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(/) IO (oJI ai (D 1,1-DichloroethyIene-Induced Pulmonary Injury* Poh-Ge,k Forkerr and Edward S. Reynolds ABSTRACT; Oral administration of J.1 -dichlorotihylent I J-DCEl products acute injury to the lungs of C57B1I6 mice. The bromhiolar epithelium it most seierely affected with damage teltctilt for Clara cells. After a 100 mg, kgldose of 1,1-DCE. Clara cells show extensile dilatation of tisteritae and degeneration of the endoplasmic reticulum. At 6 hr after administration of200 mg - LhDCEllg. both ciliated and Clara cells jet necrotic, and hronchiolar epithelial lining ex foliates. By 24 hr. pulmonary edema, hemorrhage, and focal atelectasis are also present. Pul monary injury, at 24 hr after the high dost, is associated u ith a significant hypoxia at demonttraied through a decrease in the partial pressure of oxygen in arterial blood. In spite of the teitre injury, recovery occurs and airuays display an intact epithelial lining with normal parenchymal tltments by 7 days. IXTRQDIK TON The hloroethylene, 1,1-dichloroethylene (1,1-DCE), or^ininyylliiddiinre chloride it an important industrial compound which it widely used in the plastics industry. In 1976. the United States and Japan together produced approximately 150 mil lion kilograms {1], 1,1 -DCK is most commonly used as a copolymer in the manu facture of flexible plastic films for food wraps. Levels of monomeric 1,1-DCE have been detected in food wrapped with plastic wrapping such as Saran Wrap [2], Moreover, workers in manufacturing facilities are also exposed to 1,1-DCE [3 -6] and this compound has been identified as a contaminant in the atmos pheres of submarines and spacecraft [7], Animal studies with 1,1-DCE indicate that it may be a potential hazard to -human health. Following its administration, liver [8-10] and kidney damage [11],ensue. It has been shown to be both a mutagen [12-13] and a potential ' carcinogen [14, 15]. Recently, it has been implicated as embryo- and fetotoxic i ':' ;[16] in studies with ratsand rabbits. >:VExposure to 1,1-DCE in humans is usually by inhalation or ingestion. In ex-, j ,; perimental animals, regardless of the method of administration (gastric intuba' tion, intravenous, or intraperitoneal injection) or of the vehicle (mineral oil, corn oil.' or aqueous Tween 80), the main route of elimination of unmetabolized ' monomer is through the lungs [17, 18]. In spite of the availability of this in formation, there are few reports on the pulmonary effects of 1,1-DCE In one i , ., 'long-ierm inhalation study involving rats, guinea pigs, monkeys, rabbits, and ' - ..^:':ici'dogs;the results of the toxic effects on the lung were inconclusive [10]. In an.' ^^i^MS^oiherglone-term ;investigation, bronchoalveolar adenomas were observed in ^ 'upportid by HU072I7 and ES-02102 from the National Institutes of Health, v-riwa*-rfiSSyAf Departments of Internal Medium and Pathology. Uniteriity ofTesai Medical Branch. vv- 1 ' %ij|fAdJnit C.rrr:p..j,.ct I. P.h Cri F.rhn.Pb.O.. D.pjnmtm .fPtsUUt}, Util mil) tflists MidiitI Brtiub, Gtlttu.H.Ttxtt 775)0. ^ r_ ^ ( ' ,, *' fe'Cf^IvrVMr^StieiKv.RghlithiftftCtimpjny. IntC IW2 U|Oi.j4Jl/K2A)l0<rt`>4 12*02.75 \ Will mmr.:+ - - iftSr fe ~ = M h *.`4 58 P. G. Forltert and E. S. Reynold several mice but were considered to be of limited significance [15J, Despite the varied toxic effects of this compound in experimental animals, and the commot exposure of humans to 1,1-DCE, there are no published reports on the acutt toxic actions of this compound on the Jung. In this investigation, we evaluated the acute consequences of toxic doses of 1,1-DCE on the lungs of mice. METHODS Male C57B1/6 mice (Timco Breeding Labs, Houston, TX) weighing 20 -- 25 g were maintained on a 12-hr light-dark cycle and allowed food (Wayne Lab- Blox, Allied Mills, Inc., Chicago, IL) and water ad libitum until death. At 10:00 a.M. of the first day of the experiment, 1,1-DCE (99% purity. Aid- rich Chemical Co., Milwaukee, WI) was administered by gastric intubation. Three to five animals in each group received either 100 mg/kg or 200 mg 1,1- DCE/kg in mineral oil. Control animals received only the vehicle. Mice were killed at 6,12,24,48,9(3, and 168 hr after administration of the chemical. After cervical dislocation, a midline incision was made.and a pneumothorax produced by cutting the diaphragm on both sides. The trachea was then exposed and cannulated with an intravenous cannula (Argyle Medicuc Cannula, Sher wood Medical Industries, St. Louis, MO). The lungs were inflated via the airway with 1% glutaraldehyde in 0.1 M Pipes buffer (pH 7.35. 360 mOsm) at a hydro static pressure of 15 cm H;0. After ligating the trachea, the lungs were re moved en bloc together with the heart, and immersed in the fixative for 4 hr. The tissues were then stored in 0.1 M Pipes buffer overnight. Slices of lung (1-mm thick) were obtained from each lobe, post-fixed for 2-5 hr in 1% osmium te- trot'ide in 0.1 M Pipes buffer, stained en bloc with uranyl acetate for 2-5 hr and dehydrated in a graded series of ethanol and propylene oxide. Infiltration was achieved slowly, starting from a mixture of 759? propylene oxide and 25% Epon, continued through 50% propylene oxide and 50% Epon and terminated in 100% Epon. Each step was processed for 4 hr. Slices of lung were then em bedded flat in the cap of a Beem capsule and the barrel filled with epoxy resin using a method similar to that described by Stephens and Evans [19]. The technique of embedding slices of lung tissue utilized in this study per mitted specimens to be thick sectioned (0.5 p.m) that were up io'5-mm wide. This facilitated the scrutiny of large pieces of Jung tissue and therefore, also of a number of intrapulmonary airways within a section. Polymerization was achieved at a temperature of 60C for 24 hr for thick sections. Blocks that w-ere selected for thin sectioning were cured at 60*C for an additional 48 hr. >- For light microscopy 0.5-nm stcrior.s were cut with glass knives on a Sorvall MT-2 Ultramicrotome and stained with toluidine blue. For electron micros- . copy, thin sections were cut on ar. LKB Ultroiome 111, with a Dupont diamond knife and stained with uranyl acetate and Reynolds' lead citrate [20}. Sections were examined with a Philips -400 c> 201 Electron Microscope. Blood Gases Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal) at a dose of 0.1 mg/g animal weight. The abdomen was exposed and arterial blood drawn from the abdominal aorta with a 26-gauge needle into a heparinized syringe. Blood gases were immediately determined in a blood gas analyzer (Instrumentation Laboratories, Model 813), R&S 025071 l,l-'Dithloroethylene.Induccd Lung Injury 59 er Enzymes' ` Mice were lightly anesthetized with ether and blood obtained from the inferior vena cava. The serum was analyzed for glutamic oxalacetic transaminase (SGOT) and glutamic pyruvic transaminase (SGPT). Reactions were performed by stan dard techniques, utilizing Smith Kline Instruments, Inc. "Spin Chem" reagents. Statistical Analysis Blood'gases were analyzed by the unpaired / test: 957? confidence limits for geometric means of liver transaminases were compared by the Cochran approxi mation of / values for grouped data with unequal variances {21 ]. RESULTS Administration of 100 mg 1,1 -DCE/kg resulted in a decrease of 97? of body weight within 24 hr; 200 mg/kg caused a reduction of 11% in body weight over that of control animals by the same time period. Histology Examination of the lung by light microscopy revealed differences in morphology of the small airways between control and treated animals (Fig. 1). Bronchioles in control animals showed an epithelial lining composed of low cuboidal ciliated cells and columnar nonciliaied (Clara) cells with prominent apices which pro jected into the lumen [Fig. 1(a)], With a dose of 100 mg/kg 1,1-DCE, the bronchiolar epithelium showed the presence of a few vacuolated cells at 12 hr after gavage. By 24 hr it was apparent that the primary target of 1,1-DCE on the bronchiolar epithelium was the Clara cells for they prominently displayed cytoplasmic vacuoles [Fig. 1(b)], while ciliated cells did not appear to be af fected. By 48 hr, recovery had occurred and the bronchioles displayed a normal and intact epithelial lining. The main bronchi and trachea did not display any prominent changes in their epithelial lining upon administration of 1,1-DCE. The 200 mg 1,1-DCE/kg dosage induced necrosis of both ciliated and Clara cells of the bronchiolar epithelium. Some epithelial cells in the small airways sloughed off as early as 6 hr. By 24 hr, the lesion had increased in severity and areas of bronchioles were denuded of epithelium. Residual epithelium appeared flattened and attenuated and cellular debris was present in the airway lumen [Fig. 1 (c)]. Peribronchial and perivascular edema were also observed and the latter appeared to affect the larger peribronchial vessels. In spite of the apparent severe injury, recovery was accomplished by 7 days, as the epithelium was simi.lar to that of control animals at that time. The main bronchi and trachea exhibited changes similar to those observed in `the bronchioles, with epithelial lining cells undergoing degenerative changes and exfoliation.. Ultrastructure ' .'i '5' Electron microscopic examination of the lung confirmed that the vacuolated cells present in the bronchiolar epithelium following 100 mg 1,1-DCE/kg, were nonciliated Clara cells and that the ciliated cells appeared unaffected. These tell* in the control animals possessed the characterisric morphology attributed ..V m m ;*h-v ,1^ `t; R&S 025072 a i - . a !- Figure 1 Bronchiole in the lung of control mouse (a). 100 mg/kg (bl and 200 mg/kg tc) 1J-DCE Toluidine blue stain 1000* (a)* In controls. Clara cells (CD have prominent apices which project into the lumen (Lof the airway, A small number of ciliated cells are present (arrows), (b), At 2A hr after exposure to 100 mg lJ.DCE/kg, numerous Clara cells are vacuolated (arrows). The ciliated cells (C) appear normal. (L) lumen, tck A: 24 hr after 200 mg l,l*DCE/kg numerous epithelial cells have sloughed off. The ciliated cells have flattened (arrows) and cellular debris (asterisk) is present in the lumen (L). i Jr ' `.,*7 :- ` 1,1-DicTiIoroethylene-lnduced Lung Injury 61 to Clara cells (Fig. 2). The nucleus is indented and basally located. The mito chondria are pleomorphic, varying in electron density and shape. The endoplas mic reticulum tends to form arrays encircling the mitochondria. While the smooth endoplasmic reticulum forms extensive networks in the apical portion of the cell, the rough variety tends to be concentrated in the lower half of the cell. Dense secretory granules were found in the apex of the cell close to the pljsma membrane. Following 100 mg 1.1-DCE kg, the extensive vacuolization in Clara cells consisted of dilated cisternae of both the smooth and rough endo plasmic reticulum (Fig. 3). The rough endoplasmic reticulum also appeared degranulated and polyribosomes disaggregated. Perinuclear cisternae were also distended and the nuclei assumed a more rounded profile. Mitochondria of irtected cells were swollen and showed a reduction in numbers of cristae, and the distribution of organelles in the cytoplasm appeared disorganized. Electrondense aggregates, approximately 10-15 nm in cross-section, appeared in the cy toplasm adjacent to tubules of smooth endoplasmic reticulum. These aggregates formed a fuzzy coat on their cytoplasmic surface. Transitional forms between "coated" and normal smooth endoplasmic reticulum were seen in some rubular profiles (Fig. 4). A web of filaments was often present in the cytoplasmic matrix Figure 2 Electron micrograph of bronchiolar epithelium oflung in control mouse. The ciliated cells are low and cuboidal; rhe Clara cell is columnar, has numerous mitochondria (M> and dense secretory granules (G1 and abundant rough (ER) and smooth endoplasmic reticulum (Seri (Bl) basal lamina. Uranyl acetate and lead citrate stains (6,200 x). i m -p8 t.zh,.-. 30 So OJ P. G. Forkerc and "E. S. Reynolds Figure 3 Electron micrograph ofbronchiolar epithelium of lung in mouse administered 100 mg 1,1-DCE/kg. At 24 hr after gavaae, the Clara cell shows degenerative changes. The endoplasmic reticulum shows extensive dilatation with the formation of numerous vacu oles (V). The mitochondria are swollen with rarified matricies. Perinuclear cisternae (P) have formed around the rounded nucleus. Coated tubular aggregates are present in the cytoplasm (arrows). Debris (arrowhead i of degenerated cells are seen in the lumen of the airway. Uranyl acetate and lead citrate stains (6,200X). adjacent to these aggregated tubular prohits..The aggregates were rather scattered randomly throughout a.Ttcted cells ano were also present in autophagic vacuoles. Pulmonary edema was also observed in the peribronchtal and perivascular areas. The perivascular edema appeared to be most severe in larger vessels in the peribronchial interstitium. Although interstitial edema was found surrounding small vessels, this was focal and rather minimal. Ultrastructural examination of the lungs of animals administered 200 mg 1,1 * 1,1-Dichloroethylcnc-Ioduccd Lung Injury 63 Figure 4 Electron micrograph of the area boxed in Fig. 3. The coated tubular aggregates appear similar in cross-section to coated vesicles (arrowhead) but in longitudinal section, represent tubules of endoplasmic reticulum (arrows). A filamentous web (asterisk) is frequently seen in the vicinity of these aggregates. Uranyl acetate and lead citrate stains (49,300 x). -- DCE/kg revealed more severe degenerative and inflammatory responses than those exhibited by animals gavaged with 100 mg 1,1-DCE/kg. Ciliated and non- ciliated cells displayed extensive vacuolization by 6 hr and some areas of the epithelium were denuded by this time. By 24 hr, numerous Clara cells had sloughed off exposing patches of bare interstitium and basal lamina. Ciliated cells were also similarly affected, albeit not to the degree demonstrated by Clara cells, since the residual epithelial lining was formed by flattened ciliated cells, which had Stretched out over the interstitium. These ciliated cells showed a re duction in ciliary profiles and the mitochondria'were swollen and decreased in number. Peribronchial and perivascular edema was also observed (Fig. 5). A dosage of 200 mg 1,1-DCE/kg did not consistently produce pulmonary injury to the same extent. The lesions ranged in severity from bronchioles devoid of epithelium to extensive damage involving the whole lung. The Jarter included severe' alveolar edema in conjunction with frank necrosis of bronchiolar epithelium. The endothelial lining of blood vessels was disrupted and exhibited gaps. Subendothelial blebs were also observed together with large cytoplasmic vacuoles. Areas of atelectasis and hemorrhage were seen and gaps were pres ent in the alveolar lining formed by Type I alveolar lining cells. Infiltration of leukocytes and monocytes into the lung was extensive in the latter in- ' stance. Mortality among animals given 200 mg 1,1-DCE/kg was approximate ly 25%- I. , Functional Correlation Arterial blood gases determined at 24 hr, a time coincident with peak morpho logic injury, revealed a mean decrease of 1 i% in the arterial pO, level with 100 mg 1,1-DCE/kg and a 27% decrease with 200 mg 1,1-DCE/kg (Table 1). Both doses of 1,1-DCE caused concurrent hepatic necrosis as evidenced by 30 (0 64 P. G. Forkert and E. S. Reynold: C Ci i i * _ s&ffm&fgr*,' n fsg#:- 'yv/\ pL i c V ,* E V vJ ' k* ->-?r v ,* "pc* Figure 5 Electron micrograph of bronchiole in lung of mouse administered 200 mg 1,1DCE/kg. Epithelial cells are predominantly ciliated and they have attained an elongated and flattened appearance. Interstitial edema (E) is present in areas surrounding the bronchiole and blood vessels (B). The interstitial cells (F) contain large homogeneous in clusions. Type 1 (1) and Type II (111 alveolar cells line the alveolar spaces (S). Uranyl acetate and lead citrate stains (6,200 x I. increased liver enzyme activity ir. the strum. Following 100 mg 1,1-DCE/kg, SGOT and SGPT increased f.vt- and nineteen-fold, respectively. The 200 mg 1,1-DCE/kg dosage caused 1 6-fold ipereases in SGOT and pl-fold elevations in SGPT (Table 2). DISCUSSION Preparation of lung tissue for ultrastructural examination has utilized fixation either by vascular perfusion or tracheal instillation [22-24). As our preliminary studies were focused on Clara cells in the bronchioles, the procedure of inflation fixation through the trachea was preferred, since this method facilitated preser- I :. >>!> *- / 1,1-Dkhloroethylene-Induccd Lung Injury Table V Blood gases 24 hr after 1,1-DCE Dosage Arterial pOj (mmHgl* Control 100 mg/kg 200 mg/kg 100 = 11 8S = 24 73 - 81' Values are arithmetic meins = standard devtarion of fed animals (5 in each group I, ''Significantly dintrent from control values./1 < 0.05. 65 Table 2 Glutamic oxalacetic transaminase (SGOT) and glutamic pyruvic transaminase (SGPT) in serum measured in international units per liter Dosage SGOT* SGPT* Control (7)b .100 mg 1,1-DCE/kg (5) 200 mg 1,1-DCE/kg (5) 68 r 6 314 - 68' 1063 = 491' 47-6 875 - 143' 1465 = 1245' Values are geometric means standard error of fed animals mea sured at 24 hr after gavage with 1,1 -DCE. The number of animals in each group is given in parentheses. 'Significantly different from control values. P < 0.C5. vation of Clara cell morphology, especially of rhe endoplasmic reticulum [23, 24]. Instillation fixation has been reported to cause perivascular and interstitial . edema [25]. The 100 mg 1,1-DCE/kg dosage caused more prominent peri bronchial and perivascular edema than that seen in the lungs of control animals, .and as dose increased, injury increased. Following the 200 mg 1,1-DCE/kg dose necrosis was more severe in some animals as evidenced by flooding of alveoli by edema fluid. Thus, in the case of 1,1-DCE, edema appears to be a consequence of chemical administration rather than a fixation artifact. Since pulmonary edema was present in varying degrees, blood gases were analyzed to assess the functional integrity of the lungs in response to 1,1-DCE. Furthermore, Clara cells in lungs of Hamas living at 4720 m above sea level, where the-partial pressure of oxygen in ambient air is 82 mmHg [26-27], ex hibited morphology similar to 1,1-DCE-exposed mice. Since the Clara cells in the lungs of llamas living at sea level did not display such morphology, the - differences in appearance were attributed to the environment [27]. Moreover, rats that have been subjected to acute hypoxia show hyperactivity in the Clara cell population [28]. This suggests the possibility that the changes observed following exposure to 1,1-DCE are in response to hypoxia. A-number of chemical agents have been reported to cause airway injury. Necros!s"bf bronchiolar epithelium is a consequence of administration of bromobenzene [29], although ir is unclear from the report which cell type was , invqlved.j'4-ipomeanol [30], 3-methylfuran [31] and carbon tetrachloride [32] $ all cause'selective damage to the Clara cells of the bronchiolar epithelium. In ' addition to bronchiolar injury, parenchymal changes were also observed. In the ,*'(.'/ >-/V1.,->'-, i *tvi* s mM. mm twasss ' -j i ' -- '1 66 P. G. Forkert and E. S. Reynolds mouse, 4-ipomeanol [33] causes severe pleural effusion and alveolar edema. However, Clara cell damage is the primary event and the edematous changes were secondary effects [30]. Damage following carbon tetrachloride also in volves endothelial and Type 1J alveolar cells, with resulting pulmonary edema and hemorrhage [34, 35]. It is not clear in the dose-response relationships with carbon tetrachloride-induced necrosis, whether Clara cell injury occurs at lower doses, with the exclusion of parenchymal changes. In mice, administration of 100 mg 1,1-DCE/kg primarily affected the Clara cells with no obvious changes elsewhere in the parenchyma. This parallels the lesion present in lungs of ani mals dosed with 4-ipomeanol, where necrosis of Clara cells precedes the onset of edema and congestion, as dose increases. In contrast to carbon tetrachloride where alveolar Type II cells were damaged [34], 1,1-DCE caused gaps to occur in the attenuated epithelial lining of Type I cells. However, the initial sites of reaction, at least to 4-ipomeanol and 1,1-DCE, appear to be within Clara cells. Therefore, pulmonary reactivity in response to 1,1-DCE is qualitatively similar to that produced by other chemical toxins. The presence of "coated" tubules in affected Clara cells was an obvious fea ture of the lesion induced by 1,1 -DCE. These tubular aggregates were also found in liver parenchymal cells injured by the administration of carbon tetrachloride C36], vinyl chloride [37]. and trichloroethylene [38]. It would, therefore, appear that these profiles were not specific to the Clara cell and probably represented denatured membranes [36]. However, the chemical agents administered were all chloroethylenes. Previous investigation indicated that the Clara cell is a cellular site of cyto chrome P-450 dependent mixed function oxidase activiry in the lung. Indeed, P-450 has recently been localized in the apices of Clara cells [39]. The presence of lung metabolizing enzyme systems in the Clara cells may account for sus ceptibility to chemical insult and indicates that injury' to this cellular population occurs because they can metabolize 1,1-DCE to a cytotoxic reactive electrophile. 1,1-DCE is also an acute hepatotoxin in male fasted rats. Liver necrosis in duced by 1,1-DCE is characterized by swelling of mitochondria, disintegration of nuclear chromatin, and contraction of plasma membranes [40]. In contrast to the striking involvement of the endoplasmic reticulum of the Clara cells en countered in this scudy, the endoplasmic reticulum in injured hepatocytes re mains unaffected. Indeed, among the chlorocarbon hepatotoxins, the morpholog ic pattern of injury following 1,1-DCE is unique, for the Others, including carbon tetrachloride, vinyl chloride, trichloroethylene, and halothane, all appear to preferentially injure endoplasmic reticulum within liver cells [40, 41]. This apparent paradox in the case of 1,1-DCE may reflect different pathways of metab olism in the two organs. Different pathways and/or rates of detoxification of active metabolites formed or both may account for the difference. Further in vestigation will hopefully resolve this paradox. The authors wish to rhi.il; C R. Lamke electron micrographs, RC. Aversino for sssistir.ee with the blooJ gases, anc V.M \\ eo :c:11 assistance. REFERENCES _ 1. 1ARC: 1ARC monographs on the evaluation of the carcinogenic risk of chemicals to humans. 19:439-459,19"9. 2. Gilbert J, Shepherd MJ. Startin JR et al.: Gas chromatographic determination of vinyldene chloride monomer in packaging films and infoods.J Chromat I9r:71 -;S, 1980. 1,1-Dichloroethylenc-Induced Lung Injury 67 J*'gcr RJ: Vinyl chloride monomer: Comments on its hepatotoxicity and interaction with 1,1-dichloroethylenc. Amer NY Acad Sci 246:1 50-151,1975. . 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