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4 Markers of Altered Structure or Function WHOLE LUNG The simplest methods for assessing al- terations of lung structure are examina- tion with the naked eye and whole-tissue examination with a dissecting microscope. Of course, access to relevant materials in vivo is problematic. Events on the pleural surface can be used to establish that lung disease is present (Spencer, 1977~. For example, pleural fibrosis and fibrotic adhesions between the visceral and parietal pleurae are clear indicators of exposure to toxic gases or asbestos or of multiple infections (Spen- cer, 1977~. Those alterations can be seen when the chest cavity is opened, and fi- brotic pleural plaques can be seen im- mediately or in wet tissues viewed with dissecting microscopy. . ~. .. .. The mechanisms that mecilate the pathogenesis of pleural fibrogenesis are not at all clear (Bignon et al., 1983~. It is conceivable that in- flammatory cells provoked to migrate into the pleural cavity produce factors that mediate pleural inflammation and later fibrosis. Thus, analysis of pleural fluids and cells could yield an important marker of impending disease, just as bronchoscopy has provided a window on the cells and flu- ids of the airways and parenchyma. The whole lung can be sampled with vari 83 ous techniques to establish the burden of inhaled particles. Lungs collected at autopsy provide exceptionally useful material, because anatomic regions can be sampled and quantitative values derived (Abraham, 1978; Churg and Wright, 1983~. Determination of particle types and their correlation with the presence of lung dis- ease have been valuable in increasing the understanding of etiology (Abraham, 1978~. Whether such studies will allow for the development of markers of pulmonary disease can be determined only when the nature of the lung burden in occupational and environmental settings is known. _ _ . . . . AIRWAYS A variety of inhaled agents, including oxidant gases and pathogenic microbes, can cause alterations of the large and small airways. The cells of the airways are relatively accessible with bronchos- copy, brushing, and biopsy and therefore have great potential as markers of exposure - anc injury. In studying a nasal, tracheal, or bron- chial biopsy, it is necessary to establish normal values and appearances of such en- tities as ciliary beat frequency, cell size, physiologic ion concentration, and structural features as determined by

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84 light and electron microscopy. However, the most useful markers are likely to be those related to the basic mechanisms by which airway epithelial cells respond to exposure. The tracheobronchial lining consists of a pseudostratified epithelium that contains a diverse population of cell types. The ciliated cell is one of the major cell types and is probably a nonprolifera- tive, terminally differentiated cell. The conciliated population consists of various secretory cells (mucous, Clara, and serous cells, depending on species) and a nonsecretory cell (the basal cell). The Clara, mucous, and basal cells can undergo cell division. Evidence is emerg- ing that the mucous and Clara cells can differentiate into ciliated cells, but the function of the basal cell is not yet established. The cells in the tracheobron- chial epithelium can undergo differentia- tion during vitamin A deficiency and after toxic or mechanical injury. Understanding the mechanisms of differentiation could aid in providing markers of exposure and injury. The availability of biochemical markers of the various differentiated phenotypes is essential because it pro- vides a direct indication of cellular dam- age. Mucous glycoproteins are used as markers for alterations of mucous cells; specific histochemical staining tech- niques, as well as biochemical analysis, have been used to identify and characterize secretory cell products (Rearick et al., 1987~. For ciliated cells, the presence of dynein appears to be a good chemical marker of structural effects. A low-molec- ular-weight protein identified in Clara cells appears to be a Clara cell-specific secretory product that can function as a biochemical marker of altered function (Patton et al., 1986~. A biochemical mark- er specific for basal cells has yet to be discovered. Results of in viva and in vitro studies indicate that differentiation of tracheo- bronchial epithelial cells is a multistep process (Jetten et al., 1986) and has sev- eral characteristics in common with epi- dermal differentiation. Like epidermal cells, tracheobronchial epithelial cells undergo cornification. Cornification AL4RKERS IN PULMONARY TOXICOLOGY involves the deposition of a layer of cross-linked protein beneath the plasma membrane. The extensive cross-linking between proteins is catalyzed by the enzyme transglutaminase. Biochemical and im- munologic analyses have identified the tracheal transglutaminase as type I (epi- dermal) transglutaminase. Differentia- tion of tracheobronchial cells is accom- panied by an increase in the activity of transglutaminase by a factor of 20-30 (Jetten and Shirley, 1986~. Immunohis- tochemical staining of tracheas from vita- min A-deficient hamsters with a monoclonal antibody against type I transglutaminase indicated that in vivo synthesis of this enzyme is associated with differentiation of tracheobronchial cells (Jetten and Shirley, 1986~. Squamous cell differentiation is accom- panied by an increase in cholesterol sul- fate (Rearick and Jetten, 1986~. The in- crease appears to be due to an increase in the enzyme cholesterol sulfotransferase. The high correlation between the expres- sion of that enzyme and increases in cor- nification indicate that these chemical changes play an important role in differen- tiation. Such changes could be early in- dicators of pathologic changes in easily accessible airway lining cells. . Recently, a cDNA library-a library of complementary DNA produced from an RNA template by action of RNA-dependent DNA polymerase (reverse transcriptase)- was established from rabbit tracheal epithelial cells that had undergone squa- mous cell differentiation (Smite and Jetten, in press). Two cDNAs that are abundant in squamous cells were isolated: SQ 10 and SQ 37, which identify RNAs of 1.0 and 1.25 kb, respectively. The two RNAs appear to be squamous cell-specific and can function as markers for the squamous cell phenotype. Intermediate filaments have been used in various systems as markers for specific cell types. It has been shown that Clara cells express a keratin profile charac- teristic of simple epithelium, whereas basal cells express a keratin pattern char- acteristic of stratified epithelium, such as that consisting of epidermal keratino- cytes. Squamous differentiation in vivo,

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ALTERED STRUCTURE OR FUNCTION as well as in vitro, is accompanied by qual- itative and quantitative changes in kera- tin expression (Huang et al., 1986~. Those findings suggest that keratin expression can be specific for particular cell types of the tracheobronchial epithelium and that changes in keratin expression can indicate particular pathologic altera- tions, such as metaplasia. PARENCHYMA It is in the alveoli that toxic particles and gases exert a fibrogenic influence on the lung interstitial cells that produce connective tissue. The basic cellular and biochemical mechanisms through which inhaled materials cause lung fibrosis are not well established, and it is difficult to ascribe the function of markers to a poorly understood pathologic process. However, animal models of asbestos- and silica-induced disease do offer some po- tential for elucidation ot pathogenes~s and thus provide markers of exposure, in- jury, and disease progression. Detection and analysis of inhaled particles at the alveolar level have been discussed above. The following discusses early cellular responses and methods proposed to study them. Morphometric Markers of Injury from Exposure to Inhaled Agents Proliferation of epithelial cells, fibroblasts, and macrophages can be meas- ured in viva with light and electron mi- croscopy techniques (Evans, 1982~. The very earliest responses of the various pulmonary cell types are likely to be pro- liferative if injury has taken place. For example, both oxidant gases (Evans, 1982) and chrysotile asbestos fibers (Brody and Overby, 1989) cause a rapid incorporation of tritiated thymidine into nuclei of bron- chiolar and alveolar cells. That reflects a repair phase, and the degree and extent of the incorporation can be used as a meas- ure of lung injury. Inhaled chrysotile asbestos and instilled crocidolite asbes- tos cause proliferation of epithelial cells, fibroblasts, and interstitial and alveolar macrophages (Adamson and Bowden, 85 1986; Brody and Overby, 1989). It is reas- onable to speculate that this early pro- liferative event could serve as a marker of exposure to a toxic gas or particle. Morphometry is a sensitive quantitative tool that can be of great value in evaluat- ing changes in lung tissue caused by toxic substances. Morphometry is the quantifi- cation of three-dimensional structure. Values are derived from an analysis of two- dimensional profiles based on microscopic and other imaging techniques (Weibel, 1979~. The advantages of using morphometry to study lung structure include objective measurement of changes in lung structure, identification of subtle changes in tissue structure caused by different agents or by progressive exposure to the same agent, and selective measurement of changes in specific tissue compartments. The en- hanced sensitivity of detecting a change in structure by using a morphometric analy- sis is particularly beneficial when tissue changes due to an experimental treatment are probable, but not obvious. An impor- tant application of morphometry involves studies in which subjective grading stand- ards suggest that changes have taken olace, but are not conclusive with respect to the relative toxicity of drugs, chemi- cals, or pollutants at specific exposures or doses. Morphometry eliminates the sub- jective bias that occurs with many grading techniques used to measure structural changes. It also reduces or eliminates variations in grading between and within observers. The types of morphometric information that can be obtained in a study of lung par- enchyma depend on the resolution used. Light microscopy provides adequate reso- lution to determine alveolar surface area, proportional volumes of air and tissue, and total number of alveoli. Light micros- copy is rapid, is accurate, and can be used with large numbers of specimens. Use of the mean linear intercept (MLI) makes it possible to measure alveolar surface den- sity. MLI measurements have been used to study postnatal lung growth, the normal adult human lung, and lungs with emphysema (Barry and Crapo, 1985~. Electron micros- copy is better for determining the volumes, thicknesses, and surface densities of

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86 specific alveolar compartments. Electron microscopy is required for adequate reso- lution of alveolar tissue into its compo- nents, including type I and type II epi- thelium, cellular and noncellular inter- stitium, endothelium, and inflammatory cells. Subcellular components-such as the nucleus, cytoplasm, and cytoplasmic organelles-in specific cell types can be measured with electron microscopy. Numbers of particles, cells, and subcel- lular organelles in the alveolar region can be determined morphometrically. The total number of cells in the lung and the distribution of cells among the various major types of alveolar cells have been determined in humans and several species of laboratory animals. The distribution of cell types in the alveolar interstitium has also been estimated (Barry and Crapo, 1985~. The purpose and types of questions to be answered will determine whether light microscopy alone or with electron micros- copy is needed. The advantages of light microscopy include rapidity, accuracy, and low cost. Its disadvantages include the need to determine a correction factor for tissue shrinkage due to paraffin em- bedding of tissues (although other materi- als that are now available, such as plas- tic, minimize shrinkage) and restrictions as to the types of alveolar structures that can be easily and reliably measured-air- space, tissue, and capillary volumes; alveolar surface area; mean free path lengths; and numbers of alveoli. Electron microscopy has the advantages of providing quantitative information on specific alveolar compartments and requiring no correction factors, because tissues can be embedded in plastic that allows only negligible shrinkage, as noted above. Its disadvantages include higher costs and requirements of substantially greater labor and time. Morphometry has been used most commonly to study toxic agents that cause a given kind of injury throughout the entire lung; a random lung sample can be assumed to be representative of the injury. Damage caused by high concentrations of oxygen, a gas that is highly diffusible in lung tissue, is a good example of that type of MARKERS IN PULMONARY TOXICOLOGY injury. Some inhaled toxic agents selec- tively damage the terminal bronchioles and the adjacent alveoli. Those agents include ozone and nitrogen dioxide, which occur as air pollutants in relatively low concentrations, but are more reactive than oxygen. Particles can also selectively injure the small airways in proximal al- veolar tissue. The alveoli adjacent to terminal bronchioles are the site of the lesion of centrilobular emphysema, which is at least in part caused by the particles and gases in cigarette smoke. Rigorous morphometric study of a select region of the lung, such as the terminal bronchioles or the adjacent alveoli, re- quires sampling techniques that are de- signed to assess structures that are local- ized to specific regions of the lung. Com- mon morphometric formulas and basic sam- pling procedures can be applied, but the methods of tissue selection and data anal- yses must be adapted. Morphometric studies of the effects of toxic substances on ter- minal bronchioles and adjacent alveoli have been few, because of the difficulties involved in dealing with samples from spe- cific regions. The appearance of a few studies in recent years directed at the terminal bronchioles or the proximal al- veoli indicates increasing awareness of this site of toxic injury and the develop- ment of the necessary morphometric tech- niques (Barry and Crapo, 1985; Chang et al., 1988~. PULMONARY VASCULATURE To assess the development of structural markers of pulmonary vascular change, one must understand the normal architecture of the arterial and venous circulation. Within the pulmonary circulation are four structural types of pulmonary artery in a progression from hilum to periphery- elastic, muscular, partially muscular, and nonmuscular (Reid, 1979; Meyrick and Reid, 1983~. Elastic arteries, by defini- tion, contain more than five elastic lam- inae, including the internal and external elastic laminae; muscular arteries con- tain two to five elastic laminae, partially muscular arteries have muscle in only part of the wall; and nonmuscular arteries have

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ALTERED STRUCTURE OR FUNCTION no muscle in the wall and are structurally similar to alveolar capillaries (Meyrick and Reid, 1979a). The four types can be related to external diameter: arteries greater than 2,000 fig in external diameter are elastic, those from 150 to 2,000 Am are muscular, and those less than 150 ,um are muscular, partially muscular, or nonmuscular. In addition to the conventional arteries that run and divide with the airways, there is a population of supernumerary arteries (short arterial branches) in the lung that do not (Elliott and Reid, 1965~. The super- numerary arteries from the axial pathway are more numerous than the conventional arteries and carry approximately 30% of the blood volume. It is likely, but not certain, that the supernumerary and con- ventional arteries respond similarly to . vascu ar Injury. Although not described in detail here, the pulmonary veins can be divided into structural regions similar to those of the arteries. In many cases, the pulmonary veins are easily distinguished from pul- monary arteries on the basis of their posi- tion in the lung. For example, the walls of the veins are less muscular than those of arteries for a given diameter, and the muscular coat of the bongs is not bounded on its luminal aspect by an internal elas- tic lamina (Hislop and Reid, 1973~. Two potential outcomes of exposure of the lung to toxic chemicals are progressive restructuring of the lung vasculature and chronic pulmonary hypertension. One of the few well-documented examples of that result in humans is the outbreak of chronic pulmonary hypertension in Europe associ- ated with ingestion of the drug, Aminorex. The paucity of documented examples of chem- ically induced, chronic vascular injury in the lung other than that incident raises a question as to the importance of the phe- nomenon in humane. Chronicpulmonaryhy- pertension is difficult to diagnose and might be associated with other maladies, so it could be more common that it seems to be. Indeed, animal studies have reveal- ed that chronic exposure to low doses of several agents that injure endothelium can cause progressive injury to the lung vasculature and pulmonary hypertension. 87 Moreover, the position of the lung in the circulation renders it the first vascular bed to encounter toxic metabolites of chem- icals that are produced by the liver and enter into the venous circulation. The pulmonary vasculature should therefore be considered as a target for chemical insult and chronic injury. Comprehensive studies of the structure of the pulmonary vessels have used autopsy specimens of pulmonary arteries or veins which were filled with barium gelatin be- fore airway inflation (Reid, 1979; Meyrick and Reid, 1983~. The technique allows easy identification of small arteries and veins and full distention of the vascular bed. Full and reproducible distention of the pulmonary circulation allows the use of morphometric techniques; the severity of changes can be assessed, and hyperten- sive lungs can be compared with normal lungs. The same techniques can be used in lung biopsy tissue (Rabinovitch et al., 1984), although the more subtle and earlier changes of vascular injury might be harder to identify in biopsy tissue. This section outlines how the pulmonary vasculature responds to changes in hemody- namic behavior, to direct injury, and to environmental changes, such as altera- tions in ambient oxygen. Results of pathologic studies of clini- cal and experimental lung samples have indicated some ways in which the pulmonary vasculature can respond to injury and to changesinpulmonaryhemodynamics. Patho- logic markers or structural alterations that occur in the pulmonary vessels are usually associated with the development of chronic pulmonary hypertension-e."., intimal hyperplasia, extension of muscle into smaller and more peripheral arteries than normal, increase in medial thickness of normally muscular arteries, reduction in peripheral arterial volume (seen either as a reduction in number of arteries or as a narrowing of intra-acinar arterial lu- minal diameter), recanalization of block- ed arteries, fibrinoid necrosis, dilata- tion, and plexiform lesions (Wagenvoort and Wagenvoort, 1977; Harris and Heath, 1986~. A single lung biopsy or autopsy specimen might reveal the entire range of morphologic markers of vascular injury or only a few, depending on the stimulus

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88 and the severity of the injury. For exam- ple, the finding of plexiform lesions or fibrinoid necrosis is associated with end- stage pulmonary vascular disease (Heath and Edwards, 1958; Harris and Heath, 1986~; this seemingly restricted response could reflect the paucity of cell types normally encountered in the walls of lung vessels. In Vivo Observations of Pulmonary Vasculature Disease of the pulmonary vasculature can be examined in patients both by radiog- raphy and by angiography. The radiograph- ic appearance of edema marks pulmonary endothelial injury and is dealt with ear- lier in this report. Angiographic radi- opaque mass marks thrombus formation and vascular occlusion. Rate and abruptness of narrowing and loss of vascular volume can be assessed in pulmonary arterial wedge angiograms; the latter markers have been described in association with chronic pulmonary hypertension secondary to con- genital heart defects (Rabinovitch et al., 1981). Gross Examination of Pulmonary - Vasculature The lung is supplied by two vascular beds: the bronchial, which supplies oxy- genated blood to the connective tissue around large arteries, veins, and airways; and the pulmonary, which carries venous blood to the lung capillaries for oxygena- tion. The pulmonary circulation accounts for more than 95% of the blood volume in the lung. Arterial and venous sides of the pulmonary circulation are easily iden- tified on gross examination or cut lung surfaces, because the arteries run cen- trally in the acinus (terminal bronchioli and its branches) and the veins at the edge of the acinus. Gross examination of lung slices also allows detection of thrombi, areas of atelectasis, and emphysema, and careful dissection along arterial and venous pathways can provide evidence of congenital defects in the pulmonary vasculature. MARKERS IN PULMONARY TOMCOLOGY Light Microscopic Examination of Pulmonary Arteries Heath and Edwards (1958) suggested a grading system for the progression of the structural changes of chronic pulmonary hypertension. Grade I represented medial hypertrophy; Grade II, intimal hyper- plasia; Grade III, luminal occlusion by intimal hyperplasia; Grade IV, arterial dilatation; Grade V, angiomatoid lesions; Grade VI, fibrinoid necrosis. They con- sidered Grades I-III reversible markers of vascular injury. The grades are readily recognizable in autopsy and biopsy tissue, but only recently have structural markers been correlated with hemodynamic behavior of the lungs. Rabinovitch and colleagues ( 1984) re- fined the grading system of Heath and Edwards, particularly the early rever- sible changes, by applying morphometric techniques to the lungs of patients with congenital heart defects associated with high flow. Additional structural markers of chronic pulmonary hypertension were identified with that system and graded A, B or C. The quantitative techniques are well described for structural changes in distended and nondistended arteries in autopsy and biopsy tissue (Hislop and Reid, 1973; Reid, 1979; Rabinovitch et al., 1984~. Grade A structural changes (extensions of muscle into smaller and more peripheral arteries than normal) are found in patients with an increase in only pulmonary blood flow; Grade B changes (Grade A changes plus increased medial thickness), when both pulmonary arterial pressure and blood flow are increased; and Grade C changes (Grade B changes plus reduction in peripheral arterial volume), when pulmonary vascular resistance is increased. Those correlations can be extended by examining animal models of chronic pul- monary hypertension: rats exposed to hy- poxia and rats given Crotalaria spectabilis seeds (which contain monocrotaline). The same structural changes (Grades A, B. and C) can be identified and correlated with hemodynamic behavior (Meyrick and Reid, 1978, 1 979b; Rabinovitch et al., 1979; Meyrick et al., 1980~. Such studies have shown that the two models of pulmonary

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ALTERED STRUCTURE OR FUNCTION hypertension lead to the same structural markers of pulmonary hypertension, but at different rates (Meyrick and Reid, 1983~. The structural changes after hypox- ia occur faster than those after adminis- tration of Crotalaria. The severity of each structural marker also differs between the two models (Meyrick and Reid, 1983~. For example, loss in peripheral arterial volume after administration of Crotalaria is more striking than that after hypoxia, and muscle extension is more severe after hypoxia. Pulmonary arterial pressure also occurs faster after hypoxia. Correlation of the structural changes with the hemody- namic behavior in both models reveals that the best structural correlate of increased pulmonary arterial pressure is extension of muscle into small arteries; for Crotalar- ia administration, it is increased medial thickness. The data also suggest that the speed of the changes can depend on the stimulus for the development of chronic pulmonary hypertension. With hypoxia, one of the mechanisms involved is almost certainly hypoxic vasoconstriction; with Crotalaria administration, the changes are thought to be secondary to endothelial damage, and the role of vasoconstriction is less certain. Additional evidence that en- dothelial damage can contribute to the development of pulmonary hypertension was provided by Jones and colleagues (Jones et al., 1984) who used rats exposed to hyperopic conditions. Recent data on sheep given repeated infusions of endotoxin (Meyrick and Brigham, 1986) and sheep subjected to continuous air embolization (Meyrick et al.. 1987: Perkett et al., 1988) suggest that inflammation of the lung can be a trigger. The data suggest that there are several stimuli of develop- ment of chronic pulmonary hypertension and that the structural markers of pul- monary hypertension might not be identical in each type of hypertension. Development of chronic pulmonary hypertension is likely to be seen in association warn hypoxia, hyperopia, prolonged vasocon- striction, chronic or repeated lung in- flammation, and endothelial injury. The variation in severity and appearance of structural markers of chronic pulmonary 89 hypertension and the range of initiating stimuli are borne out in the clinical set- ting (Reid, 1979; Meyrick and Reid, 1983~. For example, natives of high-altitude environments and children with persistent fetal circulation (pulmonary hypertension following right-to-left blood shunting) have marked muscle extension, and patients with chronic bronchitis and emphysema, cystic fibrosis, and primary pulmonary hypertension have a less severe change. Reduction in peripheral arterial volume is striking in patients with primary pul- monary hypertension, but blood volume is within normal limits in cases of persistent pulmonary hypertension. Plexiform lesions are found in patients with primary pulmonary hypertension and in older pa- tients with congenital heart disease, but have not been seen in the hypoxic forms of chronic pulmonary hypertension. Not only the pulmonary arterial circula- tion is limited in its response to injury, but also the veins. On the venous side of the circulation, "arterialization" of the veins occurs if pressure in the veins is chronically increased. The structural markers are a medial coat and an internal elastic lamina in the venous walls. Those changes are often accompanied by intimal fibrosis (Harris and Heath, 1986~. An increase in pressure also leads to the development of the structural markers of pulmonary hypertension on the arterial side of the circulation. Electron Microscopic Examination of Pulmonary Arteries Electron microscopy shows the major cell types in the walls of the pulmonary cir- culation to be endothelial cells, smooth muscle cells and their precursors and fi- broblasts. In experimental models of chronic pulmonary hypertension, such as the rats exposed to hypoxia (Meyrick and Reid, 1978, 1 979b) or given Crotalaria spectabilis (Meyrick et al., 1980), it has been shown that the precursor smooth muscle cells undergo hypertrophy and, at least in hypoxic animals, cell division; that accounts for the appearance of muscle in the normally nonmuscular intra-acinar pulmonary arteries (Meyrick and Reid,

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go 1983~. Encroachment of the new muscle cells and endothelial hypertrophy con- tribute to the reduction in peripheral arterial volume (Meyrick and Reid, 1983~. Ultrastructural studies also show that smooth muscle cell hypertrophy and altera- tions in connective tissue synthesis and accumulation contribute to the increased medial thickness seen in preacinar arter- ies (Meyrick and Reid, 1980~. Future Directions Few reports have suggested alterations in bloodborne mediators in patients with chronic pulmonary hypertension although Geggel and associates have suggested that measurements are possible. Increases in ristocetin cofactor activity relative to plasma von Willebrand factor have been reported in patients with primary pulmon- ary hypertension (Geggel et al., 1987~. Similarly, patients with primary pulmon- ary hypertension and monocrotaline-treat- ed rats with increased pulmonary arterial pressure have increased plasma copper concentrations (Ganey and Roth, 1987~. The origin of the latter change is unclear, but such observations deserve further attention with regard to development of markers of chronic pulmonary vascular changes. Advancements in this direction will be of obvious importance to patients, especially if changes can be detected early in the development of the disease. MARKERS IN PULMONARY TOXICOLOGY Results of recent biochemical and molec- ular studies of chronic pulmonary hyper- tension have indicated that the develop- ment of the structural changes might depend on the stimulus. For example, in the mono- crotaline model of pulmonary hypertension in rats, an increase in elastase activity in the preacinar arteries resulted in thin- ning and fragmentation of the internal elastic lamina of the thickened walls (Todorovich et al., 1986~. In the hypoxic neonatal cow, the increased thickness of the walls of the preacinar arteries includ- ed proliferation of adventitial fibro- blasts invoked by a growth factor released from the medial muscle cells (Mecham et al., 1987~. In hypoxic rats, treatment with a latherogen (beta-aminopropio- nitrile) or with an inhibitor of collagen production (cis-4-hydroxy-L-proline) caused attenuation of the structural mark- ers of pulmonary hypertension examined. as well as partial protection against the increase in pulmonary arterial pressure (Kerr et al., 1984, 1987~. Thus, markers of pulmonary vascular disease are likely to be diverse and to depend on the initial stimulus of hypertension. The introduc- tion of such techniques as in situ hybridi- zation is likely to advance our understand- ing of these alterations at the cellular and molecular level and to yield markers useful in the early detection of chronic pulmonary hypertension.