Document NGe7LpVbjXDyqOX27LYoqg8oV

Vo!. 110. No. 2, 1983 Jonuory 27, 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 675-681 LUNG-SELECTIVE IMPAIRMENT OF CYTOCHROME P-450-DEPENDENT MONOOXYGENASES AND CELLULAR INJURY BY 1,1-DICHLOROETHYLENE IN MICE Klaas R. Krljgsheld, Michael C. Lowe, Edward G. Mimnaugh, Michael A. Trush. Erika Ginsburg and Theodore E. Gram Laboratory of Medicinal Chemistry and Pharmacology National Cancer Institute National Institutes of Health Bethesda, Maryland 20205 Received December 7, 1982 The acute toxic effects of 1,1-dichloroethylene (DCE; 125 mg/kg, l.p.) on mouse lung, liver and kidney were Investigated 24 hr after Its administration. DCE caused a reduction of cytochrome P-450 levels and related monooxygenases In lung mlcrosomes with no corresponding changes In liver and kidney. Examination of the lung tissue by light microscopy revealed-necrosis restricted to the Clara cells. In contrast, liver and kidney were relatively unaffected by DCE treatment, as indicated by lack of changes in microsomal monooxygenase activities and morphology. 1,1-Diehloroethylene (DCE) Is a volatile liquid which is widely used in the plastics Industry and, as a result, human exposure usually occurs by inhalation. Acute toxicity studies in the rat revealed that DCE can cause cellular damage to liver (1,2) as well as kidney (3). Surprisingly, little attention has been paid to the possible acute pulmonary toxicity, even though Investigations into the disposition of DCE in rats (4,5) revealed that after oral, Intraperitoneal or intravenous administration, pulmonary exhalation of the unmetabolized compound accounted for a signif icant part of the administered dose. Forkert and Reynolds (6) reported microscopic evidence of lung injury in mice after oral administration of DCE. At a dose of 100 mg/kg DCE, a selective neerosis of the non-ciliated bronchiolar cells (Clara cells) was observed. Increasing the dose of DCE to 200 mg/kg resulted, however, in damage to other pulmonary cell types. | \ 0006-29U/83/020675-07S01.50/0 675 SL 065574 \fol. 110, No. 2, 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIC In addition to DCE, several other chemicals such as 4-1pomeanol (7), bromobenzene (8), carbon tetrachloride {9) and naphthalene HO) have been r ported to preferentially damage Clara cells. Since these compounds require cytochrome P-450-dependent metabolic activation in order to become toxic, these observations suggest that-Clara cells contain a relatively high concen tration of cytochrome P-450. By selective destruction of these cells one might expect that the overall activity of the microsomal cytochrome P-450 system of the lung would be dramatically diminished. In the present preliminary study in the mouse, morphological changes 24 hr after l.p. Injection of DCE are compared with changes in microsomal cytochrome P-450 and P-450-dependent enzyme activities In lung, liver and kidney. The results demonstrate an organ-specific effect of DCE on pulmonary cytochrome P-450-dependent monooxygenase activity, concomitant with selective damage to the Clara cells. MATERIALS AND METHODS: Male C57BL/6J mice (The Jackson laboratory. Bar Harbor, Maine) (20-28 g), given free access to water and laboratory cho (Zeigler Bros, Inc., 6ardner, PA), were Injected intraperitoneally with 1,1-dichloroethylene (99* purity, Aldrich Chemical Co., Milwaukee, Ul) dissolved In olive oil, 125 mg/kg body weight. Control animals received Olive oil only (5 ml/kg). Twenty four hours after administration, mice werkilled by cervical dislocation, and raicrosomes were isolated by differentia centrifugation In 150 mM KC1 -50ed Tris-HCl buffer, pH 7.4 (11) (35-40 lungs, 5 livers, end kidneys of 12-16 mice were pooled for each microsomal sample). Protein concentration was estimated by the method of Lowry et al (12), with bovine serum albumin as standard. Enzyme assays: Cytochromes P-450 and bs were determined by the methods of Omura arid'Sato (13,14). NADPH-eytOChrome c reductase activity was measured as described by Williams and Kamin (15). 7-Ethoxyresorufin 0-deethylase was estimated according to the method of Burke and Mayer (16), as previously described by Tong et al .(17). Benzphetamine N-demethylase activity was estimated by measurTngThe production of formaldehyde accordin'to the method of Nash (18). Benzol aJpyrene hydroxylase was estimated according to the fluorimetrlc method of Nebert and Gelboin (19). Because o the low renal hydroxylase activity, the concentration of the components of the Incubation mixture were doubled and the incubation time was extended to 60 minutes for kidney microsomes. Morphology: Lung, liver and kidney tissues were examined histologically. Mice (5 controls and 5 DCE-treated) were killed 24 hr after treatment by cervical dislocation. Lungs were Infused In situ with Karnovsky's fixative (20) following tracheal cannulation; the organs 6f interest were removed am. immersed in fixative. Sections of the fixed tissues were stained with hematoxylin and eosin, and evaluated double-blind by light microscopy. 676 SL 065575 Vol. no. No. 2, 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS RESULTS AND DISCUSSION: Morphology: Histological sections of lung, liver and kidney fro* control *1ce were normal, Indicating that the olive oil used as the vehicle for DCE did not cause significant morphologic alterations. In contrast," all lung specimens fro* mice receiving DCE were characterized by a highly specific and nearly complete loss of Clara cells from the linings of the bronchioles (Figures 1 and 2). This effect was extremely uniform, and encompassed the entire bronchlolar tree from the , trachea to the alveolus and Involved every segment observed. Characteristically, in tissues from DCE-treated mice the nuclei of Clara cells were pyknotlc and extremely dark-stained, whereas the cytoplasm of such cells was only faintly eosinophilic. Rarely were Clara cells still attached to bronchlolar walls; more typically, they were detached, accumu lating as luminal debris. Ciliated bronchlolar epithelial cells were not morphologically affected by the DCE. In the liver sections examined from DCE-treated mice there were occasional vacuolated hepatocytes, but no evidence of irreversible Injury, r were there any histologically apparent changes in kidney sections from mice receiving DCE. Fig. 1 Photomicrograph of bronchiole (paraffin embedment, HIE stain) from control mouse. Clara cells (arrows) are abundant and appear normal. (Magnification 409.6x) 677 Ntol. no. No. 2, 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICXJ. nss* \ 'v F<4. 2 Photomicrograph of bronchiole from mouse treated with DCE (125 >19/1(9 ip) 24 hr* prior to sacrifice. Clara cell* (arrow*) uniform)/ have pyknotlc. heavily *t*ined nuclei, and appear to be completely separated from the wall* of the bronchiole*, forming dumps of cellular debri* in the lumen. (Magnification 409.6*) Microsomal monooxygenase activities: Twenty-four hours after the int. peritoneal administration of DCE to mice (1Z5/kg, l.p.), a significant decrease was observed In both the pulmonary microsomal cytochrome. P-450 content and the related enzyme activities. By contrast, these activities were essentially unaffected in liver and kidney (Table 1). The concentrations of cytochromes P-450 and 05 in lung microsomes were reduced to approximately 50 and 601 of control, respectively. NADPH cyto chrome reductase and benzphetamine N-demethylase also declined by about 50*. Activities of 7-Ethoxyresorufin O-deethylase and benzo(a)pyrene hydro lase, cytochrome P-448-dependent enzymes, were less affected, at least at the time point studied; the hydroxylase was decreased by about 30t, while 'there was a slight decrease In O-deethylase activity which was not statisti cally significant. Although DCE elicits hepatic and renal toxicity in the rat. neither the morphology nor the monooxygenase activities in mouse liver and kidney were affected under the dose and time conditions of this Study (Table 1). In thi recent study by Forkert and Reynolds (6), also using mice, hepatic damage apparently occurred as indicated by an elevation in SCOT and SGPT levels 67S SL 065577 I* * !, n o . N o. 2, 1983 BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1: Effects of 1.1-dlchloroethylcne (125 ng/kg, 1p> on microsomal enzyme activities In Tung, liver and kidney of nice 24 hours after treatment Paraaeter Cytochrome P-450* LUNG control net 0.11 t 0.01 0.06 t 0.001* LIVER control occ 0.74 1 0.03 0.75 1 0.05 K1DMCY control OCC 0.20 t 0.02 0.20 1 0.02 Cytochrome bj* 0.11 1 0.004 0.07 1 0.004* 0.43,1 0.01 0.35 1 0.02* 0.26 t 0.01 0.24 1 0.01 HAOPH-cytochrome^ 305 t El 114 t 10* 207 1 1) 204 1 24 153 1 0 ' 144 t 6 O' reductase 'O Henzphetamlneb 2-41 1 0.20 1.30 S 0.20* 7.33 l 0.30 7.05 l 0.47 1.02 t 0.14 1.30 t 0.09 H-demethylase 7-Ethoxyrcsorufln 0-deethy1asec 26 i 5 19 t 2 412 l 41 415 i 22 1.6 1 0.3 0.9 i 0.6* Oenzof *)p/rpnt hydr5ylec'^ 3.6 1 0.1 2.4 1 0.2* 59.7 1 4.0 63.4 1 7.1 27.2 l 5.0 28.0 i 3.5 * nmoles/mg protein *> nmoles/mg proteln/mtn c pmoles/ng proteln/mtn <* for kidney mlcrosomes activity Is expressed as pmolet/*g protein/hour * Statistically different from control, p <0.01 Data are given as mean t S.E.M.; n 6-12 SL 065578 Vol. 110. No. 2, 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATION (5- and 19- fold, respectively), measured 24 hr after oral administration of DCE (100 mg/kg). Unfortunately little else is known about acute toxic effects of DCE In mice. The organ-specific effect of DCE in the present study may be attributed to the pharmacokinetic behavior of the agent and th resulting tissue exposure, since,the lung is the major organ of unmetaboli2ed DCE excretion, and a significant part of the administered dose Is excreted by this route (4,5). Interestingly, DCE caused a clear and significant rise in the P-448 dependent 7-ethoxyresorufin O-deethylase activity in kidney microsomes, an effect which was very reproducible.- We have no explanation for this findin" at present, since no changes were observed either in renal cytochrome P-450 content or in bemo(a)pyrene hydroxylase, also a cytochrome P-448-linked eniywe. Moreover, 7-ethoxyresorufin O-deethylase activites were unchanged In either liver or lung microsomes following DCE treatment. The specific pulmonary toxic effects of DCE strikingly resemble the effects of naphthalene, which decreased microsomal cytochrome P-450 content and related monooxygenase activities in mouse lung, concomitant with selective Clara cell damage and without any morphological or biochemical indication of damage to the liver (17). Thus, the present results for DCE, like those obtained for naphthalene (17), 4-iponeanol (7), carbon tetra chloride (9) and bromobenzene (8) strongly suggest that pulmonary toxins, which must be metabollcally activated to reactive intermediates to cause tissue damage, may be activated by enzymes localized within the Clara cells. Abundant work has shown cytochrome P-450 and its related monooxygenase activities to be highly concentrated In Clara cells relative to the 40 other cell types in lung. In studies employing isolated, purified (70-801 pure) lung cell types, the specific activity of several monooxygenases in Clara cells was 3-40 times higher than in type II cells (21). We are thus left with the provocative question: if monooxygenase activity Is concentrated in Clara cells which are almost totally destroyed by DCE treatment (Figures 1 end 2), why is the total lung monooxygenase activity reduced by only 50-601 6SO SL 065579 f. 110, No. 2. 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (table D and not > 951? The answer may He In the relative numbers of . .ra cells and alveolar type II cells In House lung. If type II cells are many tines more abundant than Clara cells, then the residual enzyne activity in lung after destruction of Clara cells by DCE nay be attributable to that residing in the undamaged type II cells. The answer to this question is under investigation. REFERENCES 1. Jenkins, L.J., Trabulus, M.J., and Murphy, S.D. (1972) Toxicol. Appl. Pharmacol. 23, 501-510. 2. Reynolds, E.S., Moslen, M.T., Szabo, S., Jaeger, R.J., and Murphy, S.D. (1975) Am. J. Pathol. 81. 219-236. 3. Jenkins, L.J., and Andersen, M.E. (1978) Toxicol. Appl. Pharmacol. 45, 131-141. 4. Jor.es, B.K., and Hathway, D.E. (1978) Chem. Biol. Interact. 20, 27-41. 5. Cnieco, P., Moslen, M.T., and Reynolds, E.S. (1981) Toxicol. Appl. Pharmacol. 57, 146-155. 6. Forkert, P.G., and Reynolds, E.S. (1982) Exp. Lung Res. 3, 57-68. 7. Boyd, M.R. (1977) Nature (London) 269, 713-715. 8. Reid, W.D., Hett, K.F., Gllck, J.K., and Krishna, G. (1973) Am. Rev. Respir. 01s. 107, 539-551. 9. Boyd, M.R., Statham, C.N., and Longo, N.S. (1980) J. Pharmacol. Exp. Ther. 212, 109-114. 10. Mahvl, 0., Bank, H., and Harley, R. (1977) Am. J. Pathol. 86, 559-572. 1,1 Lltterst, C.L., Mimnaugh, E.6., Reagan, R.L., and Gram, T.E. (1975) Drug Metab. Dispos. 3, 259-265. . . Lowry, O.H., Rosebroogh, N.J., Farr, A.L., and Randall, R.J. (1951) J, Biol. Chem. 193, 265-275. 13. `Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2370-2378. 14. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2379-2385. 15. Williams, C.H., and Kamin, H. (1962) J. Biol. Chem. 237, 587-595. 16. Burke, M.D., and Mayer, R.T. (1974) Drug Metab. Dispos. 2, 583-588. 17. Tong, S.S., Lowe, M.C., Trush, M.A., Mimnaugh, E.G., Ginsburg, E., HiroLata, Y, and Gram, T.E. (1982) Exp. Mol. Pathol. 37, in press. 18. Nash, T. (1953) Biochem. J. 55, 416-421. 19. Nebert, D.W., and Gelboin, H.V. (1969) Arch. Biochem. Biophys. 134, 76-89. 20. Karnovsky, M.J. (1965) J. Cell Biol. 27, 137-138A. 21. Devereux, T.R.,' end Fouts, J.R. (1981) in: Methods in Enzymology, Yol. 77, pp 147-154 (ed. W.B. Jakoby) Academic Press, Inc., New York. 681 SL 065580