Pathobiology of air pollutants

ENVIROSJIETT.4L

RESEARCH

1,

178-197

(1967)

Pathobiology

of Air HOLLIS

VA Hospital,

1056 Clermont Receizwd

G.

Pollutants1

BOREN~

St., Denver, May

Colorado

SO820

31, 1967

Results of inhalation exposure of guinea pigs to carbon followed by N02, or to NO, followed hy carbon, are presented to exemplify both specific and general problems inherent in using animal systems to determine injurious effects of environmental agents. Inhalation of carbon alone is followed by a macrophage response. Subsequent exposure to NO? results in lung destruction. Inhalation of NO, followed by carbon gives n macrophage response of lesser degree. These findings are interpreted to mean that the sequence of exposure may determine a given response. The limitations of this study indicate the nccessit.y of using approaches which control or measure multiple biologic factors operative at different levels of organization of diverse animal sgsteme. “Now, evidently a cell without an environment is :I fiction; hence no property or manifestation of a cell can he divorced from a consideration of the environment in interaction with which it has occurred or been displayed.” Paul We&, 1953

The growing realization that man’s atmosphere is being contaminated by cvrrincreasing types and amounts of pollutants has engaged the attention of people with a wide diversity of background, training, and interest. Yet, despite this range and diversity of interests, there has emerged no unifying picture of the significance of air pollutants wit’11regard to man’s health. The Symposium on Physiological Characterization of Health Hazards in Man’s Environment (Bretton Woods. N.H., August, 1966) found much information to be lacking: the effects of long-term, low-dose exposures; the effects of factors operating in combination or in sequence; the effects of agents on enzyme and subcellular levels; the intimate mechanisms of damage by even the simplest substances; t.he role of adaptation; the role of individual susceptibility; and the extremely difficult determination of what constitutes an optimum environment. The purpose of this paper is to examine critically the limitations of animal experimental studies of air pollutants in the light of current ideas, information, t,ools, and strategies of pathobiology. Pathobiology is defined by D. W. King in the preface to The Third Pathobiology Conference (Aspen, Colo., 1966) as “the union of biology (the study of life) and pathology (the study of illness).” The specific aims of this presentation are to examine the relationships of air pollutants ’ Tliis study was supported by U.S. Public Health Service ‘Bssociate Professor of Medicine, University of Colorado Pulmonary Disease Research in Medicine at VA Hospital, Colorado 80220. 178

grant OH 69267. Medical Center and 1055 Clermont Street,

head of Denver,

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to cellular injury and to consider factors, particularly biologic, which may modify these interactions. The approach chosen is to examine one of the author’s experiments as an example to be used for a discussion of the problems and limitations as well as the advantages of using animal systems. There is no attempt to review t,he broad effects of air pollutants, or even of animal exposures to such agents. The great majority of animal studies have evaluated the inhalation of a single agent. The reasons for this approach are both practical and conceptual. It is difficult to use two different agents in a single exposure chamber. There has been an understandable urge to establish ‘lmaximal permissible concentrations” and to detect substances of high toxicity. There has been an assumption that one agent is responsible for one lesion, or one disease, as was the case in some bacterial infections. Measurement of air pollutants in natural occurring conditions in Los Angeles (Hamming et al., 1960) showed varying concentrations of different pollutants, giving rise to classifying pollutants into two types (Goldsmith, 1962). One showed two peaks (7:00 AM and 8:00 PM) with carbon monoxide, hydrocarbons, nitrogen dioxide, sulphur dioxide, and particulates being involved ; the second type had one peak at noon consisting of higher concentrations of ozone, oxidants, and aldehydes. Experimental studies utilizing two agents have usually had as their aim the demonstration of synergistic effects when the agents were inhaled at the same time (Amdur, 1960; Frank et al., 1964; Goetz, 1961; LaBelle and Brieger, 1959). A study of Heppleston (1961) utilized the inhalation of different colored dusts (coal, hematite, silica) in succession, to tag the sequence of events after exposure. The rationale behind the present st.udy was that inhalation exposures to agents in sequence might elicit morphologic changes in lung which could not be found either when an agent> acted alone or at the same time as another agent. MATERIALS

AND METHODS

The two materials selected for exposures were carbon particles and nitrogen dioxide (NO,). Since the sequence of exposure was under study two experimental groups were required: one in which carbon was inhaled first, followed by NO,; the second in which NO, was inhaled, followed by carbon. Control groups consisted of animals exposed to NO2 adsorbed on carbon, to carbon alone, to NO, alone, and animals which had inhaled only normal air. Exposures were accomplished using large chambers accommodating as many as 100 guinea pigs at a time. Exposures using carbon consisted of concentrations of 18-21 thousand particles/cm 3 four hours a day three times a week. Exposure to NO, was for two hours a day, using concentrations of either 25 or 75 ppm as will be indicated. Following completion of exposure, groups of animals were sacrificed at time intervals from one week to two years. Lungs were fixed by intratracheal instillation of Zenker’s solution and processed by conventional histologic methods. Results reported concern only studies on guinea pigs. RESULTS

Lungs of animals inhaling only room air were usually normal except for the occasional occurrence of small focal accumulations of round cells. This finding,

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although of considerable importance, is only indirectly related to other observed results. Inhalation of carbon alone was followed by accumulation of many large macrophages in alveolar spaces. They contained large amounts of carbon ; much less carbon was found either free, i.e., apparently lying outside cells, or within cells of the alveolar wall. These findings persisted for the duration of the experiment (24 months after the end of exposure). There was no evidence of pulmonary fibrosis, lung destruction, or other abnormality. Animals exposed to 75 ppm NO,, without t,he precautions of limiting their activity or of administering oxygen, only rarely developed pulmonary edema. The acute effect of inhaling higher concentrations of NO, was pulmonary edema. The only long-term effect noted in all exposures to NO, alone was minimal amounts of lung destruction after two years. There was no pulmonary fibrosis, change in conducting airways, or change in number of alveolar macrophages. Inhalat,ion of carbon with adsorbed NQ? (22% by weight) was followed by moderate to marked destruction of alveolar walls with the formation of alveolar fenest,rae. Localized accumulations of collagen were found within the walls ndjacent to the fenestrae. There were no changes in conducting airways and no increase of macrophages or other cell types. When inhalation of carbon was followed by exposure t.o NO,, lung destruction was found. Inhalation of carbon was followed by increased numbers of alveolar macrophages, and subsequent inhalation of NO2 was then followed by their disappearance and the concomitant appearance of tissue destruction. Exposure to NO, was of several types: a single high concentration (75 ppm), weekly high concentrations, daily low concentrations (25 ppm), and cyclic exposures consisting of daily low concentrations for three weeks followed by one high concent’ration on the fourth week. The greatest degree of destruction was found undcl the latter condition of cyclic NO, exposure. Conducting airways were normal. There were no areas of nodular fibrosis or accumulations of cells other than alveolar macrophages. TTnder the conditions reversing the order of exposure wherein NO, (75 ppm) was inhaled first, followed by carbon, the findings were markedly different. Very few alveolar macrophages were seen, either with or without carbon. The amount of free carbon seemed increased. No destruction of lung tissue was found. There were no areas of nodular fibrosis and conducting airways were normal. DISCUSSIOK

Interpretation

of SequenceExposures

Alveolar macrophages seemto play a key role in these experiments. So little is act.ually known about their origin, fate, life span, function, and interactions with other pulmonary components in unstimulated circumstances, that interpretations of their response under the conditions of experiment must necessarily be prol-isionnl and limited. Inhalation of carbon as well as a wide variety of other particulates is followed by phagocytosis of the particulates by macrophages. Neither the basis for discriminating between substances which will be phagocytized and

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which will not (Boyden, 1963) nor the details of events following phagocytosis are clearly understood. Finding increased numbers of carbon-containing-alveolar macrophages following exposure might be explained as due to increased proliferation, increased life span, diminished rate of removal, or a combination of these factors. Further the findings may be related to ciliary or to surfactant function. Particles may be coated by surfactant (Pattle, 1967) that thereby enhances their phagocytosis. Macrophages may be removed from the lung by gaining arcess to the muco-ciliary apparatus (Kilburn, 1967). One conclusion seems inescapable: the long held conviction that particles huch as carbon are inert or harmless is probably incorrect since inhalation of such particles may prime the lung for subsequent, damage. That such may occur is indicated by the finding of lung destruction in the sequence exposure of carbon followed by NOz. A most obvious but probably incorrect explanation for this finding would be that NO, lysed the accumulated macrophages and thereby released t’heir digestive enzymes which then destroyed adjacent tissue. There i?: no evidence that NO, in these concentrations causes cell lysis, that lysosomal enzymes are increased in concentration or amount following phagocytosis of carbon, or that lung tissues are susceptible to digestion by the amounts of enzyme3 which would be released. Furthermore, the finding of greater destruction when cyclic NO, exposures follow exposure to carbon than with either higher or lower NO, concentrat.ions indicates t,hat a cellular response other than gross lpsis is involved. In t,hose exposures to carbon with adsorbed NO, the finding of lung destruction may be related to injury of alveolar macrophages. It is of interest to compare these findings with silicotic nodules, Macrophage damage by quartz is an early ercnt in silicosis (Vigliani and Pernis, 1963) with a continuing cycle of macrophage drath, followed by phagoeytosis of released silica by other mncropha.ges, eventually leading to fibrosis, presumably as a sequel to lymphocytic exudation. In the C~PC of NO, adsorbed on carbon, such continuing damage should be absent as soon as NO? is desorbed or dissolved off of the carbon. Perhaps the lack of the consequences of continued macrophage death explains the lack of fibrous nodules. However, more information is needed before a reliable mechanistic vicm of macrophage damage can be implicated in tissue destruction. Entirely different cellc, stfructures, or mechanisms may be responsible. Moreover, prolonged cspoul~rc t)o NO, alone is associated with lung destruction, though of lesser degree. The mo,*t straightforward explanation for the findings of the reycrse-requence exposures in which inhalation of NO, is followed by exposure to carbon is that NO, inhibits phagocytosis in some way. Myrvik (1967) has in Z&W evitlence t.hat exposure of alveolar macrophages to 50 ppm NO, suppresses subsequent phngocyt’osis of BCG bacilli. He speculates that such an effect may attenuate the immune response so that contamination 1vit.h organisms can orcur which may xul)sequentlv lead to the development of clelnyctl type hypersensitivity. Ehrliclr 11963) also has suggested that exposure to air pollutants might increase susceptibility to infection. Such suggestions are of importance in considering the s:ignificance of exposures to other gases as well as NO,. The relation of NO2 suppression of phngocytosie to its action in producing pulmonary edemn iy unclear,

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particularly when one realizes how little is known of the mechanisms of increased capillary permeability (Luft, 1965). In considering the general design of sequence exposure, the role of immune (Rose and Phills, 1967)) adaptive (Morrow, 1965, 1967)) and tolerant phenomena (Fairchild, 1963, 1967) should not be forgotten. The design of cross-tolerance studies is perhaps as similar to that of these experiments as is that of any other type exposure. Unfortunately little is known of the extent to which these general processes influence the response to injury. The effects of two agents as related to the development of cancer have been studied by investigations of both chemical and viral carcinogenesis. Boutwell (1964) has reviewed the two stage development of chemical carcinogenesis with that first stage of induction giving an undetected cellular alteration until the nonspecific effect of a promoter is added. Application of promoter and inductor in reverse order did not lead to skin cancer. Duran-Reynals (1963) suggested that a chemical agent activates a resident virus, or acts together with virus to produce cancer. Kotin and Wiseley (1963) showed that inhalation of influenza virus alone lead to cellular proliferation of lung, but when this was followed by ozonized gasoline squamous metaplasia was found. In view of the diverse effects of single, cyclic, sequence, and combined exposures the requirements for defining an “exposure” are apparent. In addition to describing the type of exposure, one must specify the time of day, week, and year, rapidity of onset and offset of exposure, concentrations at plateau, constant or varying, duration and regularity of exposure, and exposure-free intervals. The inadequacy of information given by the product of concentration multiplied by time to express the total impact of exposure is apparent,. Effects of agents such as pesticides, antibiotics, and cleaning agents with which animals under exposure might come in contact must be excluded. ,4n assumption has been made by many workers, particularly those trying to establish safe standards, that if no damage follows a certain exposure, exposures of less degree are certainly unimportant. Such an assumption disregards the interaction of environmental agents with each other and with such environmental forces such as sunlight, temperature, and humidity; it excludes the effects of combined and sequence exposures; and it leaves no room for individual differences due t,o genetic and physiologic variations. The present study is simple, almost trivial, but it points out that the exposure history may be a determinant of the response to environmental agents. FUTURE

STUDIES

Instead of simply listing missing information which is essential for a more complete understanding of the pathobiology of air pollutants, a procedure which would involve every major discipline, let us consider: (1) extensions of current studies which are liable to be productive; (2) the dissolution of communication difficulties; (3) the use of new tools; (4) more complete design strategies; (5) a greater variety of model systems; (6) correlations with other studies of pathobiologic processes; and (7) the combined use of multiple methods at all levels of organization. We may then appreciate the shortcomings of many studies, includ-

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ing our own example, and thereby be able to devise a more advanced approach to the study of injury by environmental agents. Extension

of Current

Studies

WC should welcome more studies of effects of air pollutants on enzyme syst.ems (Mudd, 1965), of studies such as the effect of ozone on chromosome breakage (Fether, 1958)) of the mitotic response of lung to particulate stimulation (Casarett and Milley, 1964), and of the effect of NO, (Pace et al., 1961) or SO? (Thompson and Pace, 1962) on established cell lines. Genetic influences, as indicated by enzyme anomalies such as measurement of alpha,-antit.rypsin deficiency, seem profitable to pursue. Extensive use of electron microscopy in all aspects of disease is expected. The first atlas of ultrastructure of human disease has just appeared (King, 1966). The ultrastructural and biochemical consequences of cell injury are summarized by Trump and Ericsson (1965). Descriptions of the physiological and pathological role of all cell organelles, such as that of mitochondria (Rouiller, 1960) and lysosomes (deDuve and Wattiaux, 1965) and of such relations as lysosomal preparation for mitosis (Becker and Lane, 1965) will continue to be the basis for other studie.s. The ultrast,ructural aspects of inflammatory and immune mechanisms (Zncifach et nl., 1965) will hnvc more complete exploration. The role of surfactant, considered so thoroughly by Pattle (1965), deserves continuing investigation if for no ot,her reason than that it is the first pulmonary constituent with which air pollutants interact. Its possible role in phagocytosis of inhaled particulates has already been mentioned. It may serve another protective function by acting like the mucous covering of amphibian skin or the keratinized layers of mammalian skin to shield underlying structures. Its low surface tension may facilitate the development of the extremely thin attenuated form of the cytoplasm of epithelial cells (Type A). The functions of the hypophase layer beneath the outer insoluble surface layer are unknown: it forms a part of the microenvironment, of alveolar cells and as such may be involved in cell to cell interactions; it could he involved in migration and with it’ cell ,specialization. Dissolution

of Co~~?nz/?2~cut~on Di&ulties

It is hoped that we shall use words effectively, avoid their tyranny as described so well by Paul Weiss (1962) so that we may incorporate information from both our own and related areas of interest. An unfortunate example of an information lag is afforded by the failure to coordinate information obtained by electron microscopy with that by light microscopy of lung. So great had been the argument,s as to the presence or absence of an epithelial covering of alveolar walls that much was made of its demonstration by electron microscopy. Although Frank Low, among others, is usually given due credit for his part in demonstrating alveolar epithelium, his work exhibiting continuity of this epithelium wit.h that of respiratory bronchioles, and his descript,ion of tissue space of lung (LOW, 1961) is usually forgotten. Despite an extensive review of structure, histophysiology, and cytodynamics of respiratory tissue by Bertalanffy (1964a, 196413) the results of electron micros-

TABLE NOMENCLATI-RE

OF MAJOR

CELL

Type A Synonyms

Form Sumber

Location

Type I, small alveolar cell, pulmonary epithelial cell, pulmonary surface epithelial cell Squamous, great,ly attenuated cytoplasm Sum of Type-A and endothelial cells slightly more numerous than Type B Cytoplasm: not seen with light microscope; thickness 0.05-0.3 microns; greatly extended: 10-15 microns Nucleus: 3 X 9 microns Covers alveolar surface of capillaries

Basal lamina” Junction

Present Type A and Type A Type A and Type B

Surface Kucleus

Thin extensions, no microvilli or pseudopods Ellipsoidal, coarse peripheral chromatin, like endothelial nuclei

1’hagocytoei.s Inclusions

NO NO

?uIitochondria Lysosomes Endoplasmic reticulum

Few Few Little

I TYPES

OF ALVEOLAR

Type B

WALL’

Alveolar Macrophage

Type II, large alveolar cell, Phagocyte, granular pneumonocyte, histioryte corner cell, foam cell, alveolar cell Spherical, cuboidal, Irregular, ellipsoidal elongated Numerous; excluding blood Variable cells Type-B cells comprise 40% of the cells of alveolar walls Cytoplasm: 11 microns Cytoplasm: Nucleus: 7 microns, variable; 12 X 16 spherical; nuclear microns; may be cytoplasm ratio 60% of giant cell type Nucleus: 6 X 8 microns Occur singly, separated from In alveolar space other Type B cells, extends partially or completely through alveolar wail Present ATo basal lamina Contacts alveolar Type B and Type A surface of Type-A or Type-B cells Microvilli Pseudopods Spherical, centrally located Irregular, may be multiple, may be within cell; characteristic of this cell type. Contains obscured by cytoplasmic 5-6 nucleoli inclusions Yes, ameboid when isolated Marked Lamellar bodies, extrusions; Phagocytized characteristic cytoplasmic material, including ferrilin vesicle@ Moderate number Many Moderate number Many Well developed rough endo- Little plasmic reticulum; many cytoplasmic ribosomes; Golgi complex regularly present

1’Other Cells: 1. Endothelial cells: basal lamina present; no pericytes at alveolar level; tight junction without fenestrae; many pinocytotic vesicles on both surfaces and within cytoplasm; few mitochondria; nucleus and cytoplasm like Type-il cells but endothelial cells are less superficial and cytoplasm is thicker. 2. Blood cells within capillaries. 3. Fibroblasts, histiocytes, mast cells, and leukocytes occasionally seen in alveolar septa. b Basal lamina of endothelial, Type-A, and Type-B cells enclose tissue space in which there are fibers of collagen, elastic fibers, microfibers, and “ground substance.” No endothelial lined lymphatic space. c -4 “nonmcuolnted” variant of Type-B cells consists of cells with vesicles of small size,. 184

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copy were not incorporated, and this led to confusion in nomenclature of cell t,ypes of lung. So common is this confusion that discussion of the results in our esample experiment were limited to the alveolar macrophage. Another cell type, the Type-B cell, may be of equal importance, but its characteristic structure and probable function are not generally appreciated. Since it is essential to specify what cell is meant in presenting results of experimental st.udies, the author lists t,he morphologic characteristics of the three major cell types of alveolar walls in Table 1. These findings agree with and extend those reported by Brooks (1966). It, is evident from the structures present in the Type-B cell that it is a major l)ulmonnry cel1. It is thought to be the ceil that produces surfactant; the evidence Iv-as reviewed by Clements and Tierney (1965). It appears that it has many characteristics of holocrine secretion cells as described by Brandes et al. (1965). Further investigation is indicated to determine if the Type-B cell is a secretory cell and, if so, the mechanisms of and the conditions controlling its secretion. This cell may be the precursor of alveolar macrophages, but definitive demonstration of such a relation is lacking. The present study showed that inhaled carbon was phagocytized by the Type-B cell as well as by alveolar macrophages. No phagocytosis by Type-A cells was demonstrated. We have also found that expo1;ur(’ to SO, gives an increase in number and size of Type-B cells and that this response is much less with exposure to NO,. It is hoped that recognition of its identifying characteristics and attention to its response in a variety of conditions will further delineate it,s significant physiologic and pathologic functions. Perhaps one reason for its currently being assigned a minor role in lung economy is that the many large vesicles within its cytoplasm are easily distorted and collapsed by conventional fixation. Studies of other systems exhibiting cellular injury are important in evaluating effects of environmental agents. Consider the type of observations found in studies of the damaging action of viruses, chemicals, and drugs. Extensive ultrastructural research on virus-infected cells has been summarized by Bernhard (1964). An almost complete inventory of visible patterns of virus reproduction has been achieved, yet ultrastructure changes in advanced stages of infection are nonspecific; lesions seen could as well have been produced by physical or chemical agents. Since morphologically indistinguishable viruses may give rise to completely different diseases, it is uncertain whether specific cellular re3cfions against viruses exist, and “t,he final cytocidal action of a virus is not inevitable and depends very much on the physiological environment of the cell.” Bernhard concludes, “Viruses may well cease to be pathogenic agents in the hands of thv experimental biologist and become useful genetic tools to explore the normal pathways of celIuIar synthesis, exactly as bact.eriophages have been used to rsplorc the genetics, biochemistry, and functional regulation of the bacterial cell.” We wonder whether the same might not be said about other environmental %cnts. The use of appropriate markers of cellular function, followed by exposure to an altered environment with additional tags on agents responsible for the environmental alteration, could well display new aspects of cellular function. Of the many excellent studies of cellular response to chemical injury, a small .qfimple Emited to effects of cnrhon tetrachloride on liver cells is cited. r)isSocia-

186

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tion of 79 S ribosomes into 54 S components with diminished incorporation of amino acids (Smuckler and Benditt, 1963)) loss of glucose-6-phosphatase activity with vacuolation of endoplasmic reticulum, degranulation of membranes with increased free ribosomes, and calcium-ion entry and sequestration (Reynolds, 1963) have been reported. Farber (1963) has emphasized the value of approaches which study the integrity of integrated chemical reactions as contrasted with those studying single-cell organelles. Such observations suggest that more than morphologic studies will be required to understand the action of air pollutants. Furt,her, we should determine whether a given environmental agent is altered in its passage through cellular membrane systems, pay heed to its flow throughout the body, determine whether it is metabolized to another compound having activity greater than the compound administered, as may be the case with chemical carcinogens (Miller and Miller, 1966) and then survey changes in enzyme systems at the same time as we monitor ultrastructural changes. Recent observations give renewed interest in drugs as influencing cellular structure and function as well as acting synergistically with other agents. Antibiotics alter nucleic acid and protein synthesis (Goldberg, 1965) and such commonly used drugs as phenobarbital markedly alter smooth cndoplasmic reticulum (Fouts and Rogers, 1965; Jones and Fawcett, 1966; Remmer and Mcrker, 1956). Conney and Burns (1962) have shown that drug metabolism is influenced by sex hormones, adrenalin, thyroxine, nutritional stat.us, and foreign compounds. In view of their wide use, drugs should not only be considered as environmental agents but also as possibly interacting with other environmental agents and thus having synergistic or inhibitory effects. Lung is subject to circulating agents such as drugs as well as to inhaled agents. Use of New

Tools

Although development of new tools cannot be forced, those used in studies by other disciplines can be developed for use in biologic systems. The laser hologram may be developed to a greater degree as a microscopic tool ; theoretically it could provide true three-dimensional images of unfixed specimens. Even such practical developments as the aerosol spinning disc generator, which provides monodisperse particles, are of considerable importance. Lung clearance and the effect of particle size and shape on phagocytosis may be determined. We are perhaps disappointed when we consider the shortcomings of seeing structure alone, even at the level of fine structure. Perhaps two immediately available methods of adding tags to follow a sequence of events are of greatest importance. These methods are the use of labeled antibodies in electron microscopy (Pierce et al., 1964) and autoradiography at high resolution (Cnro, 1962; Stevens, 1966). Modern methods of studying infection and immunity as related to the lung are only beginning to be used (Rose and Phills, 1967; Samter, 1965). The studies of DeWeck and Frey (1966) on immunotolerance to simple chemicals is particularly relevant to studies on air pollutants. We hope to see a variety of studies showing the role of lung in reactions similar to contant dermatitis, in sensitivity reactions of the delayed type, and possibly the production of antibody. Lymphoid collec-

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tions in lung have been recognized for a long t’ime. Wit’11 greater understanding of the histology of antibody production and of the control of lymphopoiesis (Noesal, 1962), another look at the focal accumulation of round cells in lung, which occur spontaneously and which can be produced in great numbers by the subcutaneous injection of lung homogenate exposed to NO, suspended in Frcund’s complete adjuvant, is in order. Use of More

Complete

Design

Strategies

In the design of experiments in which animals are exposed to air pollutant? attention is usually given to the age, sex, and speciesof animals to be used with due consideration being given to other aspects of the experiment, such as number of animals, exposures, sacrifice intervals, types of stud& to bc donr, and the spontaneous occurrence of diseasein the speciesstudied. More attention should bc given to the role of adaptation, especially of circadian rhythms, to measures of individuality of the animals (Sargent and Weriman, 19661 a~ well a< to the influence of daily activit,ies of the animals. Adaptation of mammals to polluted air has been rev&-cd by Morrow (1965). and t,here is ample evidence for adaptation in man (Leblanc, 1966). That the capacity of cells to adapt is much greater than is recognized is indicated by studies of recovery from effects of irradiation. Cells may .show restitution of broken chromosomes, resistance to killing, and escape from tnitotic inhibition despite cont’inued irradiation (Upton, 1963). Of the daily activities of animals which might influcncc experiment’al results, perhaps the distribution of food intake is one of the most important factors. Potter (1966) points out that there are significant enzyme differences in the lircr of rats as a result of feeding habits and fasting adaptation. The itnportance of circadian rhythms is well documented (XschofY, 1963, 1965; Pittendrigh, 1961). The effect of the light-dark cycle is especially important when rodents, which are naturally more active at night than during daytime, arc used for exposures. As Pittendrigh (1961) states, “A rose may be a rose but is a different biochemical system at noon and at midnight.” As we consider the significance of cellular control mechanisms in bot,h the normal and the injured cell, we should retnembcr that’ cyclic behavior has as its cellular ba+ their delicate balance and integration. Use of a Greater

T7ariety

of Model

Systems

The desirability of using multiple species in animal experiments is a wellrecognized method whereby results gain in generalit,y and thus become more applicable to man. However, there has been remarkably little diversity in model syst’ems used to study air pollutants. There has been no attempt at selective breeding to develop pollutant-susceptible strains as was done in mice to study chemical carcinogenesis (Boutwell, 1964). Little use has been made of newborn animals; they might allow evaluation of immunologic competence and subsequent responsesas the animals mature. Logical extensions would he to use fetal tnatcrial by placing environmental agents in amnionic fluid or to USCmarsupials such as the oppossum to obtain very immature newborn animals. Observation+ of plant

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damage by air pollutants suggests that a better understanding of multicellular organisms may come from botanical systems having simpler components with which to deal. Use of entirely different, systems such as protozoa, protophyta, and bacteria might prove of value. One wonders if Escherichia coli, “the best understood organism at the molecular level” (Wat,son, 1965), might not be exploited still further by using it as a model system to study air contaminants. Interest in the use of tissue cultures, even synchronous cultures (Zeuthen, 1964), may increase as investigations of the relationships of phases of the cell life cycle to injurious episodes are explored. The problems of dedifferentiation of tissue cultures, the effects of extracellular material and cell masses, and the question of how environment influences genes are being reexamined ; Davidson (1964) gives a quotation of Parker which is appropriate: “The potencies of living cells are far more varied than is generally believed, and . . . the properties which t.hey manifest at any particular moment are functions not only of their inherent capacities, but also of the composition of the environment, in which they live, and to which they contribute.” Correlation

with

Other Studies of Pathobiologic

Processes

Comments already made indicate the relation of studies of environmental agents to studies of viral infections, cancer, and chemical injury. The relationship to other major problems of pathology, inflammation, connect,ive-tissue function (Gould, 1963; Green and Goldberg, 1965; Mancini, 1963) and dclaprd type hypersensitivity should be emphasized. -4reas of biologic interests which are of major importance for understanding the interactions of environmental agents with cells are interactions between cells, population dynamics of cells, and cellular control mechanisms. Although of critical importance in metazoan organisms, interaction between cells is the least understood of t,hese three major areas of cell biology. Studies arc reported considering cell contact (Weiss, 1958), adhesion (Weiss, 1953) and th(, physical chemical basis of cell adhesion (Pethica, 1961). Intriguing observat,ions on experimental histogenesis are given by Moscona (1962). Weiss (1958) describes factors of contact of cells with their physical substrata, of mutual reactions of cells with one another, and of the transmission of specific agents and influences from one cell to another. He speculates that the transfer of macromolecules could be initiated by specific local reorientation at a cell surface giving a microchink, followed by subsequent transfer of large energies in a nonspecific manner. He states that “The specific component of the transfer process need exercise no major force, and the energetic component need have no specificity.” In the lung, the hypophase layer of alveolar lining may be of importance in t,his regard. Considerably greater progress has been achieved in understanding the life cycle and population dynamics of cells (Lamerton and Fry, 1963; Price, 1958) although methods of studying them are only beginning to be used to demonstrate their significance in reactions of the lung to environmental agents. The concept of renewing cell populations (Bertalanffy, 1960; Bertalanffy and Lau, 1962; Leblond et al., 1959) has been applied to lung in studies using col-

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chicine blockade (Bertalanffy, 1960; Leblond and Walker, 1956), or tritiated t,hymidine (Bertalanffy, 1964c; Leblond et al., 1959; Shorter et nl., 1966) to measure turnover rates (Bertalanffy and Leblond, 1953; Leblond et al., 1959). Factors thought to influence the rate of renewal are diurnal and seasonal variations, endocrine effects, temperat,ure, and nutritional status (Bertalanffy and Lau, 1962; Leblond and Walker, 1956). Experimental evidence substantiates the marked influence of feeding on mitotic rate (Blumenthal, 1940,195O; Hunt, 1957). To this list the relations between ccl1 proliferation and drugs (Kihlman, 1966), :+ging (Curtis, 1963) and adverse conditions (Bertalanffy and Lnu, 1962) shoultl be added. In the latter regard only one study (Casarett and Millcy, 1964) reports changes (increases) of mitosis following challenge of an air pollutant (FeOH,). Bec:ausc changes in the estrus cycle alter mitotic rates, only male rats have been studied as normal animals; (Bertalanffy and Leblond, 1964a,b; Bertalanffy and Leblond, 1953) this indicates the need for greater investigation to detcrminc whether t,hc marked sex differential in occurrence of some pulmonary rlisensc>s may be related to differences in cellular proliferation. Two elegant analyses of the patterns of cell division and t,he rc~lutionship between cell proliferation and specialization of renewing epithelium should be considered in studies of pulmonary structure and injury. Thrasher (1966) using single- and double-labeling techniques with mouse duodenum and esophagus found that progenitor cells produce only progenitor cells. Migrat.ion of these cells was sufficient to produce specializat,ion. He concludes, “Thus, environmental clnmges that occur over the short distance between the progenitor compartment and the functional compartment are sufficient to elicit changes in the metabolic pat.hways of the cell which are requisite for cell specialization and loss of proliferative function.” Whether migration of alveolar epithelial cells occurs is unknown. Do Type-B cells serve as progenitor cells for Type-A cells or alveolar mncrophages? Using mouse ear and body-skin epidermis (Gelfant, 1963) and combined labeling and blockade, Gelfant (1966) demonstrated that in addition to the usual cell population (G, population) which has a long G, period, a short GZ period, and moves through the cell cycle in the usual manner, that there are cells which remain in the G, period for several days (G, population). With stimulation, such as heat shock or cutting the ear, both cell populations progress to the next phase of the cell cycle, with the advance of the Gz population into mitosis being a primary event. Superimposed upon the behavior of stimulated G, population cells there are subgroups of cells having different and perhaps specific physiological requirements for mitosis. A cell population which has already undergone DNA synthesis and which persists for a long period before dividing may be a device whereby injury to a total population of one cell type is prevented. Heterogeneity of subgroups may influence the results of injury to tissue. The existence of an epithelial system (having a G, population with subgroups) makes one realize the ambiguity of speaking of interactions of cells with air pollutants, or any other types of injurious agents, unless one has a unique measure of the cell population studied. Of the other aspects of organization of cellular activity, biochemical nlterations have been most thoroughly considered by pathologists (Cameron, 1956; Cameron and Spector, 1961 ; Dawkins and Rees, 1959; Peters, 1963). This is easily

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understood either from the viewpoint of defining a cell or of identifying events in cell injury. Weiss (1958) reminds us, “Similitude in structural regards is no test of similarity of constitution,” and Karlson (1965) states, ‘Since structures are chemical entities, changes of structure are biochemical reactions.” It is useful to review from t,he vantage point of modern molecular biology, summarized so eloquently by Watson (1965), those biochemical reactions most important in cellular injury. Interest in control mechanisms of cells (Monod and Jacob, 1961) flows naturally from the central dogma of molecular biology and the Jacob and Monod theory of regulation of protein synthesis at the transcription level. They stated that there may be nonspecific, induced permanent alterations of genetic information which causes a whole cell lineage to be permanently repressed or derepreseed as the result of an initial transient event not involving any alteration of genes. Carcinogens were considered to act on the regulatory system instead of directly on structural genes (Pitot and Heidelberger, 1963). Cell macromolecules were considered as fundamental targets for chemical carcinogens as they store, replicate, and transfer information needed in cell growth and control (Miller and Miller, 1966). Another t,ype of regulatory system is now described (Gross, 1966) which acts at the level of translation of protein synthesis. Little is known about, the transfer of information from cytoplasm to nucleus, and there are no obserrations as to the nature of substances which may leave cytoplasm and act back on the same or on adjacent cells. Knowledge of all such control mechanisms is the chief prerequisite to an understanding of how air pollutants effect the microenvironment of cells and cells themselves. The basic importance of these considerations is given by King in the introduction to the Third Annual Pathobiology Conference (Aspen, 1966) : “Multiple external injurious agents in the cell’s environment arc constantly exerting an influence on cellular regulatory control mechanisms. These environmental conditions together with the hereditary information contained in the genome determine the cell structure and function at any one time. Lack of ability to maintain controlled homeoetasis resulting from various injuries, may either aggravate, diminish, or delete normal metabolic reactions resulting in disorganization of molecular structure.” Combined Use of Multiple Methods From Molecular to Organismal

at All Levels of Organization

In addition to the inability of purely morphologic methods to detect. macromolecular events such as the above which are believed of primary importance in initiating and perpetuating cellular abnormalities, they suffer the sampling error of examining structure at one point of time. Questions such as when did a given change occur, which of two changes happened first, what is happening now, and what is a specific result may not be answered. It is for these reasons that so much is gained by adding labels which are specific, or which introduce a measure of time. In addition, studies of experimental pathobiology, such as those of the results of environmental agents, should consider the possibilities of error of interpretation that exist when WC attempt to relate the presence or absence of a

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morphologic change to possible damage by the environmental agent. Two cases should be considered: first, that a visible change, a “lesion,” is present; and second, that there are no changes. In evaluating the likelihood that a given agent is responsible for a demonstrable lesion, many possibilities must be considered before we can interpret the lesion as being a specific and invariable consequence of the agent. The lesion may represent a manifestation of a prior unrecognized event such as genetic or developmental activity or the progression of changes initiated by another agent. It may represent an effect on cells previously altered by a different or by even the same agent. It may represent an interaction with another substance in the environment such as drugs or antibiotics. Even if there is n close relationship between the agent under study and the lesion, the effect may be from an altered product of the agent or the lesion may represent a nonspecific reaction. Nor does the failure to display a lesion exonerate the agent from exerting significant effects. Not only may the inability of a method to detect early or slight changes be present, but also the conditions of contact may not have been sufficient to permit the development of lesions as is the case in induction effect,s. On the other hand, adaptation to previous exposure may be operative. Lesions may have been produced but were reversible. Autoregulatory processes would tend to repair damage (King, 1962). A critique of biochemical measurements is equally important. They are analytic instead of synthetic. They may tell when events are happening but not where. They may tell what is happening but not its consequence. As Weiss (1962) sharply points out a cell is neither a bag of enzymes nor fragments of macromolecules; it is an ordered system. The complementary information given by biochemical and morphologic methods indicates the power of their combined simultaneous use. An example of such a study which used physiologic, biochemical, and cytologic methods is one (King et al., 1959) investigating the sequence of events in cell death of Ehrlich tumor ceils exposed to irradiation, salyrgan, and inexact replication of normal environment. Injured cells were found to pass through four discrete recognizable stages. The major changes were: (1) cessation of cell division, DNA loss from nucleus, protein loss from nucleus and cytoplasm, and minor nuclear changes; (2) cessation of respiration several hours later, interruption of oxidntivc and fermentation cycles, loss of mitochondria and increase of their size; (3) potassium loss concurrent with sodium and water entry into cells, cellular trypan blue entry, bursting of cell with release of protein, sodium, and water; 2nd (4) irregular nucleus in conglomerate mass of precipitated protein. The use of multiple techniques gave a more comprehensive view of cellular injury than COUICI have been obtained by using more elaborate techniques, but all yielded t,he same type of information. As we concentrate on cellular injury and reactions to injury, we should keep in mind that our problems become increasingly greater as we examine higher levels of organization. The role of intercellular actions, of extracellular matrices, of physiologic integrative functions at each level or organization add to and change results more than can be predicted by reason. Multiple techniques appropriate to the level of organization must be employed. A comprehensive view

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can be expected only from the combined use of multiple of organization from molecular to organismal. Utilization and strategies of experimental pathology and synthesis formation of biology is the only basis for understanding air pollutants.

approaches at all levels of the tools, techniques, with the ideas and inof the pathobiology of

SUMMARY

Many of the ideas of health hazards of air pol1utant.s are derived from experiences with exposure disasters, groups with increased susceptibility, high risk groups, and special substances of high toxicity. The concept that one agent is responsible for one disease does not permit considerations of the full health impact of an environment polluted by continuously variable and ever-increasing multiplicity of agents. The health of an individual is more than the absence of overt disease. It is the unimpaired capacity to function, t,he full expression of genetic potential, and an optimal adaptation to such a changing environment. illthough evidence is only fragmentary with respect to chronic, low-level, combined, sequential, or random exposures, significant health effects from such exposures are believed likely. One bit of evidence for this view is provided by t,he observations herein reported, whereby the combined effects of two agents together or in sequence is different from either alone and a reversal of the sequence of exposure is followed bv different effects. Inhalation of many particles have been thought harmless if they did not produce changes such as pulmonary fibrosis. However, such exposures may make the lung more susceptible t’o damage as indicat.ed by subsequent challenge by NO,. Conversely, exposure to NO, may impair phagocytosis which is basic protective mechanism of lung. Further, exposure to NO, adsorbed on carbon gives structural changes of lung not found after exposure to either agent alone. The failure to demonstrate morphologic change following exposure to a given agent does not necessarily mean that significant changes for the future have not been produced, just as the presence of an abnormality does not mean that a given agent is solely responsible for it. The assumption that failure of a given exposure to produce a,nomalies indicates that exposure of a lesser degree is harmless, excludes many factors, not t’hc least important of which is the exposure history. The exposure history of an individual to cnvironmcntal agents may determine the expression. nature. and extent of ahnormality as much as do instruction of his genome. Additional determinants of the reaction to an enviromnental change arc the actions of other influences: resident viruses, drugs, chemicals, nutrition, endocrine control. Cyclic behavior, found at all levels from organismal to enzyme, mnv modifv rc5nonsein a crucial manner. IXfficultic~s of understanding animal exposures to air pollutants make apparent many gaps in our knowledge and lags in communication. Limitations of design stra,tegy, of lmiquelv characterizing cell populations, and of using morphologic and biochcn~icnl methods arc inherent, in studies with all animal model systems. The polvcr of using combined methods of monitoring each level of organization is :mparent and the necessity to study each level is appreciated. Neither gcnctics. nor morphology, nor cytologv, nor biochemistry, nor any one appronrh nlonr iq

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sufficient to determine the intimate mechanisms of cellular injury. All taken together, if improperly applied, will not provide necessary and sufficient proofs. Only when the effects of all agents and their sequence of action are considered in the light of the imposed strategy of the experiment and the inherent biological modifiers of the system will an authentic reconstruction of cellular reaction result The basis of cellular injury is to be found in normal cell function. The critical determinants of cell function are their control systems. They are necessarily unstable, are dependent upon interactions with other systems, and are responaivc to continual change of microenvironment; yet they regulate and integrate the bioenergetic, replicative, synthetic, structural, and functional events of cell life of environment as well as perhaps the interactions of cells. Large fluctuations which become injurious represent a not too exaggerated oscillation of the usual response mechanisms. We can secure the most reliable foundation for advances in understanding disease by basing our investigat,ions upon the control mechanisms of molecular and cell biology. Equally as important as understanding the harmful effects of air pollutants upon cells is their possible use as tools to understantl cell biology, especially when acting in conjunction with other agents such :IY viruses and drugs. Every change in an individual has a physical basis and every change of structure represents a chemical reaction, the initiation of which was an alteration of a cellular regulatory mechanism. This concept of disease as an alteration of cellular control mechanism is complementary to but not the reverse of t,he concept of health as optimal function in a changing environment. REFERENCES

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