus rating is used because it was not considered appropriate to develop a weighted scoring system leading to a sin- gle numeric ranking for each assay. The table is intended as a summary of characteristics.
