Pulmonary alterations in rats due to acute phosgene inhalation

FuNDAMENTALANDAPPLIEDTOXICOLOGY8,107-114(1987)

Pulmonary

Alterations

WILLIAMD.CURRIE,*

in Rats Due to Acute Phosgene Inhalation’ GARY E.HATcH,~

AND MICHAELF.

FROSOLONO$

*Department ofRadiology, Duke University Medical Center, Durham, North Carolina 27710, THealth Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina and *Respiratory Section. The Burroughs Wellcome Company, Research Triangle Park, North Carolina

Effects 27711. 27709

Pulmonary Alterations in Rats Due to Acute Phosgene Inhalation. CURRIE, W. D., HATCH, M. F. (1987). Fundam. Appl. Toxicol. 8,107- 114. This study evaluated the relationship between low-level phosgene (COCI,) exposure and pulmonary change or damage. Male Sprague-Dawley rats were exposed to phosgene for 4 hr at concentrations of 0.125 to 1.Oppm (30,60, 120, and 240 ppm min). We examined the dose-related changes in body weight, lung wet and dry weights, lavage fluid protein concentrations (LFP), total cell count, and cell differential in rats exposed to phosgene under carefully controlled conditions. These parameters were measured at the conclusion of single acute exposures and for 3 days postexposure. Significant changes in lung weights (wet and dry) were observed following exposure to 120 and 240 ppm. min phosgene and the LFP was significantly altered at 60 ppm. min. The changes in lung wet and dry weights pooled over all times and phosgene concentrations each correlated significantly with the change in LFP induced by phosgene. The total number of cells in the lavage fluid of phosgene-exposed rats was increased, and the most sensitive cellular indicator of phosgene inhalation was the increase in the percentage of polymorphonuclear leukocytes (PMNs). These results confirm that LFP concentration and cellular differentials can be used as an index of lung damage due to phosgene. A dose-response relationship for the measured parameters was observed. Over the dosage range studied, the return of all measured parameters to near control levels within 3 days following exposure showed that the pulmonary damage was reversible or rapidly reparable. Although the acute effects were shown to be reversible, studies on chronic, low-level phosgene exposures are necessary to determine safe levels for industrial employees. G. E., AND FROSOLONO,

0 1987 Society ofToxicology

Approximately 1 million tons of phosgene (COC&) are produced in the United States each year and more than 10,000 workers are involved in manufacture and use of this important industrial intermediate (NIOSH, 1976). Many other people, e.g., chemists, glassworkers, welders, and firemen, are subject to exposure to this extremely toxic compound due to its formation from accidental combustion of chlorinated hydrocarbons. The severity of pulmonary damage tradition-

’ This report has been reviewed by the Health Effects Research Laboratory, Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

ally has been expressed based on the phosgene concentration X time (Ct) factor (Haber, 1924). The LCtso for man has been estimated at approximately 500 ppm . min. The biochemical basisfor phosgenetoxicity is not known, a factor that has hindered development of a specific medical treatment. Because of its toxicity, the potential for exposure of a large number of individuals, and the lack of a direct therapeutic regimen, phosgene must be considered a significant environmental hazard if releasedinto the atmosphere. Except for local irritation of the skin, eyes, and upper respiratory tract, phosgene has no direct action outside the lungs which, therefore, may be considered the target organ of the gas. There are two major pulmonary re-

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0272-0590187 $3.00 Copyright 0 1987 by the Society of Toxicology. All rights of reproduction in any form reserved.

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sponses to inhalation of phosgene: inflammation and edema. In humans, inhalation of a single, acute phosgene dose below 25 ppm. min can be regarded as harmless (Diller and Zante, 1982). Exposure to 25-50 ppm.min results in pulmonary inflammation (Diller, 1978). Even the most severe instances of inflammation, in the absence of other complicating factors, usually resolve within 2 weeks without lasting consequences. After exposure to 50- 150 ppm. min, there is an initial inflammatory response that may be followed by signs of pulmonary edema (Diller, 1985). Doses of phosgene > 150 ppm . min can produce clinically significant and life-threatening pulmonary edema. Except at extremely high concentrations, which produce an immediate bronchoconstriction, death from phosgene poisoning is a consequence of the pulmonary edema. The clinical latent period for development of pulmonary edema is an important problem in cases of phosgene exposure. Clinical signs of pulmonary edema can be delayed for some hours after the exposure. Once pulmonary edema develops, its progress can be extremely rapid, thereby complicating or even confounding therapeutic countermeasures. The length of the clinical latent period for pulmonary edema is inversely proportional to the phosgene dosage but does not extend beyond 24 hr. No biological parameters have been identified which can be used during the latent period to predict the onset or severity of the subsequent pulmonary edema. The effects of high concentrations of phosgene are fairly well understood and have been described in animals and humans (NIOSH, 1976; Frosolono and Pawlowski, 1977; Pawlowski and Frosolono, 1977; Polednak, 1980; Diller and Zante, 1982; Polednak and Hollis, 1985). There is, however, a lack of definitive information concerning (a) dose-response relationships over a wide range of Ct factors and concentrations and durations of exposure producing given Ct factors, (b) effects of chronic low-level exposures to phosgene, (c) changes that precede or lead to pulmonary

edema, (d) the molecular basis for the pathological changes, and (e) recovery after inhalation of the gas. The present investigation was designed to provide a framework of reference for further experiments addressing these issues as well as for evaluation of various therapeutic modalities for phosgene poisoning. Accordingly, we examine doserelated changes in body weight, lung wet weight, lung dry weight, lavage fluid protein (LFP), total cell count, and cell differential in rats exposed to phosgene under rigidly controlled conditions. MATERIALS

AND

METHODS

Sprague-Dawley rats (CD), 250-300 g, were obtained from Charles River Breeding Labs, Kingston, New York. The animals were placed in individual stainless-steel cages and exposed to phosgene in Rochester-type inhalation chambers. Phosgene, 300 ppm in nitrogen, was metered into chambers having a volume of 0.32 m3 and mixed with an air flow of 0.32 m’/min at the top of the chamber. The gas mixture passed downward through stainless-steel wire cages holding the test animals and was exhausted at the bottom through a water scrubber. Temperature and humidity in the chambers were controlled to 23.0 f 3.4”C and 60 + lo%, respectively. Even distribution of phosgene was assured by sampling from various areas while animals were in the chambers. During an exposure, the chamber was monitored continuously with a Wilks Miran 80 Infrared Analyzer and with a Hewlett-Packard 1183 1 gas chromatograph coupled to an automatic sampling system. The chamber phosgene concentrations were maintained within 2-6% of the intended target concentrations. The length of the exposures was 240 min (4 hr). The estimated phosgene dose or Ct factor (ppm.min) for these studies was obtained by multiplying the concentration of phosgene in the chamber by 240 min. Control animals were placed simultaneously in an identical chamber and allowed to breathe filtered air for 4 hr. At the end of phosgene exposures and at 24-hr intervals thereafter, the rats were killed by decapitation. The chests were opened and the lungs were quickly removed, blotted on filter paper, and weighed. The lungs were then dried to constant weight in an oven at 100°C for the determination of lung wet weight to lung dry weight ratios. For the studies on lavage fluid, exsanguination was accomplished by severing the abdominal aorta. The lungs were then collapsed, the trachea exposed, and 37°C saline (0.85%) was injected in a volume of 35 ml/kg body wt. This quantity of saline was equivalent to approximately

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FIG. 1. Dose effects of phosgene on body weight after 4 hr exposure. Rats were weighed immediately after exposure (m) or at Day 1 (O), Day 2 (A), or Day 3 (0) postexposure. Data are given as means + SE for at least 10 animals. Asterisks indicate significant differences (p ~0.05) from air-exposed controls (Williams’ test).

90% of total lung capacity determined from the allomettic data of Takezawa et al. (1980). The same saline was injected and withdrawn three times, then centrifuged at 700s for 10 min to remove cells. A portion of the lavage fluid was centrifuged at 400g for 15 min, and the pellet was resuspended in 5 ml of Geys balanced salt solution (Microbiological Associates, Bethesda, MD). An aliquot of this cell suspension was pelleted on a microscope slide using a cytocentrifuge (Shandon Southern Instruments, Inc., Sewickley, PA), dried, and stained with Diff-Quick stain (Harleco, Scientific Products) for enumeration of the cell differential. Another aliquot was diluted 1:1 with 10 mg% crystal violet in 4% acetic acid and the total cell count determined with a hemacytometer. Protein concentrations in these supematants were determined by the method of Lowry et al. (195 1). Dose-response data at all time points were evaluated by the method of Williams (1972). Log transformation (cell count data) or arcsine-square root transformation (cell percentage data) was used to achieve homogeneity of variance prior to two-way analysis of variance. Williams’ test (1972) was used to test for the effects of increasing phosgene dose using an overall type I error rate of 5%. Figures show nontransformed means and standard errors. Correlation coefficients during linear regression analysis were determined by the least-squares method.

RESULTS The effects of phosgene on the total body weight of rats after 4-hr exposures at 0.125 to l.O-ppm concentrations of phosgene are shown in Fig. 1. A minimum of 10 animals were sacrificed at 0, 1,2, and 3 days postexposure. The body weight was reduced signifi-

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cantly 1 and 2 days after exposure to 120 and 240 ppm . min phosgene and remained decreased for 3 days after the highest dose. As shown in Fig. 1, at dosages of less than 120 ppm . min phosgene the body weight was unchanged. As shown in Fig. 2 the effects of phosgene on lung wet weight were dose related. At the highest dose (240 ppm.min) the lung wet weight was increased significantly at the end of the 4-hr exposure and remained elevated for 3 days postexposure. A lung weight increase was observed on Days 2 and 3 postexposure to 120 ppm . min phosgene, and was increased significantly at all sacrifice times following 240 ppm.min phosgene. No change in lung wet weight was observed after exposure to 60 and 30 ppm . min phosgene. Measurements of rat lung dry weights after 4-hr phosgene exposures are shown in Fig. 3. The weights were increased significantly in the 120- and 240-ppm . min phosgene experiments at Days 1, 2, and 3 postexposure but not immediately after exposure. The lung dry weights had reached a maximum by Day 2 at the higher dosage of phosgene and no change in lung dry weight was observed when the dose of phosgene was less than 120 ppm . min. The total lung wet weight/body weight ratio was significantly increased immediately 1.8 l *

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FIG. 2. Dose effects of phosgene on lung wet weight after 4 hr exposure. Rats were sacrificed immediately after exposure @) or at Day 1 (O), Day 2 (A), or Day 3 (0) postexposure. Data are given as means + SE for at least 10 animals. Asterisks indicate significant differences (p < 0.05) from air-exposed controls (Williams’ test).

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FIG. 3. Dose effects of phosgene on lung dry weight after 4 hr exposure. Rats were sacrificed immediately after exposure (m) or at Day 1 (0) Day 2 (a), or Day 3 (0) postexposure. Data are given as means (SE =G0.01) for at least 10 animals. Asterisks indicate significant differences (p < 0.05) from air-exposed controls (Williams’ test).

following 240-ppm . min phosgene exposures and remained elevated for 3 days (Fig. 4). In the 120-ppm . min phosgene studies, this ratio was increased at l-3 days postexposure. These ratios were not different from control values in studies conducted with phosgene dosages below 120 ppm . min. Figure 5 relates the concentration of lung lavage fluid protein (LFP) to phosgene dose

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FIG. 4. Dose effects of phosgene on lung wet weight to body weight ratio after 4 hr exposure. Rats were sacrificed immediately after exposure (a) or at Day 1 (O), Day 2 (A), or Day 3 (0) postexposure. Data are given as means f SE for at least 10 animals. Asterisks indicate significant differences (p < 0.05) from air-exposed controls (Williams’ test).

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FIG. 5. Dose effectsof phosgene on lavage fluid protein concentration after 4 hr exposure. Rats were sacrificed immediately after exposure (m) or at Day 1 (O), Day 2 (A), or Day 3 (0) postexposure. Data are given as means * SE for at least 10 animals. Asterisks indicate significant differences (p < 0.05) from air-exposed controls (Williams’ test).

following 4-hr exposures at 0.25 to 1.O ppm phosgene. The LFP was increased significantly at all sacrifice times after exposure to 120 and 240 ppm . min phosgene. Following the 60-ppm . min phosgene, a significant increase in LFP was observed only at 1 day postexposure. At 120 and 240 ppm . min, increased LFP was observed at all sacrifice times. According to Williams’ test, the total number of cells per milliliter of lavage fluid was elevated on Days 2 and 3 after exposure to 240 ppm . min ( 1 ppm) phosgene (Fig. 6). The maximum increase in lavagable cells occurred on Day 2. The cellular differentials indicated a dose-related trend with the percentage polymorphonuclear leukocytes (PMNs, Fig. 7) increasing with increased dosage of phosgene. The percentage PMNs was elevated significantly on Days 1 and 2 at the 60ppm. min (0.25 ppm) exposure level, and at all points at the higher phosgene doses. DISCUSSION Results from this investigation describe dose-related changes in body weight, lung wet

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FIG. 6. Effect of phosgene on total cells/milliliter of lavage fluid after 4 hr exposure. Rats were sacrificed immediately after exposure (m) or at Day 1 (0) Day 2 (A), or Day 3 (0) postexposure. Data are given as means + SE for four animals. Asterisks indicate significant differences (p < 0.05) from air-exposed controls (Williams’ test).

weight, lung dry weight, LFP, total cell count, and cell differential after exposure to phosgene dosages over a range of 30-240 ppm. min. This range of Ct factors encompasses pulmonary edematogenic and nonedematogenie dosages of phosgene. We previously demonstrated, through histologic studies, that pulmonary edema was greatest when rat lung wet weight was at its maximum following exposure to 240 ppm. min (Currie et al., 1985). In the present experiments, increases in lung weights were dependent on the phosgene concentration. There were no changes in either lung wet or lung dry weights after exposure to 30 and 60 ppm . min but increases in both parameters occurred after 120 and 240 ppm . min. The maximum increases in lung wet weight were observed at Day 2 after both of the latter Ct factors. The results of the present study combined with our previous findings suggest strongly that exposures to as high as 240 ppm . min result in damage that is reversible within 3 days postexposure. Our observed increase in lung dry weight is consistent with accumulation of serum proteins in interstitial and alveolar spaces due to leakage of the pulmonary capillaries and to inflammatory responses occurring due to

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phosgene exposure. Frosolono and Pawlowski ( 1977) previously reported increased lung weights and protein content following phosgene exposures at 1000 ppm . min. Although the total cell count of the lavage fluid of phosgene-exposed rats was elevated, the most sensitive cellular indicator of the phosgene effect appeared to be the change in the percentage of polymorphonuclear leukocytes. Similar increases in PMNs have been reported following acute exposure to ozone (Coffin et al., 1968) and nitrogen dioxide (DeNicola et al., 1979). Acute injury to the lung is believed to release a variety of agents that are chemotactic for PMNs (Fruhman, 1964) and the accumulation of these cells may contribute to further injury to the lung due to release of enzymes or active oxygen species (Fox et al., 198 la,b; Harada et al., 1984; Nelson et al., 1985). All these factors would be expected to increase the lung dry weight following phosgene exposure. An increase in the lung wet weight to body weight ratio has been reported to reflect pulmonary edema (Coman et al., 1947). This ratio was increased significantly in the 120- and 240-ppm . min exposures compared with those of air controls. Inspection of Fig. 1 sug-

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Ftc. 7. Effect of phosgene on the percentage of polymorphonuclear leukocytes in lavage fluid after 4 hr exposure. Rats were sacrificed immediately after exposure (a) or at Day 1 (O), Day 2 (A), or Day 3 (0) postexposure. Data are given as means f SE for four animals. Asterisks indicate significant differences (p < 0.05) from air-exposed controls (Williams’ test).

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gests that, although the increased ratio can be attributed to increased pulmonary water levels, the loss of body weight can also be a factor. The lung wet weight to body weight ratio then is not a reliable measure of pulmonary edema when a substantial loss in body weight has occurred. An increase in the lung wet weight to dry weight ratio frequently is taken as an index of pulmonary edema. Calculations from the data in Figs. 2 and 3 show that there was a significant increase in this ratio only at Day 0 after 240 ppm , min. On subsequent sacrifice days and in all other experiments, this ratio was the same or lower than control values. In the absence of other measurements, the decrease in lung wet weight to dry weight ratio would suggest that pulmonary edema was not present in these experiments. Histological changes demonstrate the presence of pulmonary edema after phosgene exposure when the lung wet weight to dry weight ratio was not altered (Currie et al., 1985). The lung wet weight to dry weight ratio is a good indicator of pulmonary edema when lung water is considerably or massively elevated. On the other hand, under mildly edematous conditions, the percentage increase in lung wet weight may closely approximate the percentage increase in dry weight and the resultant ratio is useless as an indicator of edema. The dry weight increase presumably reflects the leakage of serum proteins into interstitial and alveolar spaces and inflammatory responses due to phosgene exposure (Currie et al., 1985). These considerations demonstrate the importance of measuring several parameters and carefully analyzing each of them in the assessment of pulmonary edema or damage. Our data, however, show that an increase in lung wet or dry weight is as good an indicator of pulmonary injury as any other single parameter after exposure to acute low doses of phosgene. The accumulation of protein in lung lavage fluid was also used as an indicator of pulmonary response to phosgene. Increases in LFP were related directly to the phosgene Ct factor. Our data indicate that

AND

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LFP is a more sensitive index of pulmonary damage than any of the gravimetric measurements: LFP was increased significantly at Day 1 after 60 ppm . min when no weight increases were observed. The correlation between LFP concentrations and wet and dry lung weights is not impressive at Day 1 postexposure due to the large relative increase observed in LFP values. The relationship between LFP and lung weights differed at the various sacrifice points following exposure and the strongest positive correlation was observed at Day 3 postexposure. Across the entire range of Ct factors in this study, however, there was a good correlation (Y = 0.86) between increases in LFP and lung wet and lung dry weights. Accordingly, the accumulation of protein in the lavage fluid is a valuable adjunct to the weight measurements and provides an additional means of detecting subtle edematogenic alterations. Diller et al. ( 1985) recently reported similar increases in LFP in rats after exposure to 50 ppm.min phosgene. The accumulation of proteins labeled with [‘311]albumin in LFP was shown by Alpert et al. (197 1) to be a good indicator of pulmonary damage. [‘3’I]Serum albumin could be detected in the lungs following ozone exposure that did not yield histologic evidence of edema. Selgrade et al. (198 1) used a similar technique to measure changes in lungs of guinea pigs exposed to NO2 . Hu et al. ( 1982) demonstrated the utility of determining LFP concentration as a rapid quantitative means of assessing small edematogenic changes in animals exposed to ozone. Although not studied in the present experiments, most of the increase in lavage fluid protein is probably serum protein. This has been documented by polyacrylamide gel electrophoresis in which accumulated lavage protein co-migrated with serum proteins (Selgrade et al.. I98 I) as well as studies by Alpert et al. ( 197 1) in which 13’1-labeled serum albumin injected into the blood stream was recovered in lavage fluid (Hu et al., 1982). Transudation of protein from capillaries or disruption of lung cells could account for the

PULMONARY

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increase in LFP (Sherwin and Carlson, 1973). Roth (198 1) and Diller et al. (1985), however, share the view that the increase in LFF’ is due to increased surfactant secretion. Our previous reports that there is a compensatory increase in pulmonary surfactant concentration following phosgene exposure is consistent with the view that at least part of the increase in LFP may be due to surfactant protein (Frosolono and Currie, 1985a,b). In summary, the present findings indicate a dose-response relationship for the various measured parameters and phosgene exposure. These relationships confirm that LFP concentration can be used as an index of lung edema or damage due to phosgene. Additionally, over the dosage range studied, the return of all narameters to near control levels within 3 days following acute exposure suggests that the observed lung alterations are reversible or rapidly reparable. The effects of chronic lowlevel exposures to phosgene on the lungs, however, still need to be explored to determine safe levels or nontoxic environmental concentrations of phosgene for industrial employees (Cucinell, 1974).

ACKNOWLEDGMENTS We thank Dr. Judith A. Graham of the Inhalation Toxicology Division of the Environmental Protection Agency for providing the inhalation exposure facility and animals used in this study. We also thank Dr. Thomas Williams of Northrop Services Inc. for conducting and monitoring the phosgene exposures, and Mr. Andrew G. Stead for assistance with statistical analyses. The excellent technical assistance by Marvin and Julia B. Nunn was greatly appreciated throughout the course of these studies. This research was supported by Grant 1 ROI ES02671-01 between the National Institute of Environmental Health Sciences and Duke University.

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