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6 Markers of Cellular and Biochemical Response In recent years, much has been learned concerning the cellular and biochemical mechanisms of lung response to both chemi- cal insult and disease. This chapter ex- amines the rapidly developing field of cellular interactions and biochemical mechanisms of respiratory response. It focuses particularly on the analysis of respiratory tract fluids. SOURCES OF RESPIRATORY TRACT MARKERS Although this report deals with several possible sources of biologic markers, the introduction of sampling techniques pecu- liar to the lung and upper respiratory tract has improved understanding of the lung in normal and diseased states. Those techniques are relatively new and still entail some problems in their application for studying biologic responses in large groups of people. This section reviews three techniques for sampling the respira- tory tract. Bronchoalveolar Lavage in Humans The technique of bronchial washing is not new; Reynolds and Newball in 1974 de- scribed a method of Bronchoalveolar ravage through a flexible fiberoptic bron 105 choscope (Reynolds and Newball, 1974~. Their general method has since become wide- ly accepted and is used in many diseases. Bronchoalveolar ravage is the subject of two recent reviews (Daniele et al., 1985; Reynolds, 1987~; the following discussion is limited to questions regarding its ap- plication to patients or populations ex- posed to pulmonary toxicants. Bronchoalveolar ravage (BAL) is usually performed on subjects who are awake. Bron- choscopy with a flexible fiberoptic bron- choscope requires only minimal premedica- tion, usually atropine, and mild sedation. During the procedure, topical anesthesia is provided with Xylocaine (lidocaine). Xylocaine alters the function of alveolar macrophages (Hoidal et al., 1979), but the dose or amount of Xylocaine in the final BAL fluid is usually far below that associ- ated with any effect on alveolar macrophage function (Reynolds, 1987~. The bron- choscope is usually passed as far as pos- sible in the right middle lobe or left upper lobe of the lung. Normal saline solution is introduced and aspirated; aspirated fluid is collected and analyzed. One of the major difficulties in inter- preting the literature on BAL findings has been the variety of techniques used for ravage. High-pressure suction (pres- sure, over 40 cm H2O) usually leads to air

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106 way collapse and poor sampling of the al- veoli and therefore to preferential sam- pling of the bronchi. Several groups have noted that changes in the volume used for ravage result in chemical and physiologic differences in the sample obtained. The first 20-60 ml of instilled fluid usually yields a sample of only the proximal air- ways, and not the alveoli. Several groups discard the fluid retrieved after the first 20 ml is instilled. When the total volume of instilled ravage fluid was 240 ml, the relative proportions of neutrophils and lymphocytes decreased from the first 120 ml to the second 120 ml in normal subjects. In patients with interstitial lung disease and presumably inflammatory cells in the alveoli, the percentages of lymphocytes and neutrophils increased in the second 120 ml (Dohn and Baughman,1985~. Differ- ent portions of the lung might yield dif- ferent proportions of cells, despite the appearance of a homogeneous disease state, as in sarcoidosis (Cantin et al., 1983) and idiopathic pulmonary fibrosis (Garcia et al., 1986~. Another major problem in BAL is that the source of cells retrieved is unknown. Early studies showed a correlation between the extent of inflammation detected with ravage and later biopsy specimens (Crystal et al., 1981; Paradis et al., 1986~. How- ever, results of functional studies have suggested that cells retrieved by BAL dif- fer from those found in the interstitium (Weissler et al., 1986~. Despite the potential wide variability in performing ravage, consensus on how to perform the technique seems to be grow- ing. A questionnaire on BAL technique was MARKERS IN PULMONARY TOMCOLOGY completed and returned by 62 centers throughout the world (Klech et al., 1986). Table 6- 1 shows good agreement. The vari- ability among centers could well decrease with time. The amount of fluid withdrawn in BAL is not standard. One usually retrieves 40-80% of the instilled fluid. The aspirated fluid is a mixture of the instilled fluid and lung fluid. There is no satisfactory way to calculate the extent of dilution of instilled fluid with lung fluid. Markers based on BAL have included endo- genous and exogenous markers. Of the endo- genous markers, albumin and total protein have been most commonly used. The results of BAL are corrected to milligrams of pro- tein or albumin. In inflammatory states, there is an increase in protein transfer across the alveolar-capillary barrier and therefore an increase in the amount of albumin in the ravage fluid. The in- crease has been detected in sarcoidosis (Baughman et al., 1983), asthma (Crimi et al., 1983), and oxygen toxicity (Davis et al., 1983~. The use of albumin is there- fore unsatisfactory in studying disease states not associated with inflammation. Another endogenous marker is urea (Rennard et al., 1986~. Urea readily crosses the alveolar-capillary membrane and therefore is in the same concentration in the lung fluid as in the peripheral blood. Although measurement of urea in aspirated BAL fluid would yield some idea of the amount of lung fluid retrieved, there are again problems. Urea passes rapidly from blood into the alveoli, so the longer the ravage tube is in place, the more urea will go into the alveolar space (Sietsema et al., 1986; TABLE ~1 Results of Survey on BAL Technique in 62 Centers in 19 Countries l echnique Proportion of Centers Using Technique. % Flexible bronchoscopy with only local anesthesia Lavage of either right middle lobe or left upper lobe Use of 10~300 ml of ravage fluid Collection of fluid by pump (low pressure) Collection of fluid in plastic vessels or silicone-coated glass vessels Use of total cell counts Use of differential cell counts 93 98 92 77 100 91 100

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108 Although those studies were done on small numbers of patients, their results suggest that BAL can be performed safely on asth- matics. However, asthmatic patients should be selected with care and carefully monitored after the procedure. Most researchers exclude from BAL pa- tients with moderate to severe airway ob- struction due to asthma. Patients with an FEV~:FVC ratio of less than 0.60:1 are also usually excluded (NHLBI, 1985; Metz- ger et al., 1985~. That approach is safer in that it reduces the likelihood that a patient will develop bronchospasm during the procedure. It has been demonstrated that patients with moderate to severe airway obstruction regularly have a poor return of instilled fluid during BAL (Finley et al., 1967; Martin et al., 1985), probably because of airway collapse during aspiration of the fluid, which is more likely in patients with severe ob- structive airway disease. BAL has been widely applied to evaluation of interstitial lung diseases. Patients with hypersensitivity pneumonitis have a marked influx of lymphocytes into their BAL fluid (Reynolds et al., 1977; Weinber- ger et al., 1978~. The cells are usually characterized as Ts/c cells, so there is a reduction in the Th/i:Ts/c ratio (Leath- erman et al., 1984~. The Ts/c lymphocyte concentration is clearly higher than that in the normal population, but might not be very different from that in subjects exposed to the same antigen but not ill. Leatherman et al. studied pigeon breed- ers and found that those with hypersensi- tivity pneumonitis had increased lympho- cytes in their BAL. They also found that asymptomatic pigeon breeders had increas- ed lymphocytes in their BAL fluid (Leath- erman et al., 1984~. In studying patients with farmer's lung, another type of hyper- sensitivity reaction, Cormier et al. (1987) found an increase in the percentage of lymphocytes with acute disease. How- ever, the increase in lymphocytes was found also in patients who continued to work on their farms but had no further symp- toms. The authors concluded that BAL lym- phocytosis had no prognostic significance for farmer's lung patients. BAL is useful in securing alveolar macro- phages (AMs) from the lung, and retrieval BLURTERS IN PULMONARY TOXICOLOGY of those cells can be useful in character- izing what the lung has been exposed to. For example, BAL fluid from workers exposed to asbestos might contain ferruginous bodies. The most striking example of changes in the cells in BAL fluid is seen in patients who have smoked cigarettes (Finch et al., 1982~. Cigarette-smoking grossly changes the number and properties of AMs retrieved in BAL fluid. There is usually a 10-fold or greater increase in the concentration of AMs retrieved from heavy smokers, compared with nonsmokers. AMs from smokers contain a large amount of amorphous material, which still appears in AMs from ax-smokers. The sur- face properties and histochemical stain- ing of the AMs have changed. They are also more biochemically active. For example, AMs from cigarette-smokers often spon- taneously release hydrogen peroxide and other oxygen radicals (Hoidal and Niewoehner, 1982; Baughman et al., l986b). In assessing the BAL fluid of patients exposed to pulmonary toxins, one must bear in mind that the changes in the AMs caused by smoking can mask other changes due to toxicants. Studies have demonstrated the utility of BAL in assessing patients with asthma. Lavage takes place immediately after chal- lenge or later. The delayed ravage has tended to be 6-8 hours after challenge, to correspond to the late phase of the asth- matic response (De Monchy et al., 1985), or 48-96 hours after challenge (Metzger et al., 1985), to determine the presence or absence of persistent abnormalities in BAL fluid. In asthmatic patients who have the biphasic response to antigen, a difference in the BAL-fluid cellular population between the early and late phases can be demonstrated (De Monchy et al., 1985~. Eosinophils seem not to appear in BAL fluid until the late phase of a reac- tion; BAL fluid from patients without a late-phase reaction does not contain eo- sinophils. That difference supports the current concept that what causes the early phase of the asthmatic response is the release of histamine from mast cells, whereas the late phase is mediated by in- flammatory cells (Booij-Noord et al., 1971).

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CELLULAR AND BIOCHEMICAL RESPONSE In summary, BAL is an interesting diag- nostic tool that allows the sampling of distal airways in a way achieved by no other method. The sensitivity of BAL for disease is not known. It is clear that BAL is not specific for disease, inasmuch as abnor- malities can be seen in the BAL fluid of asymptomatic patients (Leatherman et al., 1984; Cormier et al., 1987~. In the hands of properly trained personnel, it is a safe procedure. In high-risk patients, it might be a useful way of revealing early biologic effects or altered structure, but its role in screening for disease could be limited, because it can be applied only to select populations. Bronchoalveolar Lavage in Animals Analysis of BAL fluid for biologic mark- ers of pulmonary conditions has been useful in animal toxicity studies (Beck at al., 1982; Henderson, 1984, 1988a,b; Henderson et al., 1985a). In large laboratory ani- mals, such as dogs and nonhuman primates, in viva BAL is usually performed in a manner similar to that used in humans. A fiber- optic bronchoscope is wedged into an airway, and the bronchoalveolar space distal to the wedge is ravaged several times (commonly five or six times) with physiologic saline solution (Muggenburg et al., 1972, 1982~. Lavage volumes vary, but 10 ml is adequate. Lavage of small lab- oratory animals can be performed in viva (Mauderly, 1977) if required, but most ravages of rodents are performed on excised lungs. A syringe inserted into the trachea is used to instill the saline solution. Either the total lung or a known fraction of it is ravaged. Lavage volumes are usual- ly approximately half the total lung capac- ity of the section of the lung ravaged. The number of ravages depends on the objec- tive of the study. If the objective is to evaluate the cellular portion of the BAL fluid, numerous ravages, sometimes accom- panied by gentle massage of the lung, might be used to retrieve the maximal number of cells; Fels and Cohn (1986) have reported that the most functionally active cells are retrieved in the later ravages. If the objective is to evaluate the acellular fraction of the BAL fluid, two to four lav 109 ages might be performed to avoid exces- sive dilution of the biochemical compon- ents to be assayed. Recovery of ravage fluid in total-lung ravage in control animals is approximately 75% for the first ravage and 100% for later ravages (Henderson, 1988b). In segmental ravages in large animals, the recovery might be less than 50% on the first ravage, but approaches 100% on later ravages. Data from BAL-fluid analyses can be reported in terms of the total amount of fluid re- trieved per lung or per gram of lung (if the experimental procedure has not affect- ed lung weight) or the concentration of the constituent of interest in the fluid. BAL-fluid analysis has been used for a variety of research objectives. The most common use has been to rank various air- borne materials for potential pulmonary toxicity by determining the inflammatory lung response that follows administration of increasing amounts of them. A second important use has been to follow the prog- ress of a pulmonary condition in an animal without having to kill the animal. BAL- fluid analysis has also been used to eluci- date pathogenic mechanisms in experimen- tally induced lung disease. Examples of each kind of application are described BAL-fluid analysis has been used to rank inhaled or intratracheally instilled mineral dusts for toxicity (Moves et al., 1980; Morgan et al., 1980; Beck et al., 1981, 1982, 1987; Begin et al., 1983; Henderson et al., 1985b) and similarly to rank metallic compounds for toxicity (Henderson et al., 1979a,b; Benson et al., 1986~. The toxicity of an administered material was evaluated according to the degree of inflammation and cell injury as measured by BAL-fluid consent of neutro- phils (marker of influx of inflammatory cells), serum proteins (marker of increas- ed permeability of the alveolar-capillary barrier), lactate dehydrogenase (marker of cytotoxicity), and lysosomal enzymes (usually either beta-glucuronidase or N-acetyl-beta-glucosaminidase, markers of activation or lysis of phagocytic cells). The degree of increase in those markers in BAL fluid was shown to dis- tinguish between the pulmonary response

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110 to more fibrogenic materials (quartz and asbestos) and to less fibrogenic materials (A12O3, Fe2O3, latex beads, and fly ash); between the pulmonary response to the high- ly toxic CdC12 and to the less toxic CrCl3; and between several nickel compounds in acute pulmonary toxicity. The studies were conducted in sheep, rats, or hamsters; results were similar in all species. In several of the studies, comparisons were made between the histologic evaluation of the effects of the materials and the effects as evaluated by BAL-fluid analy- sis. The histologic evaluations confirmed the pulmonary conditions. BAL-fluid analysis is a valid means of detecting an inflammatory response in the lung. Markers in BAL fluid have also been used to measure pulmonary responses to inhaled O3 (Gush et al., 1986) and NO2 (DeNicola et al., 1981~. The most sensitive biologic markers of the inflammation induced by those gases were increased numbers of neu- trophils and, in the acellular fraction, increased protein. Other potential mark- ers of inflammatory response that could be measured in BAL fluid include additional factors released by phagocytic and epi- thelial cells, such as growth factors, arachidonate metabolites, and interleukin I, which are beginning to be used in toxi- cology (Seltzer et al., 1986; Henderson et al., 1 985a; Koren et al., 1989~. In larger animals, such as dogs and non- human primates, BAL-fluid analysis offers a means of following the course of a pul- monary condition sequentially in the same animal. By inserting the bronchoscope in different airways, one can perform BAL several times in the same animal without ravaging the same area. Or one can instill a test material into one area and a vehicle into another area and use a given animal as its own control in determining the ef- fect of the test material. Both applica- tions have been used by Bice et al. (1980a) and reviewed by Bice (1985~. The inves- tigators instilled sheep red blood cells into the left lung of a dog and followed the course of appearance of IgM- and IgG- forming cells. The right lung of the same dog received saline solution and served as a control. Significantly more antibody- forming cells were found in BAL fluid from Af'9RKERS IN PULMONARY TOX[COLOGY the immunized lung than from the control lung. One could even do a whole dose-re- sponse study in the same animal by instil- ling different amounts of the test material into different areas of the lung. In one study (Bice and Muggenburg, 1986), various numbers of sheep red blood cells were in- stilled into different areas of a dog's lung to determine the dose-response char- acteristics of the immune response. Bice et al. (1982) used BAL-fluid analy- sis to elucidate the mechanism of recruit- ment of immune cells to the lung. Two lung lobes of a dog were immunized with antigen- ically different particles (sheep and rabbit red blood cells) to determine wheth- er immune cells in the blood are recruited to the lung in an antigen-specific manner. Analysis of BAL fluid indicated that equal numbers of anti-sheep-red-blood-cell antibody-forming cells were present in both immunized lobes. The authors con- cluded that cell recruitment was not anti- gen-specific, but related to nonspecific changes in the lobes induced by antigen exposure. Another example of the useful- ness of biologic markers in BAL fluid in the elucidation of mechanisms of disease is the work of Holtzman et al. (1983), who found that an increase in airway respon- siveness in dogs was related to an influx of neutrophils and increases in prostag- landins E2 and F2a in BAL fluid. Thus, the markers of biologic events that can be found in BAL fluid are useful in toxicology. The use of BAL-fluid mark- ers has several advantages. BAL-fluid analysis results in a rapid, quantitative measure of pulmonary response that is not obtained with routine histologic evalua- tions. BAL-fluid analysis allows detec- tion of early biologic events. Investigat- ors have reported detection of inflamma- tion through BAL-fluid analysis before radiographic detection was possible (Fahey et al., 1982~. In large animals, the procedure can be used sequentially to follow the course of a biologic event . . - In a given anlma . The present limitation of the method is the lack of specificity of the markers for the site of inflammation or injury or for the lung disease. Only a general in- flammatory response can be detected. Con

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CELLULAR AND BIOCHEMICAL RESPONSE tinned research is needed to develop site- specific markers of respiratory tract injury and validation of profiles of BAL- fluid changes that are indicative of the presence of or progression toward a speci- fic lung disease. Especially useful would be markers to indicate the early stages of a progressive condition that leads to disease, such as fibrosis, emphysema, or cancer. In toxicology, such markers would allow earlier detection of late-occurring events; if they were applicable to humans, they would allow therapeutic intervention at an early stage in a disease process. Research to elucidate the mechanisms by which respiratory diseases develop should aid in obtaining the information required to select the correct markers. Bronchial Lavage Bronchial ravage, a variation of BAL, has emerged in the last few years. The need for such a procedure became obvious as people began to note the differences be- tween small- and large-volume ravage (Dohn and Baughman, 1985~. The cells retrieved in the first portions of BAL fluid are from the larger airways. Although those cells might reflect contamination in subjects with alveolar disease, the first portions might be of most interest in connection with patients in whom the disease is of the large airways. In one method of bronchial ravage, a catheter is passed through a flexible fi- beroptic bronchoscope into a main bronchus with light anesthesia. Attached to the outside of the catheter are two balloons several centimeters apart. The balloons are blown up, occluding the airway and sealing the section of bronchus between the two balloons. Fluid is then introduced into and withdrawn from the lumen between the balloons. Eschenbacher and Gravelyn (1987) used the method to expose the bron- chial wall to hypo-osmolar challenge, ravage the area, and examine the fluid for biochemical factors. Nasal Lavage The nose is the primary portal of entry of inspired air, and one of its major roles 111 is to protect the lower respiratory tract from inhaled pollutants. For example, 100% of SO2 drawn into the nose, 20-80% of O3, and 73% of NO2 are trapped there under normal conditions (Vaughan et al., 1969~. Therefore, if nasal clearance is impaired, a larger amount of pollutants could reach the lower lung. That is reason enough to study the effects of pollutants on the nasopharyngeal region; another reason is that the nasal passages contain many of the same cell types as the trachea and bronchi, but are more convenient and more accessible for studying in vivo effects of airborne toxicants (Proctor, 1982; Cole and Stanley, 1983; Koenig and Pierson, 1984~. Secretions from the nasal area have been analyzed for various proteins and cell types (Remington et al., 1964; Rossen et al., 1966; Lorin et al., 1972; Mygind et al., 1975~. In clinical trials, one cannot ensure that rhinitis will be produced in experimental subjects; even if a pollutant is noxious enough to produce a heavy secre- tion, an unexposed control group will pro- vide little secretion for comparison. An alternative is to collect specimens with nasal ravage. Nasal ravage is simple to perform, noninvasive, and nontrau- matic. In an adaptation of the technique reported by Powell et al. (1977), the sub- ject is instructed to sit upright with head tilted back and to establish palatal pres- sure. A needleless syringe is used to in- still into each nostril 5 ml of sterile phosphate-buffered saline solution (Brain and Frank, 1973~. The saline solution is held in the nasal passages for 10 seconds and then forcibly expelled and collected. Of the 10 ml instilled, about 7ml is rou- tinely recovered. Sloughed squamous, columnar, and (less often) ciliated epithelial cells are rou- tinely found in nasal ravage. Leukocytes are normally present, and their numbers increase in some disease states. An in- crease in nasal eosinophils has been used as a clinical verification of an allergic reaction (Malmberg and Holopainen, 1979; R. E. Miller et al., 1982), and nasal baso- phils have been shown to increase in aller- gic persons 10-20 minutes after antigen challenge (Bascom et al., 1988~. Neutro

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112 phils increase by a factor of 10-100 during an upper respiratory tract viral infection (Parr et al., 1984; Henderson et al., 1987~. A significant increase in nasal- lavage neutrophil numbers has also been shown to occur in response to acute expo- sure to ozone at 0.5 ppm; ozone is an oxidant air pollutant known to induce an inflam- matory response in the lungs of animals (Graham et al., 1988~. When looking at changes in the nasal- lavage cell population, one must consider the effect of earlier unidentified envi- ronmental exposures. Of 200 volunteers. 50% had fewer than 104 polymorphonuclear neutrophils (PMNs) per milliliter of nasal ravage, and 10% had over 105 PMNs per mil- liliter (Graham et al., 1988) The remain- ing ravages were evenly scattered between those extremes. Responses to a question- naire on life style and exposure suggest that increased numbers of PMNs might be associated with recent colds, with expo- sure on the previous evening to heavy ciga- rette smoke or chemicals found in Chlorox and paint stripper, with gasoline fumes, or with recent swimming in lakes or ponds. Such environmental exposures could ac- count for the variability in cell counts seen by Parr et al. (1984) when five samples were taken from the same person over a 2-week period. Potential effects of uncon- trolled environmental exposures must be taken into account in the design of a study. Pre-experiment and post-experiment sam- ples taken on the same day and instruction of subjects can reduce the confounding effects. Markers in nasal-ravage fluid that have been studied include increase in total protein, associated with cell damage and permeability change (Lorin et al., 1972; Marom et al., 1984~; increases in concen- trations of albumin and immunoglobulin G. associated with increased vascular permeability (Butler et al., 1970; Rossen et al., 1971; Brandtzaeg, 1984~; histamine and prostaglandin D2, released from mast cells in response to an allergic reaction (Naclerio et al., 1983; Eggleston et al., 1984~; increases in concentrations of sulfidopeptide leukotrienes and kininogens, after an allergic response (Creticos et al., 1984; Baumgarten et al., AL9R=RS IN PULMONARY TOXICOLOGY 1985; Togias et al., 1986~; and increases in concentrations of immunoglobulin E in hay fever (Miadonna et al., 1983; Small etal.,1985~. Substances measured in nasal-ravage fluid have an unknown dilution factor, as is the case in BAL fluid. The absolute concentration cannot be determined, and interpretations are of limited value. Pre-experiment concentrations of media- tors can vary widely between individuals and generate a "noisy" baseline. In study- ing allergic responses, Togias et al. (1986) uses four or five ravages before an experimental exposure and analyzes the first and last of them. That yields infor- mation on the pre-experiment concentra- tions and provides a more stable baseline. Analysis of nasal-ravage fluids for speci- fic mediators might thus not be practical for screening large populations. In spite of those concerns, much informa- tion can be obtained from nasal ravage. The procedure allows measurement of an effect of a pollutant on a mucosal surface, requires no anesthetic, and does not itself induce the release of mediators (Baumgarten et al., 1985) or an inflam- matory response (Graham et al., 1988~. Multiple samples from the same person are possible, as well as samples from subjects who are at risk, such as asthmatics. No special equipment is required, so this is an attractive and inexpensive approach for epidemiologic or occupational studies. Furthermore, nasal ravage can be useful in determining which air pollut- ants might induce an inflammatory response in the human respiratory tract. Increases in neutrophil, eosinophil, or basophil concentrations-which are easily measured and have been associated with health ef- fects-could be the most useful markers in the nasal ravages. More environmental and occupational studies analyzing nasal ravages for both cells and mediators in both normal and asthmatic persons are needed for a full appreciation of the value of this approach in screening. Studies of such different pollutants as sidestream tobacco smoke, cotton dust, and SO2 could be useful in that regard. Nasal ravages might be useful in studying the effects of indoor air pollu

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CELLULAR AND BIOCHEMICAL RESPONSE lion, inasmuch as nasal irritation is one of the most common complaints associated with that pollution. Studies comparing cell and mediator changes in the nasal- lavage fluid with those found in BAL fluid from pollutant-exposed people are needed to determine whether the nasopharyngeal region can be used as a diagnostic mirror of the lower respiratory tract-i.e., whether nasal symptoms can herald lower respiratory disease or provide important clues to coexisting chest disease. POTENTIAL MARKERS IN RESPIRATORY TRACT FLUIDS Both the cellular and acellular contents of nasal, bronchial, and bronchoalveolar ravages can provide markers of response to environmental exposures, as shown in Table 6-2. In the following sections, the cellular and the acellular supernatant fractions of these fluids and their poten- tial for use as biologic markers are dis- cussed in more detail. Their use as markers is summarized in Table 6-3. Cellular Content Macrophages The predominant cell in BAL fluid from normal subjects is the macrophage. In some species (Henderson, 1988a), lymphocytes can be present in small numbers. Neutro- phils, eosinophils, and mast cells might also be present as a result of an inflam- matory response. Human alveolar macrophages (AMs) are composed of several populations that can be distinguished by density. In general, denser AMs are less mature and resemble blood monocytes more than less dense AMs. The denser cells are more potent producers of a soluble factor that inhibits fibro- hlast proliferation (Elias et al.. 1985a) and of interleukin- 1 (It- 1 ~ (Elias et al., 1985b), and they are more efficient acces- sory cells for antigen-induced prolifera- tion (Ferro et al., 1987~. AM abnormali- ties in sarcoidosis might represent dif- ferences in the relative proportions of AM subpopulations, rather than intrinsic differences in the same AM subpopulations. 113 Hance and colleagues ~ 1985) have found that AMs from patients with sarcoidosis express antigens that are present on blood monocytes, but AMs from normal subjects do not. Other attributes of sarcoid AMs, such as accessory cell function (Venet et al., 1985) and spontaneous release of interferon-gamma and growth factors (Bitterman et al., 1983), are compatible with the influx of less mature AMs in sar . coldosls. Accessory Cell Function. Macrophages are important in the regulation of the immune response, acting as both promoters and suppressors of events that result in immunization or inflammation (Unanue et al., 1984~. In general, AMs that can be obtained from normal humans with ravage contain subpopulations that can increase or suppress lymphocyte proliferation. The result depends on culture conditions- the presence of other accessory cells and the amount of antigen or mitogen present (L1u et al., 1984; Toews et al., 1984; Ettensohn et al., 19864. Alterations in accessory cell function might affect path- ogenesis. In fact, as mentioned above, some studies have shown that AMs from pa- tients with sarcoidosis have accessory cell function more efficient than that in AMs from normal subjects (tom et al., 1985; Venet et al., 1985~. AMs from asth- matics are less able than AMs from normal subjects to suppress mitogen-induced lymphocyte proliferation (Aubas et al., 1984~. Changes in accessory cell function are gaining acceptance and should be used as markers of environmental exposure and perhaps as markers of susceptibility to disease. Interleukin-l. AMs can secrete Il-l, but apparently to a smaller degree than can peripheral blood monocytes (Koretzky et al., 1983; Wewers et al., 1984~. The uncertainty regarding AM Il-1 production and its control mechanisms probably de- rives from the multiplicity of AM products, some of which antagonize Il-1 production (Monick et al., 1987~. Il-1 is an important mediator of inflammation (Dinarello, 1984) and acts as a differentiation signal for several subsets of lymphocytes. In particular, I1- 1 promotes differentiation

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114 AL4R=RS IN PULMONARY TOXICOLOGY TABLE ~2 Techniques for Detecting Markers of Inflammatory and Immune Response Technique Advantages Disadvantages Bronchoalveolar Relatively safe; material Variable concentration of ravage obtained from lung (com- return fluid constituents; partmentalization); large expenshe; obtaining of normal quantities of cells and controls in large numbers fluid available; repeatable; difficult; sample can be inter large area of lung sampled stitial, alveolar, or bronchial; invasive Nasal ravage Suitable for large studies; Yields few cells; might not safe; repeatable; establish- reflect lower respiratory tract ment of reproducible base- response; response short-lived; line values possible; in- timing of sampling after expo expensive; affords both sure critical; requires patient cellular and fluid analysis; cooperation and understanding quick return to baseline; no anesthesia needed Blood and urine Inexpensive; safe; repeat- Often does not reflect collection able; large sample avail- respiratory tract response; able; widespread patient risk of exposure of investigator acceptance Measurement of Sensitive for inflammatory Leukocyte influx a non cellular influx and immune responses; easily specific response and might measured by established be transient methods; presence of in creased numbers of some leukocyte types is assoc iated with specific pul monary responses Phenotyping Specific; reproducible Expensive; pathophysiologic relevance of specific marker must be established; might re quire large number of cells Measurement of Probably important mediators Expensive; difficult with cur arachidonic acid of respiratory tract injury rent methods; importance cur metabolites, easily measured in respire- rently unknown; nonspecific; cytokines, and tory secretions; relatively samples sometimes cannot be enzymes inexpensive; sensitive stored in bulk for later analysis Examination of Sensitive; relatively spe- Measurement difficult; ex mast cell cific for inflammation pensive In vitro assay Measures antigen-specific Cumbersome; expensive; re response; risk low, well quires extrapolation to established whole-body response; relevant antigens unavailable Skin test Measures antigen-specific Some associated risk; sensi whole-body response; has tivity and specificity often been used in large popula- poor; relevant antigens una tion studies vailable Intrabronchial Measures antigen-specific Use still limited; some as test tissue response sociated risk; expensive

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CELLUL4R AND BIOCHEMICAL RESPONSE 115 TABLE ~3 Biologic Markers of Inflammatory and Immune Response in Humansa Circumstances Controlled Natural Occupational Environmental Challenge Illness Studv Studv Marker White blood cells Neutrophil-Lymphocyte Influx Nasal Y Y P P Bronchial Y P N N BALL y y y N Neutrophil release O'yradicals P Y N N Factors P Y N N Lymphocyte (BAL) Subpopulation changes Y Y Y N Functional changes Y Y Y N Macrophage (BAL) Release of factors Y Y P N Release of o~yradicals Y P P N Change in cell surface Y P P N Mast cells Influx Y Y P N Release of factors Y Y P N Eosinophils Influx Y Y Y Y Release of factors P Y N N Biochemical changes Arachidonic acid metabolites Urine N Y N N Nasal wash Y N N N BAL Y Y N N Histamine Blood N Y N N Nasal wash Y Y P - N BAL Y Y N N En~ymes, fluid Blood Y Y N N Nasal wash Y Y N N BAL Y Y P N Enzymes, cell-associated Nasal wash P P N N BAL N Y N N Functional changes Antigen-toxin challenge Skin Y Y Y Y Nasal wash Y Y P N Aerosol Y Y Y P Intrabronchial Y Y N N Rast N Y Y N In Vitro Lymphocyte Blood Y Y Y N BAL Y Y P N Monocyte-Macrophage Blood Y Y Y N BAL Y Y N N ay = yes (well documented); P = preliminary data; N = not done yet.

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122 of prostacyclin, and platelets are active producers of thromboxane A2. The most im- portant cellular sites of lipoxygenase reactions are mast cells, basophils, and neutrophils (Said, 1982; Lewis and Austen, 1984;Lewis, 1985~. Humanairwayepitheli- al cells can also selectively generate 15-lipoxygenase metabolites of arachidon- ic acid (Hunter et al., 1985~. Generally, prostaglandins of the D and F series are vasoconstrictors, whereas those of the E series and PGI2 are vasodila- tors. PGE2 acts as a vasodilator in the fetus and newborn and a weak vasoconstric- tor in the adult. Prostaglandins, unlike leukotrienes, neither increase vascular permeability nor promote chemotaxis. The principal products of the lipoxygen- ase pathway are leukotrienes. Three (LTC4, LTD4, and LTE4) are sulfidopeptide leuko- trienes and constitute the mediator ori- ginally described as slow-reacting sub- stance of anaphylaxis (SRS-A). The com- pounds induce a sustained bronchospasm that is greater in peripheral than in cen- tral airways and can play a role in bron- chial asthma (Weiss et al., 19824. It has been suggested that they can cause bron- chial hyperresponsiveness that charac- terizes the asthmatic condition (Griffin et al., 1983~. However, further research is needed to elucidate their exact role. In addition to their putative role in asth- ma, they have been shown to mediate in- creased vascular permeability in some tis- sues (Lewis and Austen, 1984), but their role in the lungs is uncertain. Studies with LTB4 have shown it to be a potent chemo- tactic agent that produces endothelial cell adherence. Arachidonic acid metabolites are not stored in tissues, but are synthesized de novo in response to stimuli. Their role as mediators of lung disease is only begin- ning to unfold. With the advent of sensi- tive radioimmunoassay and high-perfor- mance liquid chromatographic (HPLC) tech- niques, it is now possible to measure them in biologic fluids and study their produc- tion in isolated cultured cell systems. A number of recent studies have examined the presence of those compounds in BAL fluid. Murray et al. (1986) performed BAL on MARKERS IN PULMONARY TOXICOLOGY five patients with chronic stable asthma before and after local challenge with Der- mc~ophagoides pteronyssimus. The BAL fluid was analyzed for arachidonic acid metabo- lites. In the five patients, PGD2 concen- trations increased by an average of a fac- tor of 150 after local instillation of antigen. The results constitute evidence that the release of PGD2 into the airways is an early event after the instillation of D. pteronyssimus in patients who are sen- sitive to this antigen. The oxidant air pollutant ozone can pro- duce airway inflammation and hyperrespon- siveness in exposed people. Seltzer et al. (1986) exposed 10 healthy human sub- jects to air or O3 (0.4 or 0.6 ppm). Airway responsiveness to inhaled methacholine was measured before and after each expo- sure, and BAL was performed 3 hours after the exposure. An increase in the number of neutrophils was found in BAL fluid from O3-exposed subjects, especially those in whom O3 exposure produced an increase in airway responsiveness. They also found significant increases in PGE2, PGF2, and thromboxane B2 in BAL fluid from O3-exposed subjects. Hence, O3-induced hyperrespon- siveness appears to be associated with both neutrophil influx and changes in the concentrations of some cyclo-oxygenase metabolites. Other studies (Laviolette et al., 1981) have examined the production of arachidon- ic acid metabolites by human AMs recovered from smokers and nonsmokers. PGE2 and thromboxane B2 synthesis was significantly lower in AMs from smokers than in those from nonsmokers . A cigarette-smoke- induced lesion in phospholipid hydrolysis is most consistent with the findings. Inasmuch as arachidonic acid metabolites are in- volved in the regulation of immune and inflammatory responses and bronchiolar and vascular smooth muscle reactivities in the lung, it was concluded that the de- fect observed in smokers' AM can play a role in the pathogenesis of cigarette-smoke- induced diseases. Animal studies have also attempted to correlate exposure to airborne substances with increases in ravage-fluid arachidon- ic acid metabolites. Mundie et al. (1985) exposed New Zealand white rabbits to aero

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CELLUL4R AND BIOCHEMICAL RESPONSE solized cotton dust extract and performed ravage at various times after exposure. PGF2, PGE2, and thromboxane B2 were maxi- mally increased in the ravage fluid 4 hours after exposure. Results of that study, the first to demonstrate in viva release of arachidonic acid metabolites in the lung in response to inhalation of cotton dust extract, strongly suggest that the metabolites are responsible for the bron- choconstriction seen in the acute byssin- otic reaction in humans. Further studies that establish the presence of those metab- olites at sites of lung injury are needed, for their role in the pathogenesis of lung injury to be understood. Complement The complement system, which is composed of more than 20 plasma proteins, is an im- portant mediator of various inflammatory responses, such as increase in vascular permeability and chemotaxis of PMNs (Colten et al., 1981~. The system com- prises two major pathways-classical and alternative (Perez, 1984~. The classical pathway involves the union of antigen and antibody, which then binds to and activates the C1 complex. The alternative pathway involves direct contact of the subject with antigens, such as bacteria, fungi, endotoxins, immune complexes, and par- ticles (Wilson et al., 1977; Warheit et al., 1985, 1986~. Active products of the complement sys- tem, such as Cab, have been shown to promote phagocytosis. C3a and CSa, however, can cause mast cells to degranulate and release histamine, and they can increase vascular permeability. CSa is also chemotactic for PMNs and promotes the release of their lysosomal enzymes (Perez, 1984~. BAL fluid from normal persons contains components of both the classical and alter- native pathways of complement, i.e., C3, C4, C5, C6, and factor B (Reynolds and Newball, 1974; Robertson et al., 1976; Henson et al., 1979~. Although some of those components can be derived via trans- udation from serum, local production by lung fibroblasts and AMs is also possible (Colten and Einstein, 1976; Reid and Solomon, 1977; Cole et al., 1983~. During 123 inflammation, when vascular and epithel- ial membrane permeability is increased, additional complement components of plas- ma can enter alveolar spaces and airways. If it is feasible to use enumeration and analysis of inhaled particles in situ as a marker of exposure, then it should be feasible to use cellular responses to such exposure as further markers of exposure, as well as predictors of injury at the al- veolar level. At least two biologic re- sponses take place very rapidly after ex- posure to a variety of inorganic particles. The first is activation of the fifth com- ponent of complement, C5 (Warheit et al., 1985), and the second is macrophage accumu- lation, which is a consequence of the ac- tivation (Warheit et al.,1984- 1986~. Several complement components normally are found in the complex alveolar lining layer. The role of complement on alveolar surfaces is not entirely clear, but one important function appears to be the clear- ance of inhaled microbes (Larsen et al., 1982~. It has been established that C5 can be activated through the alternative path- way to produce CSa, a potent chemoattrac- tant of neutrophils and macrophages (Snyderman, 1981~. It was recently shown that the C5 on alveolar surfaces was ac- tivated by a variety of inhaled particles (Warheit et al., 1988) chrysotile asbestos being a noteworthy example. During a 3-hour exposure to asbestos, all detec- table C5 on alveolar surfaces was converted to CSa (Warheit et al., 1986~. CSa, a chemo- tactic factor, remained active in the al- veoli for about a week, and concentrations of C5 returned to normal 1-2 weeks after the 3-hour exposure (Warheit et al., 1986~. Could that biologic response be used as a marker of exposure to inorganic par- ticles, as described in Chapter 2? Alveo- lar fluids collected with BAL have been separated by appropriate biochemical techniques. If the normal extent of com- olement-denendent chemotactic activity were established, activation of C5 might well serve as a marker of exposure. The cellular response to activation should also be predictable and could serve as an additional, correlated marker. Activated C5, whether from serum com- plement or from alveolar complement, at

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124 tracts neutrophils and macrophages (Warheit et al., 1986~. Several studies have shown that it is possible to predict whether macrophages will be attracted to specific bacteria or inorganic particles on the basis of their capacity to activate C5 in vitro (Warheit et al., 1988~. For example, chrysotile asbestos and crocido- lite asbestos are good activators of C5; after inhalation, they attract macro- phages to alveolar duct bifurcations. However, ash from Mount Saint Helens in- duces no detectable CSa production and attracts few macrophages that have been stimulated to migrate by CSa. It is con- ceivable that such cells, easily recovered from the lung, could serve as markers of exposure if their biology were better understood. There is a vast literature on macrophage physiology and function aimed at develop- ing a better understanding of macrophages (Fels and Cohn, 1986~. Those cells are avid phagocytes, so determination of their particle burden has proved to be an ex- tremely useful marker of exposure (Brody, 1984~. However, in humans, recovery of macrophages from the lung is usually too late to yield an early marker of exposure. It might therefore be important to consider initial complement activation and the later macrophage response in estimating exposure in humans. Once the macrophages have responded to inhaled agents, it is reasonable to con- clude that the cells will release a variety of products (Fels and Cohn, 1986), many of which are known to have profound effects on pulmonary cells and tissues. Three well-known examples of such products are oxygen radicals, arachidonic acid metab- olites, and growth factors for fibro- blasts. Whether any or all of them could serve as useful markers of exposure and injury is yet to be determined. But it is reasonable to suggest that they all could become markers of pulmonary insult. Recently, investigators have begun to look at complement activity in BAL fluid from patients with pulmonary disease. For example, Lambre et al. (1986) demon- strated the presence of C3b and Bb (the activated forms of the proteins C3 and B) in BAL fluid from patients with pulmonary MARKERS IN PULMONARY TOXICOLOGY sarcoidosis. Complement activity in la- vage fluid decreased in patients receiving corticosteroid therapy. That suggests that complement activity in the alveolar spaces might be a good marker of the activi- ty of the disease in lasting sarcoidosis. There have also been reports of the pres- ence of C3b in ravage fluid from patients with idiopathic pulmonary fibrosis (Robbing et al., 1981~. Results of such studies suggest that the complement system can play a role in the pathogenesis of those diseases. Further studies are needed, however, to validate the use of complement activity as a marker of lung injury and disease. Growth Factors and Monokines In addition to their role as the primary phagocytes in the lung, AMs synthesize diverse substances that exhibit a broad range of biologic activities, including mediators (monokines) that regulate the growth or activation of other cells. Pul- monary AMs. on activation, release two primary growth factors for lung fibro- blasts: fibronectin and AM-derived growth factor, or AMDGF (Bitterman et al., 1986~. Fibronectin, a 440,000-dalton glyco- protein, has been shown to act as a "compe- tence factor"; it delivers a growth-pro- motlng signal to nonreplicating lung fi- broblasts early in the G1 phase of the cell cycle. AMDGF, an 1 8,000-dalton peptide, provides the second (progression signal) of two required signals in G1 to induce fibroblasts to divide. Although fibronec- tin is a normal constituent of the alveolar epithelial lining fluid, Rennard and Crys- tal (1982) have shown that its concentra- tion is 2-5 times higher in patients with fibrotic lung disorders. In fact, pulmon- ary AMs from most patients with intersti- tial fibrosis have been shown to release both fibronectin and AMDGF (Bitterman et al., 1986~. However, the exact role of these growth-modulating signals in the pathogenesis of chronic interstitial disorders remains to be elucidated. AMs release interleukin 1 (I1- 1), a mono- kine that is a lymphocyte-activating fac- tor, in response to various immune or in- flammatory stimuli. I1- 1, a protein of

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CELLULAR AND BIOCHEMICAL RESPONSE 125 12,000-18,000 daltons, is thought to be that acute lung injury occurs if specific important in modulating T- and B-cell enzyme-substrate systems known to gener . . . . .. ~ ate oxygen metabolites are intratracheal- ly instilled into rat lungs. For example, if xanthine and xanthine oxidase (which activation and in other 1nt lammatory processes (Wewers et al., 1984~. Many of the substances that induce the secretion of Il-1 can also stimulate AMs to secrete interferon-gamma. Interferon- gamma augments T-cell replication, ap- parently by inducing 11-2 receptor expres- sion (Johnson and Farrar, 1983~. Clearly, stimulated AMs can release a wide array of potent biologic mediators. However, factors governing the selective release of those mediators into the low- er respiratory tract are still poorly understood. Oxygen Radicals Considerable evidence accumulated in recent years suggests that oxygen-derived free radicals are an important cause of tissue injury in many disease processes (Freeman and Crapo, 1982~. The lung is prone to oxidant stress with a variety of sources, and it has been suggested that a reactive oxygen species plays a role in the development of acute lung injury and the etiology of chronic lung disease (Johnson et al., 1981~. As described previously, oxidants can be generated in the lower respiratory tract via the action of PMNs and AMs involved in local inflammatory reactions. On recogni- tion of a phagocytic or soluble stimulus, both neutrophils and macrophages experi- ence a "respiratory burst" that is charac- terized by an increase in oxygen consump- tion, activation of the hexose monophos- phate shunt, and the generation of reactive oxygen species, including O2-, H2O2, and OH.. That burst of activity is related to the stimulation of membrane-bound reduced nicotinamide adenine dinucleotide phos- phate (NADPH) oxidase (Babior, 1978~. Those oxygen-derived products normally play a major role in phagocyte-mediated bactericidal activity, but it is conceiv- able that they contribute to host tissue injury when their production is stimulated inappropriately. A number of models that attempt to assess the effects of oxygen radicals on the lung have been developed. Johnson et al. ( 1981 ) demonstrated generate ()2 ) was administered 1ntra- tracheally, there was increased vascular permeability with minor edema formation and focal hemorrhage after 4 hours. Those pathologic changes could be inhibited by simultaneous instillation of superoxide dismutase (SOD). However, when glucose and glucose oxidase (which generate H202) were instilled into the airways, there was a marked increase in vascular per- meability, edema, hyaline membrane forma- tion, hemorrhage, and neutrophil influx. Those changes are consistent with the human pathologic changes referred to as diffuse alveolar damage and associated with adult respiratory distress syndrome (ARDS). The changes could be inhibited with catal- ase, but not SOD. Furthermore, if either lactoperoxidase (LPO) or myeloperoxidase (MPO) was instilled with the glucose- glucose oxidase system, severe lung injury occurred and frequently progressed to diffuse pulmonary fibrosis by 4 days. The data suggest that a product of MPO (or LPO), H202, and halide (perhaps HOC1) plays an important role in the development of pulmonary fibrosis. Oxygen-derived free radicals and their metabolites can cause acute lung injury and progressive lung injury with pulmonary fibrosis. In another series of experiments, Johnson and Ward (1982) instilled phorbol myristate acetate (PMA), a potent initia- tor of the respiratory burst, intratra- cheally into neutrophil-depleted rats. They found that the instillation caused acute lung injury that was inhibited by catalase, but not by SOD; again, H2O2 was implicated as the cause of the damage. The source of the toxic H2O2 in the model ap- pears to be PMA-stimulated AMs. Some in- vestigators have recently found that AMs retrieved from some cigarette-smokers spontaneously release H2O2 (Greening and Lowrie, 1983; Baughman et al., 1 986b). That suggests that cigarette smoke can activate these cells (Hoidal and Niewoeh- ner, 1982~. Although activated phagocytes can release substantial amounts of potent

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126 oxidants to surrounding tissues, they are by no means the only source of reactive oxygen species in the lung. Reduction of O2 to active O2 metabolites occurs as a byproduct of cellular metabolism during microsomal and mitochondrial electron transfer reactions (Cohen and Cederbaum, 1979~; considerable amounts of O2- are generated by NADPH-cytochrome P-450 reductase reactions (Kameda et al., 1979~. Because those metabolites are potentially cytotoxic, they might mediate or promote actions of various pneumotoxins. Such mechanisms have been proposed for paraquat- and nitrofurantoin-induced lung injury (Sesame and Boyd, 1979; Shu et al., 1979~. Similarly, Freeman and Crapo (1981) demonstrated that hyperoxia increases the steady-state concentrations of O2- and H2O2 in lung tissue and that mi- trochodria contribute importantly to this phenomenon. Those observations lend sup- port to the hypothesis that lung damage during hyperopia is mediated by increased production of oxygen radicals. In conclusion, reactive oxygen species in the lung can be generated by multiple and diverse processes and appear to play a role in the onset of acute lung injury and possibly in the development of chronic lung disease. Additional studies are ne- cessary, to define the precise targets of oxygen metabolites in the lung and the specific biochemical mechanisms by which the oxidants damage lung cells. Enzymes Increases in enzymatic activities in BAL fluid have been used as markers of pul- monary responses to inhaled toxicants, particularly in animals (Beck et al., 1982; Henderson, 1988a,b). Extracellular lac- tate dehydrogenase (LDH), a cytoplasmic enzyme, is used as a marker of cytotoxici- ty, because LDH is not found extracellular- ly except in the presence of damaged or lysed cells. This marker has been used in numerous studies, for example, in hamsters exposed to mineral dusts (Beck et al., 1982), in rats and mice exposed to diesel exhaust (Henderson et al., 1988), and in sheep exposed to asbestos (Begin et al., 1983~. Another cytoplasmic enzyme that MARKERS IN PULMONARY TOXICOLOGY has been assayed in BAL fluid is glutath- ione reductase (Henderson et al., 1988~. Lysosomal enzymes, such as N-acetylglu- cosaminidase (Beck et al., 1982) and beta- glucuronidase (Henderson, 1988b), appear to be good indicators of increased phago- cytic activity in response to inhaled par- ticles. The extent of the increases in BAL- fluid lysosomal enzyme activities appears to correspond to the toxicity of the in- haled particles (Beck et al., 1 982; Henderson et al., 1985a) and exceeds the degree of increase in LDH by several fold (Henderson et al., 1985b). The increase in lysosomal enzymes relative to LDH can be used to estimate how much of the increase in lysosomal enzymes is due to lysed cells (which would cause concomitant release of lysosomal enzymes and LDH) and how much is due to stimulated phagocytic cells. Acid phosphatase, also a lysosomal enzyme, does not increase in BAL fluid in response to inhaled particles (Henderson et al., 1985b). Either that enzyme is not in the same lysosomal storage site as beta-glucu- ronidase and similar hydrolytic enzymes or it is rapidly broken down in the epithel- ial lining fluid, once released. Increases in alkaline phosphatase ac- tivity have been detected in BAL fluid from NO2-exposed hamsters (DeNicola et al., 1981). A lung-specific form of this enzyme has been reported to be released from Type II pneumocytes (Reasor et al., 1978; Miller et al., 1986~. A histochemical stain spe- cific for the enzyme has been used as a mark- er of Type II cell proliferation (B. E. Milleretal.,1987~. Proteolytic activity and antiproteolyt- ic activity in BAL fluid are of interest, because an imbalance between the two could lead to breakdown of lung tissue, such as that seen in emphysema (Janoff, 1972; Starkey and Barrett, 1977~. Proteolytic enzymes detected in BAL fluid include col- lagenase, PMN elastase, metalloprotein- ase, plasminogen activator, and acid pro- teinases (Barrett, 1977a,b; Harper, 1980; Gadek et al., 1980; Pickrell, 19814. An- tiproteinases in BAL fluid are alpha'- antiproteinase, alpha2-macroglobulins, and bronchial antiproteinase. Acid pro- teinase activity is associated with lyso- somes and is released with other lysosomal

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CELLULAR AND BIOCHEMICAL RESPONSE enzymes in response to inhaled toxic par- ticles (Wolff et al., 1988~. Protein and Protein Products Protein in BAL fluid is measured as a marker of increased permeability of the alveolar-capillary barrier and is a common component of the inflammatory response. Bell and Hook (1979) reported that 80% of the soluble protein in human BAL fluid could be accounted for by 19 plasma pro- teins. The protein content indicated a preferential transfer of smaller proteins across the alveolar-capillary barrier. IgG and IgA constituted a higher fraction of total protein in BAL fluid from smokers than in serum (Bell et al., 1981~. Trans- ferrin was the only nonimmunoglobulin protein with a higher concentration in ravage fluid than in serum. Serum proteins in BAL fluid from animal studies have prov- ed to be sensitive markers of the inflam- matory response (Alpert et al., 1971; Bignon et al., 1975; DeNicola et al., 1981; Beck et al., 1982; Lehnert et al., 1986~. The amino acid hydroxyproline is amarker of collagen and has been interpreted as a marker of collagen breakdown. Hydroxy- proline content of BAL fluid has been meas- ured as a marker of breakdown or remodeling of pulmonary collagen in ozone-exposed rats (Pickrell et al., 1987~. The increase in hydroxyproline in BAL fluid appeared to parallel developing pulmonary fibrosis in hamsters and rats exposed to diesel exhaust (Heinrich et al., 1986; Henderson etal., 1988~. MOLECULAR MARKERS Exposure to environmental toxicants can cause damage in single cells at the level of DNA, and that damage can lead to the development of many diseases, includ- ing cancer. Toxicant-induced changes in specific (although often unidentified) genes are thought to be the initial events in the development of disease. Identifica- tion of genes involved in the development of specific diseases can lead to improved diagnosis, understanding, and treatment, but is not essential. In lieu of disease- specific molecular markers that could be 127 used to study the relationship between toxicant exposure and the development of disease, the general interaction between toxicants and DNA can serve as a source of molecular markers of exposure, effect, and susceptibility. The use of molecular markers, defined here as alterations in DNA or RNA, to identify cellular responses or responsiveness to environmental toxi- cants theoretically can provide informa- tion useful in determining the magnitude of exposure, the effects of exposure on human health, and the mechanisms of re- sponse. This section discusses some gener- al considerations in the use of molecular markers, defines some general types of molecular markers, identifies specific markers for potential use in pulmonary toxicology or the study of carcinogenesis, and identifies subjects for research that could lead to the identification of new molecular markers. Molecular markers can be highly sensi- tive and specific indicators of cell damage or change. Detection of toxicant-induced alterations and use of them as indicators of toxicant exposure, effect, or suscep- tibilitv depend on several factors. in cluding the frequency of the alteration, which in turn can affect the sample size required for its detection; the availabil- ity of sufficient material (DNA, RNA, or cells) for analysis; and the accessibil- ity of the cells at risk (can they be obtain- ed noninvasively, or are invasive proced- ures required?. The sample size required for detection of toxicant-induced alterations is a major consideration in the choice or use of a marker. The minimal sample size required for a given assay depends on the sensitivi- ty of the assay and on the fraction of cells in a sample that contain the specific change of interest. Changes found in a large fraction of cells in a sample will be detectable with a much smaller sample than changes found in only few cells in a sample. For example, many assays that involve the analysis of a DNA change re- quire about 5-10 ,ug of DNA from cells con- taining the change of interest. That amount of DNA can be obtained from 1 o6 al- tered cells. Obtaining 1 o6 altered cells might require a sample of as few as 106

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128 cells, if the change occurred in all cells after exposure or if the cells being used all came from a specific exposure-induced lesion. But a sample of 10~2 cells could be required, if the change occurred with a frequency of 106, which is the observed frequency of induction of some single- gene mutations (Baker et al., 1974~. A sample of 106 cells is readily obtainable with BAL or even with a small tissue biopsy, but a sample of 10~2 cells is more difficult to obtain. In addition to the frequency of the change under investigation, accessibility and availability of sample material affect the choice and use of an assay. Common changes found in cells in BAL fluid. be- cause of their greater accessibility and availability, are much easier to detect than changes (even common ones) that occur only in cells of the deep lung. Assays that depend on invasive sampling procedures can be useful if discrete lesions are being biopsied. However, the routine use of invasive sampling procedures before a lesion is identified usually cannot be justified. The use of molecular markers as indicat- ors of exposure might therefore be limited to cases in which changes are of a general nature, in which changes occur in readily accessible cells, or in which discrete exposure-induced lesions are being biop- sied. Changes that are rare or cell-speci- fic can be difficult or impractical to detect if large tissue samples or invasive sampling procedures are required. How- ever, molecular markers potentially can play an important role in mechanistic studies of disease. Potential molecular markers can be di- vided into several categories, including those based on genetics (modifications of DNA bases, changes in DNA sequence or structure, and changes in extent or pattern of gene expression) and those based on their ability to detect toxicant exposure, effect, or susceptibility or their ability to identify the toxicant involved. Markers of toxicant exposure could be used as screens for exposure to a given toxicant. Depending on the assay and the markers involved, markers theoretically could be used to indicate simply that ex A~RKERS IN PULMONARY TOXICOLOGY posure to a toxicant occurred, to estimate the extent of exposure, or to identify the toxicant. Markers of exposure should be readily detectable and measurable in an accessible population of cells. In addi- tion, toxicant-induced changes should be detectable soon after exposure, and the persistence of a given marker should be determined. Finally, for a marker to be useful as an indicator of exposure to a specific toxicant, it should be charac- terized sufficiently for its presence to be attributed to a given toxicant with reasonable certainty. Molecular markers could also be used to study the biologic effects of exposure to specific toxicants. They could be used to monitor or charac- terize the development of toxicant-speci- fic responses, such as alterations in gene expression. Such analyses would permit studies in the early stages of response, before the development of toxicant-in- duced lesions or disease. Molecular mark- ers theoretically could be used to identify people with an increased risk of the ef- fects of particular toxicants; that would make it possible to minimize their exposures. The formation of DNA adducts after ex- posure to chemicals is an example of an exposure-related modification of DNA (Poirier and Beland, 1986a; NRC, 1989~. DNA adducts form when chemicals or metabol- ically activated derivatives of them bind covalently to DNA. The presence of adducts can be detected chemically (Belinsky and Anderson, 1987; Gupta, 1987) or immuno- logically (Santella et al., 1987~. The most sensitive assay for the detection of DNA adducts is the 32p postlabeling assay (Gupta, 1987~. For maximal sensitivity, the assay requires 5-10 ,ug of DNA (Gupta, 1987), which can be obtained from a sample of 106 cells. Sufficient cells are general- ly available for this assay, because DNA adducts can be found in cells in a variety of readily available biologic samples, such as blood (e.g., adducts of lymphocyte DNA) and BAL fluid (e.g., adducts of mac- rophage DNA), depending on the type of exposure. DNA adducts are more useful as indicators of exposure or dose than as markers of ef- fect; sequence-specific or gene-specific

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CELLUL4R AND BIOCHEMICAL RESPONSE adduct formation has yet to be demonstrat- ed. However, care must be taken even in the use of adduct concentrations as indi- cators of total exposure, in that the con- centrations in tissues can be affected by a variety of biologic responses, includ- ing adduct repair and cell turnover, which vary from one tissue to another (Belinsky and Anderson, 1987~. A correlation between the concentration of DNA adducts or the presence of specific adducts and the development of disease remains to be proved. The presence of a common adduct induced by a particular treatment might have little biologic con- sequence, whereas the presence of a rare adduct induced by the same treatment could be highly significant (Poirier and Beland, 1986b). Identification of exposure-spe- cific adducts and demonstration of an as- sociation between specific adducts and toxicant-induced disease will expand the use of adducts from indicators of exposure and estimators of total dose to specific tools for identifying toxicants and es- timating risk of disease. Another type of DNA modification that could be affected by cell responses to various exposures is DNA methylation. Changes in patterns of DNA methylation potentially could be used as indicators of cellular response or of cellular respon- siveness to particular toxicants. Site- specific changes in the extent of DNA meth- ylation have been shown to regulate gene expression (Razin and Riggs, 1980; Feinberg and Vogelstein, 1983~. Some toxi- cants could result in changes in DNA meth- ylation patterns and cause exposure-re- lated alterations in gene expression. Changes in DNA methylation can be detected either as changes in gene expression or as changes in the restriction-enzyme sen- sitivity of the genes involved, because of altered methylation of restriction- enzyme recognition sequences (Razin and Riggs, 1980~. Identification of changes in DNA methylation requires identifica- tion of the affected genets) and analysis of the methylation changes in the genes, in that alterations in the extent of DNA methylation at the whole-cell level are difficult if not impossible to detect. Furthermore, large numbers of cells (more 129 than 106) containing the same changes in methylation would be needed, so biopsies of developing lesions would usually be required. That approach is more likely to be useful in retrospective studies of mechanisms of cellular response than as a source of markers of response. Changes in DNA sequence or structure could be a source of exposure-related mo- lecular markers. Structural damage to DNA, such as double-strand or single- strand breaks, can be detected with filter elusion assays and alkaline or neutral elusion (Bradley et al., 1982~. Because those assays, like the assay for DNA ad- ducts, detect General cell damage, a random sample of 10 cells would provide enough DNA for analysis. Structural DNA damage can also be detected by examining exposed cells for chromosomal aberrations. After exposure to some toxicants, chromosomal aberrations have been detected in macro- phages isolated by BAL(Au et al., 1988~. The lack of gene or sequence specificity of the assays makes them most useful as indicators of exposure to particular toxi- cants or as estimators of total dose. Changes in DNA sequence resulting from point mutations or deletions are likely to be the initiating events of some expo- sure-related cellular responses, such as tumor development. One indicator of those changes at the cellular level is the production of mutations after exposure. Many toxicants have been shown to be muta- gens in assays that use mammalian cells or bacteria in vitro (Ashby, 1982~. One means of measuring in viva mutations in man uses lymphocytes and changes in the hypoxanthine - guanine phosphoribosyl transferase (HPRT) gene (Albertini, 1980~. Cells that are deficient in HPRT can proliferate in the presence of the toxic purine analogue 6-thioguanine, whereas HPRT-normal cells cannot. HERT mutants have been detected (with autoradi- ography) by their ability to form colonies in a 6-thioguanine medium (Morley et al., 1983) or by their incorporation of [3H]thy- midine in the presence of 6-thioguanine (Albertini, 1980~. Lymphocytes from per- sons exposed to toxicants could be examined for 6-thioguanine resistance, although measurable increases in the frequency of

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130 mutants would be expected only in cases that resulted in systemic exposure to toxi- cants or their metabolites. Alternative- ly, the assay could be adapted for use with macrophages isolated by BAL. The use of toxicant-induced mutations as markers of DNA damage would provide information on exposure and total dose. The identification of toxicant-induced changes in DNA sequence at the molecular (as opposed to cellular) level is important in understanding the etiology of some toxi- cant-induced diseases, but the changes are not likely to be a useful source of mark- ers of toxicant exposure or of early stages of disease. Detection of changes in the base sequence of specific genes requires that the altered DNA be isolated and ex- amined, with radiolabeled molecular probes, for specific changes in DNA se- quence (Ready et al., 1982~. The sensiti- vity limits of that type of assay require the presence of a minimum of 1 picogram~pg) of DNA with the sequence of interest (Thomas, 1983~. For example, if the gene of interest were a single-copy gene encoded by 5,000 base pairs of DNA, 1 pg of DNA from the altered gene could be obtained from a minimum of 2 x 105 altered cells. Detec- tion of point mutations within the sequence of a particular gene generally requires that the gene be cut into multiple frag- ments with restriction enzymes for analy- sis by gel electrophoresis, so up to 1 o6 cells might be required to yield 1 pg of DNA with the sequence of interest. As noted above, isolation of so many cells from an exposure-induced lesion by noninvasive methods is not likely. Sampling of cells with ravage will yield more than 106 total cells, but most of the cells will not con- tain the change of interest. That approach is most likely to be useful in retrospec- tive analyses of mechanism, not in surveys of exposure effect. Changes in the amount or pattern of gene expression that result from exposure to some toxicants might be most amenable to the use of molecular analysis as a measure of effect. If the expression of specific (identified) genes is induced, amounts of mRNA in the target cells might be greatly increased (or reduced). The detection and measurement of mRNA by Northern or dot AL4RKERS IN PULMONARY TOXICOLOaY blot analysis requires the presence of 1 pg of the sequence of interest (Thomas, 1983~. However, the expression of a speci- fic gene can result in the production of large amounts of mRNA for the gene of inter- est; that decreases the number of cells required for detection. For example, in- duction of the ovalbumin gene in the ovi- duct gland cell of chickens results in the production of more than 3,000 ovalbumin mRNA fragments per cell, compared with the noninduced number of 2 copies per cell (Roop et al., 1978~. That amplification (by a factor of 1,500) reduces the number of cells required to obtain 1 pg of oval- bumin mRNA from about 5 x 105 to about 300. A gene- and exposure-specific response could therefore be followed with molecular markers if specific (identified) genes were overexpressed after exposure to par- ticular toxicants, if molecular probes for the genes were available (i.e., if the genes had been isolated and molecularly cloned), and if the cells containing the overexpressed genes were readily avail- able in sufficient numbers (e.g., in ravage fluid) from exposed persons. If molecular probes for specific genes of interest were not available for use in the assay described above, exposure- or disease-specific changes in gene expres- sion could be examined, provided that as- says for the gene productts) were avail- able. Poly(A)-containing RNAs isolated from affected tissues could be translated in vitro with a reticulocyte lysate system (El-sorry et al., 1982), and the amount and nature of protein product could be analyzed to detect exposure- or disease- related changes. The assays for altera- tions in gene expression, although poten- tially useful in understanding the mechan- ism of response to a toxicant once a re- sponse has occurred, are not likely to be useful for surveys of exposure or effect. Gene-specific changes, such as specific sequence changes or modifications of DNA, generally occur too infrequently to be useful as markers of toxicant exposure and often occur in cells accessible only with invasive sampling procedures once lesions have been identified. However, some gene-specific changes could be useful as general markers of toxicant exposure.

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CELLULAR AND BIOCHEMICAL RESPONSE For example, exposures that result in in- flammation involve recruitment and ac- tivation of macrophages that express genes for interleukin- 1 and c-sis (Wewers et al., 1984; Mornex et al., 1986~. Molecular probes are available for those genes, so their expression or changes in their ex- pression could be detected with mRNA iso- lated from macrophages in ravage fluid. Changes in the expression of the genes could serve as general indicators of ex- posure. The exposure-related changes in gene expression would also provide infor- mation useful in understanding the mechan- ism of toxicant effects. Available markers potentially could be used to detect specific exposure-in- duced effects. The ability to detect changes at different stages of disease will depend on the availability of suffi- cient cells for analysis. Specific expo- sure-induced effects might be detectable at the molecular level only at more ad- vanced stages of disease and are likely to be more useful in the characterization of a disease than in its diagnosis. Genes for pulmonary surfactant apoprotein (White et al., 1985), for collagen (Misku- lin et al., 1986), and for cytochrome P- 450 enzymes involved in oxidative metabo- lism (Nebert and Gonzalez, 1987) have been cloned. Changes in the expression or mo- lecular structure of those genes after toxicant exposure could be identified. Exposures-such as chronic exposure to cigarette smoke-that affect Type II cells result in alterations in surfactant pro- duction (LeMesurier et al., 1981~. Those changes could be characterized at the mo- lecular level with available probes. Simi- larly, the induction and expression or overexpression of genes for collagen could be examined after exposures that result in excess collagen deposition. Finally, the toxicant-specific induction and ex- pression of genes for cytochromes P-450 could be monitored with cloned probes. The success or feasibility of each of those analyses is subject to the same restriction of cell availability as described above. The analysis of cancer development after toxicant exposure is another endeavor in which molecular probes could be useful for understanding the mechanism of re 131 sponse and possibly as a diagnostic tool. Alterations in the number of copies or expression of cellular oncogenes have been identified in several pulmonary cancers. For example, amplification of c-myc has been found at a late stage in the develop- ment of some small-cell lung carcinomas (Little et al., 1983; Saksella et al., 1985), mutationally activated K-ras has been found in some lung carcinomas (Santos et al., 1984; Stowers et al., 1987), and overexpression of erb-B has been described in some non-small-cell lung carcinomas (Cerny et al., 1986; Gamou et al., 1987~. Theoretically, exfoliated tumor cells could be identified and characterized from ravage fluid with in situ hybridization, if tumor-specific oncogene changes were established. In addition, toxicant-spe- cific oncogene activation could be charac- terized in developing lung tumors. Some examples of carcinogen-specific oncogene activation have been described. Induction of mammary tumors in rats with nitrosometh- ylurea resulted in H-ras activation in 86% of developing tumors (Zarbl et al., 1985~. Similarly, 74% of lung tumors in rats ex- posed to tetranitromethane had an activat- ed K-ras (Stowers et al., 1987~. Those and other examples of carcinogen-specific oncogene activation suggest that analyses of oncogene activation in tumors after environmental exposures could play a role in increasing understanding of the etiolo- gy of exposure-related tumor development. In conclusion, there is clearly a need for more markers that can be used to detect and characterize at the molecular level both general and specific cell responses to exposure. Studies at the molecular level will continue to be most useful in understanding mechanisms of cellular response to toxicants. However, it might be possible to develop specific molecular orobes that could be used to diagnose or characterize specific diseases or other responses to exposure. Molecular probes have proved useful in the diagnosis and characterization of some infectious dis- eases and in sickle-cell anemia and alpha- and beta-thalassemia. Molecular markers that could identify individual susceptibility to disease or toxicant sensitivity are also needed.

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132 Molecular probes have been or are being developed for several diseases, including sickle-cell anemia and thalassemias (Dozy et al., 1979; Wilson et al., 1982; Pirastu et al., 1983), retinoblastoma (Friend et al., 1986), Huntington disease (Carlock et al., 1987), Duchenne muscular dystrophy (Monaco et al., 1986, 1987), cystic fibro- sis (Dorin et al., 1987), Lesch-Nyhan syn- drome (Brennand et al., 1982), phenylketo- nuria (Woo et al., 1983), antithrombin III deficiency (Prochownik et al., 1983), and alpha~-antitrypsin deficiency (Kidd et al., 1983~. Results of studies of chron- ic obstructive pulmonary diseases suggest a genetic basis (Kauffmann, 1984) and might therefore lead to the discovery of mole- cular markers of susceptibility. An in- creased risk of cigarette-smoking-induced bronchiogenic carcinoma appears to be A[9RKERS IN PULMONARY TOXICOLOGY associated with a highly inducible cyto- chrome P-450 phenotype (Jaiswal et al., 1985; Gonzalez et al., 1986~. Further correlations between that phenotype and development of other pulmonary diseases are needed. Molecular analyses of other pulmonary diseases or individual respon- siveness to toxicants might enable iden- tification of persons at greater risk of developing toxicant-specific diseases and lead to the development of markers of susceptibility. The development and use of molecular markers to identify cellular responses or responsiveness to environmental toxi- cants and to characterize pulmonary-dis- ease will be important in increasing under- standing of the mechanisms involved in the development of pulmonary disease and in its prevention and treatment.