Ozone Toxicity as a Model of Lung Fibrosis

Environ Health 1976; 31:37-41

3 Creasia DA, Nettesheim P, Kim JCS. Stimulation of DNA synthesis in the lungs of hamsters exposed intermittently to nitrogen dioxide. J Toxicol Environ Health

1977; 2:1173-81

4 Saltzman BE. Colorimetric microdetermination of nitrogen dioxide in the abnosphere. Anal Chem 1954; 26:1949-55 5 Henderson RF, Rebar AH, Pickrell JA, Newton GJ. Early damage indicators in the lung III. Biochemical

and cytological response of the lung to inhaled metal salts. Toxicol Appl Pharmacol 1979; 50:123-36

6 Barrett AJ. The many forms and functions of cellular proteinases. Fed Proc 1980; 39:9-14 7 Dunhill MS. Quantitative methods in the study of pulmonary pathology. Thorax 1962; 17:320-28 8 Piclcrell JA. Harris DL, Mauderly JL, Hahn FF. Altered collagen metabolism is radiation-induced interstitial pulmonary fibrosis. Chest 1976; 69:311-16 DISCUSSION

Dr. Thurlbeck: Could there be a problem with the llnear intercept measurements. 0$ they do not seem to be changing with age? Dr. Pickrell: Our rats were up to 15 months of age. They are laboratory reared and speclfic pathogen free. Measurements were reproducible when compared between two or three different people ln the laboratory.

Ozone Toxicity as a Model of Lung Fibrosis* C. E. Crou, M.D.• F.C.C.P.; T. W. Huterberg. M.S.; K. M • .Refaer, M.D.; and ] . A. Last, Ph.D.

•From the Department of Internal Medicine and California Primate Research Center, University of California, Davis. Reprint requuts: Dr. Crou, Room 27, PCC, UCD PrqfesriOnal Buildlng, Daoi6, California 95618

Wlhen rodents are exposed to either high or nearW ambient levels of ozone (03 ) for a period of days to weeks, a fibroblastic response can be observed around the terminal bronchiolar and proximal alveolar duct regions of lung parenchyma. 1 ·• Five years ago at these meetings we presented a preliminary biochemical assessment of the effects of 0 3 on lung tissue prolyl hydroxylase activity, total lung hydroxyproline content, and lung collagen synthesis, as reflected by the in vivo incorporation of (SH]proline into hydroxyproline and protein. These studies indicated that relatively low levels of 0 3 (0.5-0.8 ppm [1.0-1.6 mg/m 8] for 7-60 days) could influence several parameters of lung collagen biosynthesis. 5 In the present presentation, the results of more sophisticated studies are presented with regard to the question of whether or not exposure of rats to 0 3 causes biochemically de6nable lung fibrosis and, if so, whether or not this might be a useful model for studying lung fibrosis. Two-month-old, chronic respiratory disease-free, Sprague-Dawley male rats exposed to 1.5 ppm 0 3 for one week do indeed show fibrogenic responses around terminal airways. This is about as high a dose of 0 8 as can be administered to rats over this period without deaths. There is an extensive cellular inflammatory reaction in the peribronchiolar region of the lung. The increased cellularity and fibrous-like peribronchiolar depositions are readily apparent with hematoxylin and eosin staining. When sections are stained with Van Gieson's stain for collagen and Gomori's silver stain, the fibrous nature of the peribronchiolar interstitial deposits is easily appreciated. Although we have done no definitive, quantitative morphologic assessments comparing the collagenous-like materials seen with Van Gieson's stain with the- putative retirulin seen with the Gomori silver stain, we have the impression that the [ 1HJ proline

'

~~ Remove

left lobe

total protein

Mince into 2mm piacu

~

Incubate

1,2,3

h

/

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luno mince

/Ano~~~~H)~ a,~~ total proline

pqcipitation Anolyza for of hof'noolnate 1 [H] hydroxyproline

Hours of incubation

F'IGUBB 1. Schematic for determination of lung collagen biosynthesis

52S 2310 ASPEN LUIIC COIIFEREIICE

CHEST, 80: 1, JULY, 1981 SUPPLEMENT

silver staining is relatively more intense shortly after initiation of 0 8 exposures, and that material which

is positive with the Van Gieson stain appears later and in areas that were formerly stained with the silver stain. Although quantitative evaluations are needed to validate this impression, the silver impregnation stain could be preferentially staining newly synthesized collagen. Figure 1 depicts the system we use to study lung coHagen biosynthesis in the rat. In vivo exposure to 0 8 is followed by in vitro determination of lung collagen synthesis. Lung minces are prepared and washed; replicate samples are incubated with radioactive proline in culture medium. After incubation of replicate vials for one, two, and three hours, the tissue is homogen· ized, treated with 5% Cl8 CCOOH, and centrifuged. The supernatant is assayed for soluble proline content and radioactivity to determine the proline pool size and specmc activity. The pellet is analyzed for protein and for [&H]hydroxyproline as a measure of collagen synthesis by the lung. • The collagen synthesis rate is derived from linear regression analysis of plots of acid-precipitable hydroxyproline synthesis versus time. In a typical experiment (Fig 1) groups of three rats were exposed to 0.8 ppm 0 2 or filtered air for three weeks. The slopes of the lines are the respective collagen synthesis rates; the groups exposed to 0 3 have higher collagen synthesis rates. This aaaay technique has been used to establish a dole-response relationship between exposure level of 0 1 and collagen synthesis rates. Table 1A depicts such results• for 7, 14 and 21 day 0 8 exposures. Histologic seotlona of lung from the same rats were graded for 0 1 effects without any knowledge of the experimental conditions on an arbitrary scale of 1 to 12 ( 12 being maxbnal), and the resulting scores were compared to the collagen synthesis rates. There was a good correlation between the histologic scoring of 0 3 flbrogenic eHecta and the biochemical determination of in vitro Talde 1-t....,. Colla••n S,-nalaala by Rala E•poud aoOaone A. Dole-reaponae relatioMhlpa: Collapn Bynthe1i11 rate (%of control value)

Daya

Expoeed

0.6ppm O.Sppm

o,

o,

1.1 ppm

o.

1.6 ppm 2.0ppm

o.

o.

300

7

160

160

106

200

14

160

176

200

275

21

160

175

220

280

B. Lo111-term efrectl of expOtmre to 0.5 ppm: Total collagen content Collalen Bynthe~U! rate (mg of hydroxyproline per Expoeed (% of control value) lung, %of control value)

Daya 8

107

126

80

148

120

60

131

116

88

137

124

CHEST, 80: 1, JULY, 1981 SUPPLEMENT

collagen synthesis rates in this short-term 0 8 -induced injury model There also seemed to be a correlation between the levels of 0 8 to which the rats were exposed in vivo and the measured lung collagen synthesis rates in mtro. Table IB presents data8 on the relationship between biosynthesis rate and days of continuous exposure to 0.5 ppm of 0 8 • At all times points evaluated between 3 and 90 days, exposed rats had higher collagen synthesis rates than did matched air-breathing controls. Not only was the biosynthesis rate increased, but this increase was also reflected by a signiDcantly increased content of total lung collagen, as measured by total lung hydroxyproline. Collagen content is elevated in the lungs of all rats exposed to 0 3 for up to three months. The collagen being scored by hydroxyproline content may be related to the development of lung fibrosis, although other interpretations are also possible. 8 Conventional wisdom would have it that modiDcation of firobgenesis can best be effected by attempting to suppress the inflammatory stage of the injury process. Various treatment regimens have been administered to the whole animal in order to see whether or not the degree of excess lung collagen biosynthesis in lung minces could be altered. In one experiment, we exposed rats to 1.5 ppm 0 8 continuously for a week and con· currently treated them during the exposure with high levels of steroids. Steroid treatment appeared to cause a signiJlcant decrease in the collagen synthesis rate towards the control values. We have taken all of our data from short-term exposures to different 0 3 concentrations and plotted the relationships between 0 8 levels and lung collagen synthesis. Correlation coefBclents for these relationships of 0.76 to 0.98 Indicate good agreement between levels of 0 3 exposure and lung collagen biosynthesis rates. In .rummary, 0 3-induoed augmentation of lung collagen deposition occurs predominantly in a peribronchiolar location, unlike the distribution of lung flbrosls seen in idiopathic pulmonary flbrosis or after paraquat or bleomycin administration. This does not necessarily mean that there are any basic differences in the underlying mechanisms of the lung flbrogenic responses. Over a several-fold range of 0 8 concentration, there ls a reproducible relationship between 0 3 levels and collagen synthesis rate by lung .minces. The precision between replicate animals in such assays is very high, so that standard deviations from mean synthesis rates are small. This is a helpful attribute If the Investigator desires to study the effects of pharmacologic intervention on lung fibrosis. Finally, the relatively short time frame for the response, within one week, lends Itself to detailed studies of underlying biochemical and pathophysiologic mechanisms during the entire developmental stage of lung fibrosis. 1 We conclude that further exploitation of this model will allow one to gain further insights into the pathophysiology of lung fibrosis. The physiologic correlates of 0 3 exposure appear to be unlike those of more usual pulmonary fibrotic processes. •·• Comparisons of this

THE ENVIRONMENT AND THE LUNI 53S

model with other experimental animal models of acute lung fibrosis, such as that induced by intra-tracheal bleomycin,10 should also prove interesting. Especially fascinating would be carefully documented comparisons of structure and function of fibrotic lungs containing deposits of collagen in different anatomical areas. We believe that the 0 8 model of lung fibrosis will be especially helpful in this regard.

REFERENCES 1 Gorski OJ. Ozone toxicity studies. ill. Chronic injury to lungs of animals following exposure at a low level. Arch Environ Health 1967; 16:514-22 2 Freeman G, Juhos LT, Furiosi NJ, Mussenden R, Stephens RF, Evans MJ. Pathology of pu1monary disease from exposure to interdependent ambient gases (nitrogen dioxide and ozone). Arch Environ Health 1974; 29:203-10 3 Schwartz LW, Dungworth DL, Mustafa MG, Tarkington BG, Tyler WS. Pulmonary responses of rats to ambient levels of ozone. Lab Invest 1976; 34:565-78 4 Last JA, Greenberg DB, Castleman WL. Ozone-induced alteratioos in collagen metabolism of rat lungs. Toxicol Appl Pharm 1979; 51:247-58 5 Hussain MA, Mustafa MG, Otow C.K, Cross CE. Ozoneinduced increase of lung proline hydroxylase activity and hydroxyproline content. Chest 1976; 69:273-75 6 Last JA, Greenberg DB. Ozone-induced alterations in

Occupational Lung Diseases State of the Art Hans Weill, M.D., F.C.C .P. •

presented in this session illustrate the T hescopepapers of investigations which are being carried

out on the breadth of workplace inhalants and their health effects. A number of these depend upon availability of occupational populations for study. Gaining access to such occupational groups requires convincing both management (and their lawyers), as well as workers (and their unions) that even in this somewhat polarized, regulated and litigious society, it is in their seH-interest to participate in such investigations. In this overview, I would like to summarize several examples of how studies of populations exposed to mineral and organic dusts, or chemical gases and vapors, have contributed to our knowledge of both dose-related and non-dose-related determinants of these conditions, and raise some of the questions which remain.

MINERAL Dusts The oldest of the recognized pneumoconioses is silicosis which has for centuries been associated with the dust exposures encountered in hard rock and metal Professor of Medicine, Tulane University School of Medicine New Orleans. Reprint requem: Dr. Weill, 1700 Perdido Street, New 0

Orleans 70112

54S 23RD ASPEN LUNG CONFERENCE

collagen metabolism of rat lungs. II. Long-term exposures. Toxicol Appl Pharmacol1980; 55:108-14 7 Reiser KM, Last JA. Quantitation of specific collagen types from lungs of small mammals. Anal Biocbem 1980;

104:87-98

8 Bartlett D, Faullrner CS, Cook K. Effect of chronic ozone exposure on lung elasticity in young rats. J Appl Physiol1974; 37:92-96 9 Frank R, Brain JD, Knudson DE, Kronmal RA. Ozone exposure, adaptation and changes in lung. Environ Res 1979; 19:449-59 10 Starcbel- BC, Kuhn C, Overton JE. Increased elastin and collagen content in the lungs of hamsters receiving an intratracheal injection of bleomycin. Am Rev Respir Dis 1978; 117:299-305 DISCUSSION

Dr. Renzetti: Have there been physiologic studies to suggest an obstructive lesion, as one woold predict from the localization?

Dr. Cross: There are physiologic studies in anim.ol$ and in people which show airway effects. The problem is that things are happening in two parts of the lung at the same time, the parenchyma and the peripheral airways, and the physiologic changes haven't been put together as carefully as they could be.

mining, and subsequently in such diverse occupations as foundry and pottery work, tunneling, abrasive soap making, and sandblasting. Recently, there has been a resurgence of interest as the result of the recognition of accelerated silicosis in silica Hour mills where finely ground silica produces a high proportion of respirable particles. In addition, a silicosis risk was among the earliest fears following the eruption of Mt. St. Helens; the potential biologic effects of volcano ash are considered elsewhere in this special issue. Our findings in studies of silicotic sandblasters are concordant with the general view that the primary determinant of silicosis is the dose of free crystalline silica capable of penetrating to the alveolar component. 1 The form of the disease is importantly influenced by airborne concentration and length of exposure, the components of dose. Slowly progressive silicosis can result from a working lifetime of exposure in mining and other occupations where silica dust levels are moderate. Accelerated and acute silicosis have been recognized since the 1930's as the result of abrasive soap making, tunneling and sandblasting where airborne concentrations of fine silica particles are extremely high. The mechanism of silicosis is probably related to the toxic effect of silica on the macrophage which impairs function and may result in cell death with subsequent release of cellular enzymes. The relationship between enzyme release and stimulation of fibroblasts remains unclear. It has been noted that both autoimmune antibodies and clinical manifestations of such collagen vascular disease as systemic lupus, scleroderma

CHEST, 80: 1, JULY, 1981 SUPPLEMENT