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Biochemical studies on pulmonary response to inhalation of methylene chloride
Toxicology Letters, 7 (1980) 41-45 o Elsevier/North-Holland Biomedical Press
41
BIOCHEMICAL STUDIES ON PULMONARY RESPONSE TO INHALATION OF METHYLENE CHLORIDE SAURA SAHU*, DONNIE LOWTHER and ANDREW ULSAMER
Health Sciences Labomtories, Consumer Product Safety Commission, 200 C Stieet, S. W., Washington, DC 20204 (U.S.A.) (Received May 9th, 1980) (Accepted May 26th, 1980)
SUMMARY
Biochemical response to the toxic lung damage induced by inhalation of methylene chloride was studied. Significant increases in protein, hexose, sialic acid, lactate dehydrogenase, acid and alkaline phosphatase content were observed in the cell-free lavage effluents from lungs of exposed rats compared to the control animals. This was interpreted as increased cell damage accompanied by enhanced pulmonary secretions, perhaps of glycoproteins and mucins, as a result of inhalation toxicity. INTRODUCTION
Normal lungs secrete biochemical macromolecules for their mechanical as well as immunological protection. Abberations of these pulmonary secretions are believed to be responsible for a variety of obstructive lung diseases. It is quite likely that in pathophysiological conditions, such as in chronic toxic inhalation exposures, alteration in quantity and/or composition of normal pulmonary secretions may perhaps affect the overall function of this important organ. Unfortunately, however, our knowledge of these biochemical parameters indicative of lung injury is extremely limited at the present time. Identification of these biochemical parameters for monitoring toxic lung injury would be of practical and clinical significance. Toxicity of methylene chloride, a solvent widely used in a variety of consumer products, has been well documented for inducing fatty liver [l] , However, little is known about its toxic effects on the lungs. Only recently, we have shown that inhalation of this halogenated hydrocarbon leads to *To whom all correspondence should be addressed. Opinions expressed in this article are those of the authors. They do not necessarily represent an official position of the Consumer Product Safety Commission.
42
increase in lung phospholipids [ 21 and hyaluronic acid [ 31, indicative of pulmonary injury. As a part of our long-term program of biochemical studies on pulmonary response to inhalation toxicity, we are searching for pulmonary macromolecules, secreted by the lungs in response to induced inflammation. The present report describes an increased secretion of certain enzymatic and non-enzymatic biochemical macromolecules into the lungs as a result of the inhalation toxicity of methylene chloride. METHODS
Animal exposure
Sprague-Dawley rats (Charles River Breeding Laboratory, Wilmington, MA) of both sexes (10 rats/sex) were exposed by inhalation to 3700 * 230 ppm methylene chloride (certified ACS grade, Fisher Scientific Co., Fair Lawn, NJ) for 5 h/day, 5 days/week, for 4 weeks in an inhalation chamber as previously described [ 31. The chamber atmosphere was constantly monitored by taking samples at 1 S-min intervals and quantitating the methylene chloride concentration by gas chromatography [ 31. An equal number of rats kept in clean room air were used as controls. Collection of pulmonary secretions
Pulmonary secretions of the exposed rats as well as of the control animals were obtained by lung lavage as previously described [3,4] . The lungs were lavaged through the trachea 3 times, each time with 5 ml of ice-cold isotonic saline. Lavage fluid contaminated with blood was discarded. It was centrifuged at 300Xg for 16 min to remove cells and was processed as described before [3]. Enzyme assay
Acid and alkaline phosphatase as well as lactate dehydrogenase assayed according to the method of Bergmeyer et al. [ 51.
were
Analytical methods
Protein was estimated by the method of Lowry et al. [6], hexose by the phenol-sulfuric acid reaction [ 71, and sialic acid by the thiobarbituric acid method, as previously described [g--11] . RESULTS AND DISCUSSION
Histochemical, autoradiographic and electron-microscopic studies indicate that a common pulmonary response to lung damage induced by toxic agents is a proliferation of alveolar Type II epithelial cells by replacing the Type I cells [12,13]. The alveolar Type II cells are of vital importance to the lungs because of their involvement in the synthesis, storage and secretion onto the alveolar surface of a highly surface-active complex mixture of substances,
43
commonly known as the pulmonary surfactant, which prevents alveolar collapse and allows normal respiration by reducing alveolar surface forces. The dramatic change in alveolar cell population, induced by toxic lung damage, should be accompanied by alteration in pulmonary bi~hem~t~. Unfortunately, however, it is not known what effect, if any, toxic agents inducing lung injury play in the synthesis, storage and secretion of the pulmonary surfactant in particular and pulmonary secretions in general. Lung lavage is a valuable technique for obtaining pulmonary secretions directly from the lungs. The biochemical parameters measured in the cellfree lavage effluent are most likely to reflect the change in -Pomona biochemistry induced by toxic lung unjury. Therefore, in the present report we have selectively chosen biochemical parameters which are likely to represent pulmonary injury and can be measured easily in the cell-free lavage fluid. Alveolar secretions, a component of which is the pulmonary surfactant, are a complex mixture of lipids, glycopro~i~ and other carbohydrate containing materials [14]. The lung also secretes respiratory mucus for airway clearance and maintenance of proper water balance. Since respiratory glycoproteins and mucins are rich in carbohydrate content, any change in the protein, hexose and sialic acid content in the lung lavage of animals exposed to toxic inhalation is likely to reflect alteration in the content of these respiratory macromolecules. Acid phosphatase [15-181 and alkaline phosphatase [15, M-20] have been shown to be present in the alveolar Type II cells as well as in the isolated lamellar organelles [ 181, Presence of lactate dehydrogenase in the Type II cells has been demonstrated [20,21] . Presence of these soluble enzymes in the cell-free lavage effluent should indicate alveolar Type II cell damage. In the present report we have chosen to measure the above biochemical parameters - protein, hexose, siahc acid, lactate dehydrogenase, acid and alkaline phosphatase - in the cell-free lung lavage fluid as indicators of the change in pulmonary biochemistry as a response to the pulmonary damage induced by inhaIation of mehtylene chloride. The effect of methylene chloride inhalation on the biochemical parameters in the cell-free lavage effluent from the rat lungs is shown in Table I. As can be seen there is a significant increase in these parameters in the lavage fluid of exposed rats compared to the control animals. The activities of acid phosphatase and lactate dehydrogenase increased almost three-fold and alkaline phosphatase two-fold indicating an infl~mato~ response and cell damage due to the inhalation toxicity. The increase in the protein, hexose and sialic acid contents is representative of an increase in the pulmonary secretions, perhaps of glycoproteins and mucins. The studies reported in the present paper clearly demonstrate the potential usefulness of measuring enzymatic and non-enzymatic biochemical parameters in the lung Iavage fluid as indicators of pulmonary injury induced by inhalation of toxic agents.
44 TABLE I EFFECT OF METHYLENE CHLORIDE INHALATION PARAMETERS IN RAT LUNG LAVAGE ---_______ ~____. _ ____~ ~~~ Biochemical parameters Ratio of contents (Exposed/Control) Protein Hexose Sialic acid Acid phosphatase Alkaline phosphatase Lactate dehydrogenase
1.62 1.47 2.83 3.28 2.17 2.94 -
ON THE BIOCHEMICAL
2 0.14 f 0.12 i 0.26 f 0.29 + 0.18 f 0.26 ---.-.-
.._.-.-. .-_.. .._I_-- ~_
REFERENCES
6 7 8 9 10 11 12 13 14
16 16
J.B. Morris, F. Smith and R. German, Studies on methylene chloride-induced fatty liver, Exp. Mol. Pathol., 30 (1979) 386-393. P.L. Weisenfeld and A.G. Ulsamer, Effects of methylens chloride inhalation on rat lung phospholipids, Toxicol. Appl. Pharmacol., 48 (1979) A6. S. Sahu and A.G. Ulsamer, Hyaluronic acid - an indicator of pulmonary injury?, Toxicol. Lett., 5 (1980) 283-286. S. Sahu and W. Lynn, Metabolism of arachidonic acid in rabbit alveolar macrophages, Inflammation, 2 (1977) 191-197. H.U. Bergmeyer, K. Gawehn and M. Grassl, Enzymes as biochemical reagents, in H.U. Bergmeyer (Ed.), Methods in Enzymatic Analysis, 2nd ed., Vol. 1, Academic Press, New York, 1974 pp. 425-522. 0. Lowry, N. Rosebrough, A. Farr, and R. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. M. Dubois, K. Gilles, J. Hamilton, P. Rebers and F. Smith, Calorimetric method for determination of sugars, Anal Chem., 28 (1956) 350-356. L. Warren, Thiobarbituric acid assay of sialic acids, J. Biol. Chem., 234 (1959) 1971-1976. S. Sahu and W. Lynn, Hyaluronic acid in pulmonary secretions of patients with asthma, B&hem. J., 173 (1978) 565-568. S. Sahu and W. Lynn, Characterization of a high-molecular-weight pulmonary glycoprotein, Biochem. J., 177 (1979) 163-158. S. Sahu and W. Lynn, Isolation and characterization of proteoglycans from porcine lungs, J. Biol. Chem., 264 (1979) 4262-4266. H. Witschi, Proliferation of Type II alveolar cells: a review of common responses in toxic lung injury, Toxicology, 6 (1976) 267-277. H. Witschi and M.G. Cote, Primary pulmonary responses to toxic agents, CRC Crit. Rev. Toxicol., 6 (1977) 23-66. J. Clements and R. King, Composition of the surface active material, in R. Crystal (Ed.), The Biochemical Basis of Pulmonary Function, Marcel Dekker, New York, 1976, pp. 363-384. C. Kuhn, Cytochemistry of alveolar epithelial cells, Am. J. Pathol., 53 (1968) 809833. B. Corrin, A. Clark and H. Spencer, Ultrastructural localization of acid phosphatase in the rat lung, J. Anat. 104 (1969) 66-70.
46 17 18 19 20 21
C. Meban, Localization of phosphatidic acid phosphatase activity in granular pneumonocytes, J. Cell Biol., 63 (1972) 249-262. R. DiAugustine, Lung concentric laminar oganelles, J. Biol. Chem., 249 (1974) 634-693. S. Buckingham, W. McNary and S. Sommers, Pulmonary alveolar cell inclusions: their development in the rat, Science, 146 (1964) 1192-1193. G. Vijeyaratnam and B. Cork, Pulmonary histiocytosis simulating desquamative interstitial pneumonia in rate, J. Pathol., 108 (1972) 106-113. Said S., R. Klein, L. Norrel and Y. Maddox, Lactate dehydrogenase activity in lung, Science, 162 (1966) 667-669.
41
BIOCHEMICAL STUDIES ON PULMONARY RESPONSE TO INHALATION OF METHYLENE CHLORIDE SAURA SAHU*, DONNIE LOWTHER and ANDREW ULSAMER
Health Sciences Labomtories, Consumer Product Safety Commission, 200 C Stieet, S. W., Washington, DC 20204 (U.S.A.) (Received May 9th, 1980) (Accepted May 26th, 1980)
SUMMARY
Biochemical response to the toxic lung damage induced by inhalation of methylene chloride was studied. Significant increases in protein, hexose, sialic acid, lactate dehydrogenase, acid and alkaline phosphatase content were observed in the cell-free lavage effluents from lungs of exposed rats compared to the control animals. This was interpreted as increased cell damage accompanied by enhanced pulmonary secretions, perhaps of glycoproteins and mucins, as a result of inhalation toxicity. INTRODUCTION
Normal lungs secrete biochemical macromolecules for their mechanical as well as immunological protection. Abberations of these pulmonary secretions are believed to be responsible for a variety of obstructive lung diseases. It is quite likely that in pathophysiological conditions, such as in chronic toxic inhalation exposures, alteration in quantity and/or composition of normal pulmonary secretions may perhaps affect the overall function of this important organ. Unfortunately, however, our knowledge of these biochemical parameters indicative of lung injury is extremely limited at the present time. Identification of these biochemical parameters for monitoring toxic lung injury would be of practical and clinical significance. Toxicity of methylene chloride, a solvent widely used in a variety of consumer products, has been well documented for inducing fatty liver [l] , However, little is known about its toxic effects on the lungs. Only recently, we have shown that inhalation of this halogenated hydrocarbon leads to *To whom all correspondence should be addressed. Opinions expressed in this article are those of the authors. They do not necessarily represent an official position of the Consumer Product Safety Commission.
42
increase in lung phospholipids [ 21 and hyaluronic acid [ 31, indicative of pulmonary injury. As a part of our long-term program of biochemical studies on pulmonary response to inhalation toxicity, we are searching for pulmonary macromolecules, secreted by the lungs in response to induced inflammation. The present report describes an increased secretion of certain enzymatic and non-enzymatic biochemical macromolecules into the lungs as a result of the inhalation toxicity of methylene chloride. METHODS
Animal exposure
Sprague-Dawley rats (Charles River Breeding Laboratory, Wilmington, MA) of both sexes (10 rats/sex) were exposed by inhalation to 3700 * 230 ppm methylene chloride (certified ACS grade, Fisher Scientific Co., Fair Lawn, NJ) for 5 h/day, 5 days/week, for 4 weeks in an inhalation chamber as previously described [ 31. The chamber atmosphere was constantly monitored by taking samples at 1 S-min intervals and quantitating the methylene chloride concentration by gas chromatography [ 31. An equal number of rats kept in clean room air were used as controls. Collection of pulmonary secretions
Pulmonary secretions of the exposed rats as well as of the control animals were obtained by lung lavage as previously described [3,4] . The lungs were lavaged through the trachea 3 times, each time with 5 ml of ice-cold isotonic saline. Lavage fluid contaminated with blood was discarded. It was centrifuged at 300Xg for 16 min to remove cells and was processed as described before [3]. Enzyme assay
Acid and alkaline phosphatase as well as lactate dehydrogenase assayed according to the method of Bergmeyer et al. [ 51.
were
Analytical methods
Protein was estimated by the method of Lowry et al. [6], hexose by the phenol-sulfuric acid reaction [ 71, and sialic acid by the thiobarbituric acid method, as previously described [g--11] . RESULTS AND DISCUSSION
Histochemical, autoradiographic and electron-microscopic studies indicate that a common pulmonary response to lung damage induced by toxic agents is a proliferation of alveolar Type II epithelial cells by replacing the Type I cells [12,13]. The alveolar Type II cells are of vital importance to the lungs because of their involvement in the synthesis, storage and secretion onto the alveolar surface of a highly surface-active complex mixture of substances,
43
commonly known as the pulmonary surfactant, which prevents alveolar collapse and allows normal respiration by reducing alveolar surface forces. The dramatic change in alveolar cell population, induced by toxic lung damage, should be accompanied by alteration in pulmonary bi~hem~t~. Unfortunately, however, it is not known what effect, if any, toxic agents inducing lung injury play in the synthesis, storage and secretion of the pulmonary surfactant in particular and pulmonary secretions in general. Lung lavage is a valuable technique for obtaining pulmonary secretions directly from the lungs. The biochemical parameters measured in the cellfree lavage effluent are most likely to reflect the change in -Pomona biochemistry induced by toxic lung unjury. Therefore, in the present report we have selectively chosen biochemical parameters which are likely to represent pulmonary injury and can be measured easily in the cell-free lavage fluid. Alveolar secretions, a component of which is the pulmonary surfactant, are a complex mixture of lipids, glycopro~i~ and other carbohydrate containing materials [14]. The lung also secretes respiratory mucus for airway clearance and maintenance of proper water balance. Since respiratory glycoproteins and mucins are rich in carbohydrate content, any change in the protein, hexose and sialic acid content in the lung lavage of animals exposed to toxic inhalation is likely to reflect alteration in the content of these respiratory macromolecules. Acid phosphatase [15-181 and alkaline phosphatase [15, M-20] have been shown to be present in the alveolar Type II cells as well as in the isolated lamellar organelles [ 181, Presence of lactate dehydrogenase in the Type II cells has been demonstrated [20,21] . Presence of these soluble enzymes in the cell-free lavage effluent should indicate alveolar Type II cell damage. In the present report we have chosen to measure the above biochemical parameters - protein, hexose, siahc acid, lactate dehydrogenase, acid and alkaline phosphatase - in the cell-free lung lavage fluid as indicators of the change in pulmonary biochemistry as a response to the pulmonary damage induced by inhaIation of mehtylene chloride. The effect of methylene chloride inhalation on the biochemical parameters in the cell-free lavage effluent from the rat lungs is shown in Table I. As can be seen there is a significant increase in these parameters in the lavage fluid of exposed rats compared to the control animals. The activities of acid phosphatase and lactate dehydrogenase increased almost three-fold and alkaline phosphatase two-fold indicating an infl~mato~ response and cell damage due to the inhalation toxicity. The increase in the protein, hexose and sialic acid contents is representative of an increase in the pulmonary secretions, perhaps of glycoproteins and mucins. The studies reported in the present paper clearly demonstrate the potential usefulness of measuring enzymatic and non-enzymatic biochemical parameters in the lung Iavage fluid as indicators of pulmonary injury induced by inhalation of toxic agents.
44 TABLE I EFFECT OF METHYLENE CHLORIDE INHALATION PARAMETERS IN RAT LUNG LAVAGE ---_______ ~____. _ ____~ ~~~ Biochemical parameters Ratio of contents (Exposed/Control) Protein Hexose Sialic acid Acid phosphatase Alkaline phosphatase Lactate dehydrogenase
1.62 1.47 2.83 3.28 2.17 2.94 -
ON THE BIOCHEMICAL
2 0.14 f 0.12 i 0.26 f 0.29 + 0.18 f 0.26 ---.-.-
.._.-.-. .-_.. .._I_-- ~_
REFERENCES
6 7 8 9 10 11 12 13 14
16 16
J.B. Morris, F. Smith and R. German, Studies on methylene chloride-induced fatty liver, Exp. Mol. Pathol., 30 (1979) 386-393. P.L. Weisenfeld and A.G. Ulsamer, Effects of methylens chloride inhalation on rat lung phospholipids, Toxicol. Appl. Pharmacol., 48 (1979) A6. S. Sahu and A.G. Ulsamer, Hyaluronic acid - an indicator of pulmonary injury?, Toxicol. Lett., 5 (1980) 283-286. S. Sahu and W. Lynn, Metabolism of arachidonic acid in rabbit alveolar macrophages, Inflammation, 2 (1977) 191-197. H.U. Bergmeyer, K. Gawehn and M. Grassl, Enzymes as biochemical reagents, in H.U. Bergmeyer (Ed.), Methods in Enzymatic Analysis, 2nd ed., Vol. 1, Academic Press, New York, 1974 pp. 425-522. 0. Lowry, N. Rosebrough, A. Farr, and R. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. M. Dubois, K. Gilles, J. Hamilton, P. Rebers and F. Smith, Calorimetric method for determination of sugars, Anal Chem., 28 (1956) 350-356. L. Warren, Thiobarbituric acid assay of sialic acids, J. Biol. Chem., 234 (1959) 1971-1976. S. Sahu and W. Lynn, Hyaluronic acid in pulmonary secretions of patients with asthma, B&hem. J., 173 (1978) 565-568. S. Sahu and W. Lynn, Characterization of a high-molecular-weight pulmonary glycoprotein, Biochem. J., 177 (1979) 163-158. S. Sahu and W. Lynn, Isolation and characterization of proteoglycans from porcine lungs, J. Biol. Chem., 264 (1979) 4262-4266. H. Witschi, Proliferation of Type II alveolar cells: a review of common responses in toxic lung injury, Toxicology, 6 (1976) 267-277. H. Witschi and M.G. Cote, Primary pulmonary responses to toxic agents, CRC Crit. Rev. Toxicol., 6 (1977) 23-66. J. Clements and R. King, Composition of the surface active material, in R. Crystal (Ed.), The Biochemical Basis of Pulmonary Function, Marcel Dekker, New York, 1976, pp. 363-384. C. Kuhn, Cytochemistry of alveolar epithelial cells, Am. J. Pathol., 53 (1968) 809833. B. Corrin, A. Clark and H. Spencer, Ultrastructural localization of acid phosphatase in the rat lung, J. Anat. 104 (1969) 66-70.
46 17 18 19 20 21
C. Meban, Localization of phosphatidic acid phosphatase activity in granular pneumonocytes, J. Cell Biol., 63 (1972) 249-262. R. DiAugustine, Lung concentric laminar oganelles, J. Biol. Chem., 249 (1974) 634-693. S. Buckingham, W. McNary and S. Sommers, Pulmonary alveolar cell inclusions: their development in the rat, Science, 146 (1964) 1192-1193. G. Vijeyaratnam and B. Cork, Pulmonary histiocytosis simulating desquamative interstitial pneumonia in rate, J. Pathol., 108 (1972) 106-113. Said S., R. Klein, L. Norrel and Y. Maddox, Lactate dehydrogenase activity in lung, Science, 162 (1966) 667-669.