- Email: [email protected]
Some effects of trichloroethylene on mouse lungs and livers
Gen. Pharmac. Vol. 15, No. 2, pp. 139 144, 1984 Printed in Great Britain. All rights reserved
0306-3623/84 $3.00 + 0.00 Copyright ~. 1984 Pergamon Press Ltd
SOME EFFECTS OF TRICHLOROETHYLENE ON MOUSE LUNGS A N D LIVERS GAlL D. LEwis, ROLLAND C. REYNOLDS and ALICE R. JOHNSON University of Texas Health Science Center, 5323 Harry Hines Boulevard, Dallas TX 75235 (Tel: (214) 688-3111) (Received 17 June 1983) Abstract--1. Repeated administration of trichloroethylene (TCE) to mice by either i.p. injection or by inhalation increased the activity of hepatic microsomal NADPH cytochrome-c reductase. 2. The NADPH cytochrome c reductase activity in microsomes isolated from lungs of animals treated with TCE by inhalation was decreased relative to controls (untreated animals). 3. TCE inhalation was associated with pathologic changes in lungs, but not in livers of the treated animals. The duration of exposure is probably an important factor however, since animals exposed for only I hr per day exhibited neither pathologic changes in the lungs nor an alteration of enzyme activity. 4. These findings indicate that inhalation of TCE, without prior treatment with inducers, can enhance activity of the hepatic mixed function oxidase system. 5. The reduced activity of the pulmonary mixed function oxidase system in animals that inhaled TCE may reflect injury to the lungs.
INTRODUCTION N u m e r o u s investigations have focused on the toxicity, mutagenicity, and carcinogenicity of halogenated and unsubstituted hydrocarbons. The most extensively studied agents include simple aliphatic compounds, such as vinyl chloride, carbon tetrachloride, and chloroform, and aromatic compounds, such as benzene and the polycyclic hydrocarbons, benzo(a)-pyrene and 3-methylcholanthrene. While it is well known that many of these compounds damage the liver, some of them are also inhaled and may damage the lungs as well. Trichloroethylene (TCE) is a widely used industrial and commercial solvent which is structurally similar to vinyl chloride. It is also frequently abused by solvent sniffers (James, 1963; Litt and Cohen, 1969; Garriott and Petty, 1980). The hepatoxicity of T C E is well known and has been studied in a number of animal species (Orth and Gillespie, 1945; Smith, 1966; Baerg and Kimberg, 1970; Carlson, 1974; Allemand et al., 1977). T C E is also mutagenic (Greim et al., 1975; Henschler, 1977) and weakly carcinogenic (Henschler, 1977; Van Duuren, 1977). Administration of T C E by various routes is reported to induce liver microsomal enzymes in animals that have been pretreated with phenobarbital, and this combination of drugs is also thought to increase the toxicity of T C E (Carlson, 1974; Molsen et al., 1977; Allemand et al., 1978). Since exposure to T C E in man usually occurs by inhalation, we studied the effects of this agent on pulmonary cytochrome c reductase in mice exposed by inhalation. For comparison, we also examined this enzyme from livers of mice treated either by inhalation or by i.p. injection. N A D P H cytochrome c reductase activity was measured in microsomal fractions of lung or liver tissue as an index of the activity of the mixed function oxidase system. Tissues were examined by light and electron microscopy for damage caused by TCE. The
object of this study was to determine whether T C E alone (without previous induction) can affect the activity of hepatic or pulmonary mixed function oxidases, and whether inhalation of this agent can injure the lungs directly. MATERIALS AND METHODS
Materials
Trichlorethylene (reagent grade) was obtained from Mallinckrodt, 1,1,1-trichloroethane (reagent grade) from Baker Chemical Co., and NADPH and cytochrome c (horse heart) were obtained from Sigma Chemical Co. Male Swiss Webster mice were purchased from Simonsen Laboratories. Treatment o f animals
Mice with an average weight of 25 g were used for all experiments. Pilot studies indicated that the LDs0 of TCE administered by i.p. injection is 0.65 g/kg and that the LDs0 of l,l,l-trichloroethane is 1.15 g/kg. The doses selected for serial treatment were dictated by the necessity to keep the animals alive during the 5 days required for experiments. Two series of experiments were performed. In the first, 20 mice were divided into control (vehicle-treated), TCEtreated and 1,1,1-trichloroethane-treated groups. The animals were injected intraperitoneally with 0.2 ml of vehicle (25% Tween 80 in saline) or drugs on alternate days for a 5-day period. Each animal received a total of 3 injections. The TCE-treated animals were injected with 0.33 g/kg and the 1,1,1-trichloroethane-treated animals were injected with 0.38 g/kg at each treatment. The trichloroethane group was an additional control, since most of this compound is excreted unchanged into expired air (Hake et al., 1960; Ikeda and Ohtsuji, 1972). Animals in all groups were killed by cervical dislocation 24 hr after the last injection, and the livers were removed for isolation of microsomes as described below. In the second series of experiments, mice were exposed to TCE by inhalation in a closed system. Pyrex bell jars covered tightly with several layers of cotton cloth were used as inhalation chambers. The bottom of each jar was lined with gauze covered by wire mesh. The animals were exposed to 10,000ppm of TCE by saturating the gauze with the 139
GAlL D. LEWm et al.
140
appropriate amount of drug. Eighty-five mice were randomly divided into 3 groups. The treated animals were exposed for either 1 or 4 hr daily, and control animals were placed in the jars for 4 hr per day but not exposed to TCE. All three groups were treated daily for 5 consecutive days. Twenty-four hours after the last exposure, the animals were killed by cervical dislocation, and lungs and livers were removed for isolation of microsomes.
Preparation of microsomes The procedure used for isolation of microsomes from mouse tissues was similar to that of Hook et al. (1972). The tissues were removed immediately after death, washed in cold saline and chilled on ice. The organs were perfused with cold saline to remove blood, minced and homogenized in 3 volumes of 0.25 M sucrose (pH 7.4) in a fitted glass vessel with a motor-driven Teflon pestle. In order to obtain sufficient material for microsome isolation, 2-3 livers from each group were combined, and 5 pairs of lungs were combined. The homogenates were centrigued at 9000g for 20 min. The pellet was discarded, and the supernatants were centrifuged at 100,000 g for 60 rain. The pellet obtained after high speed centrifugation was resuspended in 0.15 M KC1 (pH 7.4), homogenized, and centrifuged again at 100,000g for 60 min. All centrifugation steps were done at 4-'C. The washed pellets were resuspended in 0.25 M sucrose and assayed for cytochrome c reductase activity by the method of Masters et al. (1967). The reduction of cytochrome c was initiated by addition of NADPH. Protein content of each rnicrosomal sample was measured by the method of Lowry (1951).
Microscopy of tissues Livers and lungs of control and TCE-treated animals were examined by light and transmission electron microscopy. Immediately after removal of livers, thin slices were cut and fixed in half-strength modified Karnovsky's fixative (Karnovsky, 1965). Lungs were inflated by infusion of the fixative into the trachea before slices were made. The tissues were post-fixed in 1% osmium tetroxide in phosphate buffer (0.1 M), embedded in araldite, and dehydrated with graded concentrations of acetone. Sections from each organ were examined by light microscopy and transmission electron microscopy.
RESULTS
Enzyme activities A d m i n i s t r a t i o n o f 0.33 g/kg T C E to mice by i.p. injection over a 5-day p e r i o d increased the activity o f hepatic microsomal NADPH cytochrome c reductase c o m p a r e d to c o n t r o l or 1 , 1 , 1 - t r i c h l o r o e t h a n e - t r e a t e d a n i m a l s (Table 1). As expected, there was no increase in e n z y m e activity in m i c r o s o m e s isolated f r o m livers o f mice t r e a t e d with 1,1,1-trichloroethane. E x p o s u r e o f the a n i m a l s to 10,000 r p m T C E by i n h a l a t i o n also i n c r e a s e d the h e p a t i c e n z y m e activity. A n i m a l s that were t r e a t e d for 4 h r / d a y for 5 consecutive days h a d an increase in e n z y m e activity c o m p a r a b l e to t h a t in a n i m a l s t h a t were injected with T C E . The mice t h a t were e x p o s e d for only 1 h r / d a y , h o w e v e r , h a d e n z y m e activities similar to t h o s e in c o n t r o l animals. T h e a v e r a g e liver w e i g h t / b o d y weight ratios were similar in all three g r o u p s o f animals. T h e s e d a t a are given in Table 2. In c o n t r a s t , m i c r o s o m a l N A D P H c y t o c h r o m e c r e d u c t a s e activity in lungs o f a n i m a l s that h a d inhaled T C E was significantly less t h a n t h a t in c o n t r o l p r e p a r a t i o n s . T a b l e 3 s h o w s t h a t the e n z y m e activity in lungs f r o m a n i m a l s e x p o s e d for l h r / d a y was d e c r e a s e d to 77% o f c o n t r o l ( u n t r e a t e d ) p r e p a r a t i o n s , a n d the activity in lungs t h a t were e x p o s e d for 4 h r / d a y d e c r e a s e d to 67% o f control. T h e difference b e t w e e n the 1 a n d 4 hr e x p o s u r e p r e p a r a t i o n s was n o t statistically significant, a n d there was n o difference in the lung w e i g h t / b o d y weight ratios a m o n g the groups.
Acute effects of T C E W h e n the mice were first placed in the c h a m b e r s , the i n h a l a t i o n o f T C E c a u s e d a s h o r t p e r i o d o f excitation, r e s p i r a t o r y s t i m u l a t i o n a n d even c o n vulsions in s o m e o f the animals. E v e n t u a l l y all mice s u c c u m b e d to the a n e s t h e t i c effects o f T C E while in the c h a m b e r . R e c o v e r y was c o m p l e t e w i t h i n an
Table 1. Effect of trichloroethylene on liver microsomal enzyme activity NADPH cytochrome c reductase (nmol/min/mg Percent Group protein) control Control (vehicle) 186.5 - 21.0 100 Trichloroethylene 287.1 + 23.6* 154* 1,1,1-Trichloroethane 177.5 _+9.1 95 Animals in each group were treated with i.p. injections on alternate days for 5 days prior to isolation of microsomes. Data are means +_SEM of 3 4 separate determinations. Asterisk (*) indicates significant difference (P < 0.05) compared to control group by Student's unpaired t-test. Table 2. Effect of trichloroethylene on liver microsomal enzyme activity NADPH cytochrome c reductase Liver/body (nmol/min/g Percent Group weight ratio liver) control Control 5.7 117.9 + 10.8 100 One hour 5.8 137.9 +_ 14.4 117 Four hours 5.8 190.0 _+ 15.0" 161" Mice in each group were exposed to air or TCE (10,000 ppm). Controls (n = 25) were in the chamber 4 hr; TCE-treated animals were exposed for 1 or 4 hr daily for 5 days. Data are means + SEM of 10 separate determinations. Asterisk (*) indicates significant difference from control by Student's unpaired t-test (P < 0.01). One and four hour groups were significantly different (P < 0.05).
Effects of TCE on mouse lungs and livers
141
Table 3. Effect of trichloroethylene of lung microsomal enzyme activity NADPH cytochrome Lung/body c reductase Percent Group weight ratio (nmol/min/g lung) control Control 6.5 2.1 +_0.1 100 One hour 6.9 1.6 + 0.2 77 Four hours 6.6 1.4 _+0.1" 67* Tissue was obtained from the same mice depicted in Table 2. Data are means _ SEM of 4 separate determinations. Asterisk (*) indicates significant difference (P < 0.01) from control group by Student's unpaired t-test.
hour after removal from the T C E atmosphere. The duration of excitatory effects and the anesthesia decreased with subsequent exposure to TCE. Pathologic changes in tissues Light microscopic examination of livers from mice treated with T C E by either i.p. injection or by inhalation showed no obvious pathology, and the morphology was similar to that in control animals. Lungs from animals exposed to T C E by inhalation, however, showed subtle pathologic changes. For example, platelet thrombi were noted in light microscopic sections of lungs from animals exposed to T C E for 4 hr/day (Fig. 1). These were not seen in sections from untreated animals. Additional changes in the lungs of TCE-treated animals were appreciated when sections were examined by transmission electron microscopy. Figure 2 shows vacuolization of bronchiolar epithelial cells in a section from an animal treated with T C E 4 hr daily. These changes were not seen, however, in lungs from animals exposed to T C E for only 1 hr/day or in control animals. DISCUSSION Trichloroethylene (TCE) is a solvent used in many industrial processes, and it is found in a number of commercial products. It was also once used as a general anesthetic agent. Currently, exposure to this
agent occurs via the pulmonary route either inadvertently through solvents or intentionally in abuse situations. The metabolism and acute toxicity of T C E has been well studied. T C E is metabolized by the hepatic mixed function oxidase system to a reactive epoxide intermediate (Henschler, 1977; Van Duuren, 1977; Politzer et al., 1981). This intermediate can rearrange to form chloral, which is then converted by N A D ( H ) containing dehydrogenases to trichloroethanol and trichloroacetic acid prior to excretion (Van Duuren, 1977; Ikeda et al., 1980). The electrophilic trichlorethylene epoxide can react with cellular nucleophiles, such as proteins and D N A to cause additional acute cytotoxic effects (Henschler, 1977; Van Duuren, 1977; Allemand et al., 1978). An alternate route for T C E metabolism in mice was reported by Hathway (1980). He proposed that, due to saturation of the pathway leading to chloral formation, T C E epoxide is rearranged to dichloroacetyl chloride. This intermediate can be metabolized to dichloroacetic acid, or it can react directly with nucleophilic macromolecules to cause cytotoxicity. T C E has been shown to bind covalently to liver microsomal proteins both in vivo and in vitro (Van Duuren and Banerjee, 1976; Bolt and Filser, 1977; Uehleke and Poplawski-Tabarelli, 1977; Allemand et al., 1978) and thus alter microsomal enzyme activity
Fig. 1. Platelet thrombus in lung from a mouse exposed to 10,000 ppm of TCE for 4 hr/day (magnification x 240).
142
GML D. LEWISet al.
Fig. 2. Panel A shows the vacuolization of bronchiolar epithelial cells (indicated by arrows) in lung from a mouse exposed to 10,000ppm of TCE for 4 hr daily (magnification x 3500). Panel B shows a section from a control animal that was not exposed to TCE (magnification x 3500).
(Moslen et al., 1977; Pessayre et al., 1979; Costa et al., 1980). Pretreatment of animals with inducers of the mixed function oxidase system, such as phenobarbital or 3-methylcholanthrene, greatly enhances the toxicity of TCE (Carlson, 1974; Allemand et al., 1978; Pessayre et al., 1979). The binding of TCE and its subsequent hepatotoxicity can be blocked by inhibitors of the mixed function oxidase system, such as SKF-525A or COC12, or by the addition of glutathione (Van Duuren and Banerjee, 1976; Van Duuren, 1977; Allemand et al., 1978). Exposure of humans to TCE usually occurs by
inhalation. However, the pulmonary metabolism of this compound has not been well studied. Dalbey and Bingham (1975) reported that addition of TCE to the air supply of isolated perfused rat or guinea-pig lungs resulted in appearance of trichloroethanol in the blood perfusate and lung homogenates. Formation of this metabolite was enhanced by pretreatment of the animals with phenobarbital. Exposure of rats to ~4C-labeled TCE by inhalation resulted in irreversible binding of radioactivity in the lung, although the total amount bound was less than that bound by liver or kidney (Bolt and Filser, 1977).
Effects of TCE on mouse lungs and livers Our experiments show that inhalation of TCE over a period of several days can affect the enzyme activity of lung microsomes and that prolonged exposure damages bronchiolar cells. These effects occurred without the prior induction of the mixed function oxidase system, and the effect on enzyme activity was the opposite of that observed in liver. Exposure to TCE by either inhalation or i.p. injection increased the activity of hepatic NADPH cytochrome c reductase. Since this effect occurred in animals that were exposed for 4 hr/day and not in those exposed for 1 hr/day, the duration of exposure appears to be an important factor. In contrast, TCE inhalation caused a decrease in lung microsomal NADPH cytochrome c reductase activity. Since the lung would be the first organ affected by inhaled TCE, the loss of enzyme activity may reflect damage to the lung cells. The differences in lung and liver enzyme activities probably reflect a differential pathologic effect on those organs. There were no obvious pathologic changes in livers of mice treated with TCE by either route. Others reported, however, that TCE causes liver damage when it is administered following pretreatment with inducers of the mixed function oxidase system (Moslen et al., 1977; Allemand et al., 1978). Our experiments show that TCE increases hepatic enzyme activity without prior induction and without causing damage to the liver. Chronic inhalation of TCE does appear to damage the lungs. Pathologic changes such as thrombus formation and vacuolization of bronchiolar epithelial cells occurred in the same group of animals that had reduced microsomal NADPH cytochrome c reductase activity. Since the morphologic changes were not seen in lungs of animals treated for only 1 hr/day, the duration of exposure to TCE probably determines the extent of injury. The localization of the mixed function oxidase system in the lungs remains unknown. The Clara cell has been suggested as a site for the metabolism of xenobiotics because of its high content of cytochrome P450 (Boyd, 1977; Serabjit-Singh et al., 1980). Since large numbers of Clara cells are found in the terminal bronchioles (Jeffrey and Reid, 1975; Boyd, 1977), TCE-induced injury in this area, as we described, may cause a decrease in the enzyme activity. TCE can damage cultured endothelial cells, as indicated by the release of lactic dehydrogenase (Lewis et al., 1982). However, since we used microsomes prepared from the entire lung, the changes in NADPH cytochrome c reductase activity cannot be attributed to injury to one particular type of cell. In conclusion, administration of TCE by inhalation to mice increases the activity of the hepatic mixed function oxidase system, as measured by increased NADPH cytochrome c reductase activity. In the same animals, however, TCE inhalation decreases enzyme activity in lung microsomes. Since damage to bronchiolar epithelium was observed only in animals where enzyme activity was decreased, the pulmonary cytotoxicity may account for the change in enzyme activity. Thus, repeated and prolonged exposure to TCE can damage the lungs and potentially can influence the handling of xenobiotics by the lungs.
143 SUMMARY
The effects of trichloroethylene (TCE) on the microsomal mixed function oxidase system were studied in mice. NADPH cytochrome c reductase activity was measured in microsomes isolated from lungs and livers of untreated animals and those that were exposed to TCE. Tissues from control and treated animals were examined by light and electron microscopy for pathologic changes. TCE or 1,1,l-trichloroethane, a closely related compound that is not metabolized, was administered by i.p. injection over a period of 5 days, and hepatic microsomal NADPH cytochrome c reductase activities were measured. As anticipated, 1,1,1-trichloroethane did not influence the enzyme activity, but TCE enhanced it by approximately 50%. Administration of TCE by inhalation had a similar effect, although the duration of exposure appeared to influence the degree of enzyme induction. Animals that inhaled TCE for 4hr/day for 5 days had an increase in hepatic microsomal enzyme activity of approximately 60%. Animals that inhaled this agent for only 1 hr/day, however, did not have increased enzyme activities. TCE inhalation had the opposite effect on the lungs. Animals that inhaled this agent for 4 hr/day had reduced microsomal NADPH cytochrome c reductase compared to animals that inhaled it for only 1 hr/day or to control animals. TCE caused no obvious pathologic changes in the livers of mice treated by either inhalation or injection. However, animals in which the lung microsomal enzyme activities were reduced had pathologic changes, such as the formation of platelet thrombi and bronchiolar epithelial vacuolization. Thus, the decrease in pulmonary microsomal enzyme activity by TCE may reflect direct damage to the lungs. Acknowledgements--This work was supported by a grant from NHLBI (HL 18826). Gail Lewis is the recipient of a training award (GM 07062) from NIH. The authors gratefully acknowledge the assistance of Mrs Anna Siler with preparation of specimensfor light and electron microscopy. REFERENCES
Allemand H., Pessayre D., Descatoire V., Degott C., Feldmann (3. and Benhamou J-P. (1978) Metabolic activation of trichloroethylene into a chemically reactive metabolite toxic to the liver. J. Pharmac. exp. Ther. 204, 714-723. Baerg R. D. and Kimberg D. V. (1970) Centrilobular hepatic necrosis and acute renal failure in "solvent sniffers". Ann. Int. Med. 73, 713-720. Boyd M. R. (1977) Evidence for the Clara cell as a site of cytochrome P450-dependentmixed-function oxidase activity in lung. Nature 269, 713-715. Bolt H. M. and Filser J. G. (1977) Irreversible binding of chlorinated ethylenes to macromolecules. Envir. Hlth Perspect. 21, 107 112. Carlson G. P. (1974) Enhancement of the hepatotoxicity of trichloroethylene by inducers of drug metabolism. Res. commun. Chem. Path. Pharmac. 7, 637-640. Costa A. K., Katz I. D. and Ivanetich K. M. (1980) Trichloroethylene: its interaction with hepatic microsomal cytochrome P450 in vitro. Biochem. Pharmac. 29, 433439. Dalbey W. and Bingham E. (1978) Metabolism of trichloroethylene by the isolated perfused lung. Toxic. appl. Pharmac. 43, 267-277.
144
GAlL D. LEWIS et al.
Garriott J. C. and Petty C. S. (1980) Death from inhalant abuse: toxicological and pathological evaluation of 34 cases. Clin. tox. 16, 305-315. Greim H., Bonse G., Radwan Z., Reichert D. and Henschler D. (1975) Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmac. 24, 2013 2017. Hake C. L., Waggoner T. B., Robertson D. N. and Rowe V. K. (1960) The metabolism of 1,1,1-trichloroethane by the rat. Arch. Envir. Hlth 1, 101-105. Hatbway D. E. (1980) Consideration of the evidence for mechanisms of l,l,2-trichloroethylene metabolism, including new identification of its dichloroacetic acid and trichloroacetic acid metabolites in mice. Cancer Lett. 8, 263-269. Henschler D. (1977) Metabolism and mutagenicity of halogenated olefins--a comparison of structure and activity. Envir. Hlth Perspect. 21, 61-64. Henschler D. (1977) Metabolism of chlorinated alkenes and alkanes as related to toxicity. J. Envir. Path. Toxic. 1, 125-133. Hook G. E. R., Bend J. R., Hoel D., Fouts J. R. and Gram T. E. (1972) Preparation of lung microsomes and a comparison of the distribution of enzymes between subcellular fractions of rabbit lung and liver. J. Pharmac. exp. Ther. 182, 474~490. Ikeda M. and Ohtsuji H. (1972) A comparative study of the excretion of Fujiwara reaction-positive substances in urine of humans and rodents given trichloro- or tetrachloro-derivatives of ethane and ethylene. Br. J. Ind. Med. 29, 99-104. Ikeda M., Miyake Y., Ogata M. and Ohmori S. (1980) Metabolism of trichloroethylene. Biochem. Pharmac. 29, 2983-2992. James W. R. L. (1963) Fatal addiction to trichloroethylene. Br. J. Ind. Med. 20, 47-49. Jeffrey P. K. and Reid L. (1975) New observations of rat airway epithelium: a quantitative and electron microscopic study. J. Anat. 120, 295-320. Karnovsky M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137-138a. Lewis G. D., Reynolds R. C. and Johnson A. R. (1982) Effects of trichloroethylene in mice and human cell
cultures. Fedn Proc. Fedn Am. Socs exp. Biol. 41, 1574. Litt I. F. and Cohen M. I. (1969) "Danger... vapor harmful": spot-remover sniffing. New Engl. J. Med. 281, 543-544. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Masters B. S. S., Williams C. H. Jr and Kamin H. (1967) The preparation and properties of microsomal TPNHcytochrome c reductase from pig liver. Meth. Enzym. 10, 565-573. Moslen M. T., Reynolds E. S. and Szabo S. (1977) Enhancement of the metabolism and hepatotoxicity of trichloroethylene and perchloroethylene. Biochem. Pharmac. 26, 369-375. Moslen M. T., Reynolds E. S., Boor P. J., Bailey K. and Szabo S. (1977) Trichloroethylene-induced deactivation of cytochrome P450 and loss of liver glutathione in vivo. Res. commun. Chem. Path. Pharmac. 16, 109-120. Orth O. S. and Gillespie N. A. (1945) A further study of trichloroethylene anaesthesia. Br. J. Anaesth. 19, 161 173. Pessayre D., Allemand H., Wandscheer J. C., Descatoire V., Artigou J.-Y. and Benhamou J.-P. (1979) Inhibition, activation, destruction, and induction of drugmetabolizing enzymes by trichloroethylene. Toxic. appl. Pharmac. 49, 355-363. Politzer P., Trefonas P., Politzer I. R. and Elfman B. (1981) Molecular properties of the chlorinated ethylenes and their epoxide metabolites. Ann. N.Y. Acad. Sci. 367, 478~492. Serabjit-Singh C. J., Wolf C. R. and Philpot R. M. (1980) Cytochrome P450: localization in rabbit lung. Science 207, 1469-1470. Smith G. F. (1966) Trichloroethylene: a review. Br. J. Ind. Med. 23, 249-262. Uehleke H. and Poplawski-Tabarelli S. (1977) Irreversible binding of 14C-labelled trichloroethylene to mice liver constituents in vivo and in vitro. Arch. Toxic. 37, 289 294. Van Duuren B. L. (1977) Chemical structure, reactivity, and carcinogenicity of halohydrocarbons. Envir. Hlth Perspect. 21, 1223. Van Duuren B. L. and Banerjee S. (1976) Covalent interaction of metabolites of the carcinogen trichloroethylene in rat hepatic microsomes. Cancer Res. 36, 2419 2422.
0306-3623/84 $3.00 + 0.00 Copyright ~. 1984 Pergamon Press Ltd
SOME EFFECTS OF TRICHLOROETHYLENE ON MOUSE LUNGS A N D LIVERS GAlL D. LEwis, ROLLAND C. REYNOLDS and ALICE R. JOHNSON University of Texas Health Science Center, 5323 Harry Hines Boulevard, Dallas TX 75235 (Tel: (214) 688-3111) (Received 17 June 1983) Abstract--1. Repeated administration of trichloroethylene (TCE) to mice by either i.p. injection or by inhalation increased the activity of hepatic microsomal NADPH cytochrome-c reductase. 2. The NADPH cytochrome c reductase activity in microsomes isolated from lungs of animals treated with TCE by inhalation was decreased relative to controls (untreated animals). 3. TCE inhalation was associated with pathologic changes in lungs, but not in livers of the treated animals. The duration of exposure is probably an important factor however, since animals exposed for only I hr per day exhibited neither pathologic changes in the lungs nor an alteration of enzyme activity. 4. These findings indicate that inhalation of TCE, without prior treatment with inducers, can enhance activity of the hepatic mixed function oxidase system. 5. The reduced activity of the pulmonary mixed function oxidase system in animals that inhaled TCE may reflect injury to the lungs.
INTRODUCTION N u m e r o u s investigations have focused on the toxicity, mutagenicity, and carcinogenicity of halogenated and unsubstituted hydrocarbons. The most extensively studied agents include simple aliphatic compounds, such as vinyl chloride, carbon tetrachloride, and chloroform, and aromatic compounds, such as benzene and the polycyclic hydrocarbons, benzo(a)-pyrene and 3-methylcholanthrene. While it is well known that many of these compounds damage the liver, some of them are also inhaled and may damage the lungs as well. Trichloroethylene (TCE) is a widely used industrial and commercial solvent which is structurally similar to vinyl chloride. It is also frequently abused by solvent sniffers (James, 1963; Litt and Cohen, 1969; Garriott and Petty, 1980). The hepatoxicity of T C E is well known and has been studied in a number of animal species (Orth and Gillespie, 1945; Smith, 1966; Baerg and Kimberg, 1970; Carlson, 1974; Allemand et al., 1977). T C E is also mutagenic (Greim et al., 1975; Henschler, 1977) and weakly carcinogenic (Henschler, 1977; Van Duuren, 1977). Administration of T C E by various routes is reported to induce liver microsomal enzymes in animals that have been pretreated with phenobarbital, and this combination of drugs is also thought to increase the toxicity of T C E (Carlson, 1974; Molsen et al., 1977; Allemand et al., 1978). Since exposure to T C E in man usually occurs by inhalation, we studied the effects of this agent on pulmonary cytochrome c reductase in mice exposed by inhalation. For comparison, we also examined this enzyme from livers of mice treated either by inhalation or by i.p. injection. N A D P H cytochrome c reductase activity was measured in microsomal fractions of lung or liver tissue as an index of the activity of the mixed function oxidase system. Tissues were examined by light and electron microscopy for damage caused by TCE. The
object of this study was to determine whether T C E alone (without previous induction) can affect the activity of hepatic or pulmonary mixed function oxidases, and whether inhalation of this agent can injure the lungs directly. MATERIALS AND METHODS
Materials
Trichlorethylene (reagent grade) was obtained from Mallinckrodt, 1,1,1-trichloroethane (reagent grade) from Baker Chemical Co., and NADPH and cytochrome c (horse heart) were obtained from Sigma Chemical Co. Male Swiss Webster mice were purchased from Simonsen Laboratories. Treatment o f animals
Mice with an average weight of 25 g were used for all experiments. Pilot studies indicated that the LDs0 of TCE administered by i.p. injection is 0.65 g/kg and that the LDs0 of l,l,l-trichloroethane is 1.15 g/kg. The doses selected for serial treatment were dictated by the necessity to keep the animals alive during the 5 days required for experiments. Two series of experiments were performed. In the first, 20 mice were divided into control (vehicle-treated), TCEtreated and 1,1,1-trichloroethane-treated groups. The animals were injected intraperitoneally with 0.2 ml of vehicle (25% Tween 80 in saline) or drugs on alternate days for a 5-day period. Each animal received a total of 3 injections. The TCE-treated animals were injected with 0.33 g/kg and the 1,1,1-trichloroethane-treated animals were injected with 0.38 g/kg at each treatment. The trichloroethane group was an additional control, since most of this compound is excreted unchanged into expired air (Hake et al., 1960; Ikeda and Ohtsuji, 1972). Animals in all groups were killed by cervical dislocation 24 hr after the last injection, and the livers were removed for isolation of microsomes as described below. In the second series of experiments, mice were exposed to TCE by inhalation in a closed system. Pyrex bell jars covered tightly with several layers of cotton cloth were used as inhalation chambers. The bottom of each jar was lined with gauze covered by wire mesh. The animals were exposed to 10,000ppm of TCE by saturating the gauze with the 139
GAlL D. LEWm et al.
140
appropriate amount of drug. Eighty-five mice were randomly divided into 3 groups. The treated animals were exposed for either 1 or 4 hr daily, and control animals were placed in the jars for 4 hr per day but not exposed to TCE. All three groups were treated daily for 5 consecutive days. Twenty-four hours after the last exposure, the animals were killed by cervical dislocation, and lungs and livers were removed for isolation of microsomes.
Preparation of microsomes The procedure used for isolation of microsomes from mouse tissues was similar to that of Hook et al. (1972). The tissues were removed immediately after death, washed in cold saline and chilled on ice. The organs were perfused with cold saline to remove blood, minced and homogenized in 3 volumes of 0.25 M sucrose (pH 7.4) in a fitted glass vessel with a motor-driven Teflon pestle. In order to obtain sufficient material for microsome isolation, 2-3 livers from each group were combined, and 5 pairs of lungs were combined. The homogenates were centrigued at 9000g for 20 min. The pellet was discarded, and the supernatants were centrifuged at 100,000 g for 60 rain. The pellet obtained after high speed centrifugation was resuspended in 0.15 M KC1 (pH 7.4), homogenized, and centrifuged again at 100,000g for 60 min. All centrifugation steps were done at 4-'C. The washed pellets were resuspended in 0.25 M sucrose and assayed for cytochrome c reductase activity by the method of Masters et al. (1967). The reduction of cytochrome c was initiated by addition of NADPH. Protein content of each rnicrosomal sample was measured by the method of Lowry (1951).
Microscopy of tissues Livers and lungs of control and TCE-treated animals were examined by light and transmission electron microscopy. Immediately after removal of livers, thin slices were cut and fixed in half-strength modified Karnovsky's fixative (Karnovsky, 1965). Lungs were inflated by infusion of the fixative into the trachea before slices were made. The tissues were post-fixed in 1% osmium tetroxide in phosphate buffer (0.1 M), embedded in araldite, and dehydrated with graded concentrations of acetone. Sections from each organ were examined by light microscopy and transmission electron microscopy.
RESULTS
Enzyme activities A d m i n i s t r a t i o n o f 0.33 g/kg T C E to mice by i.p. injection over a 5-day p e r i o d increased the activity o f hepatic microsomal NADPH cytochrome c reductase c o m p a r e d to c o n t r o l or 1 , 1 , 1 - t r i c h l o r o e t h a n e - t r e a t e d a n i m a l s (Table 1). As expected, there was no increase in e n z y m e activity in m i c r o s o m e s isolated f r o m livers o f mice t r e a t e d with 1,1,1-trichloroethane. E x p o s u r e o f the a n i m a l s to 10,000 r p m T C E by i n h a l a t i o n also i n c r e a s e d the h e p a t i c e n z y m e activity. A n i m a l s that were t r e a t e d for 4 h r / d a y for 5 consecutive days h a d an increase in e n z y m e activity c o m p a r a b l e to t h a t in a n i m a l s t h a t were injected with T C E . The mice t h a t were e x p o s e d for only 1 h r / d a y , h o w e v e r , h a d e n z y m e activities similar to t h o s e in c o n t r o l animals. T h e a v e r a g e liver w e i g h t / b o d y weight ratios were similar in all three g r o u p s o f animals. T h e s e d a t a are given in Table 2. In c o n t r a s t , m i c r o s o m a l N A D P H c y t o c h r o m e c r e d u c t a s e activity in lungs o f a n i m a l s that h a d inhaled T C E was significantly less t h a n t h a t in c o n t r o l p r e p a r a t i o n s . T a b l e 3 s h o w s t h a t the e n z y m e activity in lungs f r o m a n i m a l s e x p o s e d for l h r / d a y was d e c r e a s e d to 77% o f c o n t r o l ( u n t r e a t e d ) p r e p a r a t i o n s , a n d the activity in lungs t h a t were e x p o s e d for 4 h r / d a y d e c r e a s e d to 67% o f control. T h e difference b e t w e e n the 1 a n d 4 hr e x p o s u r e p r e p a r a t i o n s was n o t statistically significant, a n d there was n o difference in the lung w e i g h t / b o d y weight ratios a m o n g the groups.
Acute effects of T C E W h e n the mice were first placed in the c h a m b e r s , the i n h a l a t i o n o f T C E c a u s e d a s h o r t p e r i o d o f excitation, r e s p i r a t o r y s t i m u l a t i o n a n d even c o n vulsions in s o m e o f the animals. E v e n t u a l l y all mice s u c c u m b e d to the a n e s t h e t i c effects o f T C E while in the c h a m b e r . R e c o v e r y was c o m p l e t e w i t h i n an
Table 1. Effect of trichloroethylene on liver microsomal enzyme activity NADPH cytochrome c reductase (nmol/min/mg Percent Group protein) control Control (vehicle) 186.5 - 21.0 100 Trichloroethylene 287.1 + 23.6* 154* 1,1,1-Trichloroethane 177.5 _+9.1 95 Animals in each group were treated with i.p. injections on alternate days for 5 days prior to isolation of microsomes. Data are means +_SEM of 3 4 separate determinations. Asterisk (*) indicates significant difference (P < 0.05) compared to control group by Student's unpaired t-test. Table 2. Effect of trichloroethylene on liver microsomal enzyme activity NADPH cytochrome c reductase Liver/body (nmol/min/g Percent Group weight ratio liver) control Control 5.7 117.9 + 10.8 100 One hour 5.8 137.9 +_ 14.4 117 Four hours 5.8 190.0 _+ 15.0" 161" Mice in each group were exposed to air or TCE (10,000 ppm). Controls (n = 25) were in the chamber 4 hr; TCE-treated animals were exposed for 1 or 4 hr daily for 5 days. Data are means + SEM of 10 separate determinations. Asterisk (*) indicates significant difference from control by Student's unpaired t-test (P < 0.01). One and four hour groups were significantly different (P < 0.05).
Effects of TCE on mouse lungs and livers
141
Table 3. Effect of trichloroethylene of lung microsomal enzyme activity NADPH cytochrome Lung/body c reductase Percent Group weight ratio (nmol/min/g lung) control Control 6.5 2.1 +_0.1 100 One hour 6.9 1.6 + 0.2 77 Four hours 6.6 1.4 _+0.1" 67* Tissue was obtained from the same mice depicted in Table 2. Data are means _ SEM of 4 separate determinations. Asterisk (*) indicates significant difference (P < 0.01) from control group by Student's unpaired t-test.
hour after removal from the T C E atmosphere. The duration of excitatory effects and the anesthesia decreased with subsequent exposure to TCE. Pathologic changes in tissues Light microscopic examination of livers from mice treated with T C E by either i.p. injection or by inhalation showed no obvious pathology, and the morphology was similar to that in control animals. Lungs from animals exposed to T C E by inhalation, however, showed subtle pathologic changes. For example, platelet thrombi were noted in light microscopic sections of lungs from animals exposed to T C E for 4 hr/day (Fig. 1). These were not seen in sections from untreated animals. Additional changes in the lungs of TCE-treated animals were appreciated when sections were examined by transmission electron microscopy. Figure 2 shows vacuolization of bronchiolar epithelial cells in a section from an animal treated with T C E 4 hr daily. These changes were not seen, however, in lungs from animals exposed to T C E for only 1 hr/day or in control animals. DISCUSSION Trichloroethylene (TCE) is a solvent used in many industrial processes, and it is found in a number of commercial products. It was also once used as a general anesthetic agent. Currently, exposure to this
agent occurs via the pulmonary route either inadvertently through solvents or intentionally in abuse situations. The metabolism and acute toxicity of T C E has been well studied. T C E is metabolized by the hepatic mixed function oxidase system to a reactive epoxide intermediate (Henschler, 1977; Van Duuren, 1977; Politzer et al., 1981). This intermediate can rearrange to form chloral, which is then converted by N A D ( H ) containing dehydrogenases to trichloroethanol and trichloroacetic acid prior to excretion (Van Duuren, 1977; Ikeda et al., 1980). The electrophilic trichlorethylene epoxide can react with cellular nucleophiles, such as proteins and D N A to cause additional acute cytotoxic effects (Henschler, 1977; Van Duuren, 1977; Allemand et al., 1978). An alternate route for T C E metabolism in mice was reported by Hathway (1980). He proposed that, due to saturation of the pathway leading to chloral formation, T C E epoxide is rearranged to dichloroacetyl chloride. This intermediate can be metabolized to dichloroacetic acid, or it can react directly with nucleophilic macromolecules to cause cytotoxicity. T C E has been shown to bind covalently to liver microsomal proteins both in vivo and in vitro (Van Duuren and Banerjee, 1976; Bolt and Filser, 1977; Uehleke and Poplawski-Tabarelli, 1977; Allemand et al., 1978) and thus alter microsomal enzyme activity
Fig. 1. Platelet thrombus in lung from a mouse exposed to 10,000 ppm of TCE for 4 hr/day (magnification x 240).
142
GML D. LEWISet al.
Fig. 2. Panel A shows the vacuolization of bronchiolar epithelial cells (indicated by arrows) in lung from a mouse exposed to 10,000ppm of TCE for 4 hr daily (magnification x 3500). Panel B shows a section from a control animal that was not exposed to TCE (magnification x 3500).
(Moslen et al., 1977; Pessayre et al., 1979; Costa et al., 1980). Pretreatment of animals with inducers of the mixed function oxidase system, such as phenobarbital or 3-methylcholanthrene, greatly enhances the toxicity of TCE (Carlson, 1974; Allemand et al., 1978; Pessayre et al., 1979). The binding of TCE and its subsequent hepatotoxicity can be blocked by inhibitors of the mixed function oxidase system, such as SKF-525A or COC12, or by the addition of glutathione (Van Duuren and Banerjee, 1976; Van Duuren, 1977; Allemand et al., 1978). Exposure of humans to TCE usually occurs by
inhalation. However, the pulmonary metabolism of this compound has not been well studied. Dalbey and Bingham (1975) reported that addition of TCE to the air supply of isolated perfused rat or guinea-pig lungs resulted in appearance of trichloroethanol in the blood perfusate and lung homogenates. Formation of this metabolite was enhanced by pretreatment of the animals with phenobarbital. Exposure of rats to ~4C-labeled TCE by inhalation resulted in irreversible binding of radioactivity in the lung, although the total amount bound was less than that bound by liver or kidney (Bolt and Filser, 1977).
Effects of TCE on mouse lungs and livers Our experiments show that inhalation of TCE over a period of several days can affect the enzyme activity of lung microsomes and that prolonged exposure damages bronchiolar cells. These effects occurred without the prior induction of the mixed function oxidase system, and the effect on enzyme activity was the opposite of that observed in liver. Exposure to TCE by either inhalation or i.p. injection increased the activity of hepatic NADPH cytochrome c reductase. Since this effect occurred in animals that were exposed for 4 hr/day and not in those exposed for 1 hr/day, the duration of exposure appears to be an important factor. In contrast, TCE inhalation caused a decrease in lung microsomal NADPH cytochrome c reductase activity. Since the lung would be the first organ affected by inhaled TCE, the loss of enzyme activity may reflect damage to the lung cells. The differences in lung and liver enzyme activities probably reflect a differential pathologic effect on those organs. There were no obvious pathologic changes in livers of mice treated with TCE by either route. Others reported, however, that TCE causes liver damage when it is administered following pretreatment with inducers of the mixed function oxidase system (Moslen et al., 1977; Allemand et al., 1978). Our experiments show that TCE increases hepatic enzyme activity without prior induction and without causing damage to the liver. Chronic inhalation of TCE does appear to damage the lungs. Pathologic changes such as thrombus formation and vacuolization of bronchiolar epithelial cells occurred in the same group of animals that had reduced microsomal NADPH cytochrome c reductase activity. Since the morphologic changes were not seen in lungs of animals treated for only 1 hr/day, the duration of exposure to TCE probably determines the extent of injury. The localization of the mixed function oxidase system in the lungs remains unknown. The Clara cell has been suggested as a site for the metabolism of xenobiotics because of its high content of cytochrome P450 (Boyd, 1977; Serabjit-Singh et al., 1980). Since large numbers of Clara cells are found in the terminal bronchioles (Jeffrey and Reid, 1975; Boyd, 1977), TCE-induced injury in this area, as we described, may cause a decrease in the enzyme activity. TCE can damage cultured endothelial cells, as indicated by the release of lactic dehydrogenase (Lewis et al., 1982). However, since we used microsomes prepared from the entire lung, the changes in NADPH cytochrome c reductase activity cannot be attributed to injury to one particular type of cell. In conclusion, administration of TCE by inhalation to mice increases the activity of the hepatic mixed function oxidase system, as measured by increased NADPH cytochrome c reductase activity. In the same animals, however, TCE inhalation decreases enzyme activity in lung microsomes. Since damage to bronchiolar epithelium was observed only in animals where enzyme activity was decreased, the pulmonary cytotoxicity may account for the change in enzyme activity. Thus, repeated and prolonged exposure to TCE can damage the lungs and potentially can influence the handling of xenobiotics by the lungs.
143 SUMMARY
The effects of trichloroethylene (TCE) on the microsomal mixed function oxidase system were studied in mice. NADPH cytochrome c reductase activity was measured in microsomes isolated from lungs and livers of untreated animals and those that were exposed to TCE. Tissues from control and treated animals were examined by light and electron microscopy for pathologic changes. TCE or 1,1,l-trichloroethane, a closely related compound that is not metabolized, was administered by i.p. injection over a period of 5 days, and hepatic microsomal NADPH cytochrome c reductase activities were measured. As anticipated, 1,1,1-trichloroethane did not influence the enzyme activity, but TCE enhanced it by approximately 50%. Administration of TCE by inhalation had a similar effect, although the duration of exposure appeared to influence the degree of enzyme induction. Animals that inhaled TCE for 4hr/day for 5 days had an increase in hepatic microsomal enzyme activity of approximately 60%. Animals that inhaled this agent for only 1 hr/day, however, did not have increased enzyme activities. TCE inhalation had the opposite effect on the lungs. Animals that inhaled this agent for 4 hr/day had reduced microsomal NADPH cytochrome c reductase compared to animals that inhaled it for only 1 hr/day or to control animals. TCE caused no obvious pathologic changes in the livers of mice treated by either inhalation or injection. However, animals in which the lung microsomal enzyme activities were reduced had pathologic changes, such as the formation of platelet thrombi and bronchiolar epithelial vacuolization. Thus, the decrease in pulmonary microsomal enzyme activity by TCE may reflect direct damage to the lungs. Acknowledgements--This work was supported by a grant from NHLBI (HL 18826). Gail Lewis is the recipient of a training award (GM 07062) from NIH. The authors gratefully acknowledge the assistance of Mrs Anna Siler with preparation of specimensfor light and electron microscopy. REFERENCES
Allemand H., Pessayre D., Descatoire V., Degott C., Feldmann (3. and Benhamou J-P. (1978) Metabolic activation of trichloroethylene into a chemically reactive metabolite toxic to the liver. J. Pharmac. exp. Ther. 204, 714-723. Baerg R. D. and Kimberg D. V. (1970) Centrilobular hepatic necrosis and acute renal failure in "solvent sniffers". Ann. Int. Med. 73, 713-720. Boyd M. R. (1977) Evidence for the Clara cell as a site of cytochrome P450-dependentmixed-function oxidase activity in lung. Nature 269, 713-715. Bolt H. M. and Filser J. G. (1977) Irreversible binding of chlorinated ethylenes to macromolecules. Envir. Hlth Perspect. 21, 107 112. Carlson G. P. (1974) Enhancement of the hepatotoxicity of trichloroethylene by inducers of drug metabolism. Res. commun. Chem. Path. Pharmac. 7, 637-640. Costa A. K., Katz I. D. and Ivanetich K. M. (1980) Trichloroethylene: its interaction with hepatic microsomal cytochrome P450 in vitro. Biochem. Pharmac. 29, 433439. Dalbey W. and Bingham E. (1978) Metabolism of trichloroethylene by the isolated perfused lung. Toxic. appl. Pharmac. 43, 267-277.
144
GAlL D. LEWIS et al.
Garriott J. C. and Petty C. S. (1980) Death from inhalant abuse: toxicological and pathological evaluation of 34 cases. Clin. tox. 16, 305-315. Greim H., Bonse G., Radwan Z., Reichert D. and Henschler D. (1975) Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmac. 24, 2013 2017. Hake C. L., Waggoner T. B., Robertson D. N. and Rowe V. K. (1960) The metabolism of 1,1,1-trichloroethane by the rat. Arch. Envir. Hlth 1, 101-105. Hatbway D. E. (1980) Consideration of the evidence for mechanisms of l,l,2-trichloroethylene metabolism, including new identification of its dichloroacetic acid and trichloroacetic acid metabolites in mice. Cancer Lett. 8, 263-269. Henschler D. (1977) Metabolism and mutagenicity of halogenated olefins--a comparison of structure and activity. Envir. Hlth Perspect. 21, 61-64. Henschler D. (1977) Metabolism of chlorinated alkenes and alkanes as related to toxicity. J. Envir. Path. Toxic. 1, 125-133. Hook G. E. R., Bend J. R., Hoel D., Fouts J. R. and Gram T. E. (1972) Preparation of lung microsomes and a comparison of the distribution of enzymes between subcellular fractions of rabbit lung and liver. J. Pharmac. exp. Ther. 182, 474~490. Ikeda M. and Ohtsuji H. (1972) A comparative study of the excretion of Fujiwara reaction-positive substances in urine of humans and rodents given trichloro- or tetrachloro-derivatives of ethane and ethylene. Br. J. Ind. Med. 29, 99-104. Ikeda M., Miyake Y., Ogata M. and Ohmori S. (1980) Metabolism of trichloroethylene. Biochem. Pharmac. 29, 2983-2992. James W. R. L. (1963) Fatal addiction to trichloroethylene. Br. J. Ind. Med. 20, 47-49. Jeffrey P. K. and Reid L. (1975) New observations of rat airway epithelium: a quantitative and electron microscopic study. J. Anat. 120, 295-320. Karnovsky M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137-138a. Lewis G. D., Reynolds R. C. and Johnson A. R. (1982) Effects of trichloroethylene in mice and human cell
cultures. Fedn Proc. Fedn Am. Socs exp. Biol. 41, 1574. Litt I. F. and Cohen M. I. (1969) "Danger... vapor harmful": spot-remover sniffing. New Engl. J. Med. 281, 543-544. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Masters B. S. S., Williams C. H. Jr and Kamin H. (1967) The preparation and properties of microsomal TPNHcytochrome c reductase from pig liver. Meth. Enzym. 10, 565-573. Moslen M. T., Reynolds E. S. and Szabo S. (1977) Enhancement of the metabolism and hepatotoxicity of trichloroethylene and perchloroethylene. Biochem. Pharmac. 26, 369-375. Moslen M. T., Reynolds E. S., Boor P. J., Bailey K. and Szabo S. (1977) Trichloroethylene-induced deactivation of cytochrome P450 and loss of liver glutathione in vivo. Res. commun. Chem. Path. Pharmac. 16, 109-120. Orth O. S. and Gillespie N. A. (1945) A further study of trichloroethylene anaesthesia. Br. J. Anaesth. 19, 161 173. Pessayre D., Allemand H., Wandscheer J. C., Descatoire V., Artigou J.-Y. and Benhamou J.-P. (1979) Inhibition, activation, destruction, and induction of drugmetabolizing enzymes by trichloroethylene. Toxic. appl. Pharmac. 49, 355-363. Politzer P., Trefonas P., Politzer I. R. and Elfman B. (1981) Molecular properties of the chlorinated ethylenes and their epoxide metabolites. Ann. N.Y. Acad. Sci. 367, 478~492. Serabjit-Singh C. J., Wolf C. R. and Philpot R. M. (1980) Cytochrome P450: localization in rabbit lung. Science 207, 1469-1470. Smith G. F. (1966) Trichloroethylene: a review. Br. J. Ind. Med. 23, 249-262. Uehleke H. and Poplawski-Tabarelli S. (1977) Irreversible binding of 14C-labelled trichloroethylene to mice liver constituents in vivo and in vitro. Arch. Toxic. 37, 289 294. Van Duuren B. L. (1977) Chemical structure, reactivity, and carcinogenicity of halohydrocarbons. Envir. Hlth Perspect. 21, 1223. Van Duuren B. L. and Banerjee S. (1976) Covalent interaction of metabolites of the carcinogen trichloroethylene in rat hepatic microsomes. Cancer Res. 36, 2419 2422.