Introduction

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1,1-Dichloroethylene (DCE), a monomeric intermediate used in the manufacture of plastic products, elicits a pneumotoxic lesion that selectively targets Clara cells of the bronchiolar epithelium (Forkert and Reynolds, 1982). Previous studies have confirmed that the mechanism responsible for the lung damage is mediated by cytochrome P450-dependent metabolism of DCE to reactive intermediates (Okine and Gram, 1986; Forkert et al., 1987). The primary metabolites formed from DCE in rat liver microsomal incubations have been identified as 2,2-dichloroacetaldehyde, DCE epoxide, and 2-chloroacetyl chloride (Liebler and Guengerich, 1983; Costa and Ivanetich, 1984; Liebler et al., 1985, 1988). In more recent studies, it was found that the major metabolites formed in liver and lung microsomal incubations containing GSH were the conjugates 2-(S-glutathionyl)acetyl glutathione [B] and 2-S-glutathionyl acetate [C] (Dowsley et al., 1995, 1996) (Fig. 1). These metabolic products are believed to be formed from conjugation of GSH with the DCE epoxide (Dowsley et al., 1995). Acetal, the hydrate of 2,2-dichloroacetaldehyde, was also produced in the microsomal incubations. However, S-(2,2-dichloro-1-hydroxy)ethyl glutathione [A], the GSH-conjugated product of 2,2-dichloroacetaldehyde, was not detected. 2-Chloroacetic acid and S-(2-chloroacetyl)glutathione [D], the hydrolysis and GSH-conjugated products of 2-chloroacetyl chloride, respectively, were detected but were found at minimal amounts in the microsomal incubations (Dowsley et al., 1995, 1996). These results indicated that the DCE epoxide is the major metabolite formed in vitro. In recent studies, we demonstrated that the DCE epoxide-derived conjugate [C] was the major metabolite generated in vivo, whereas conjugate [B] was formed at minimal amounts (Forkert, 1999). These results indicated that the epoxide is the most plausible candidate for mediating the toxic effects of DCE.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Scheme of the proposed pathway of DCE metabolism. GSH conjugates are identified as follows: [A], S-(2,2-dichloro-1-hydroxy)ethyl glutathione; [B], 2-(S-glutathionyl)acetyl glutathione; [C], 2-S-glutathionyl acetate; [D], S-(2-chloroacetyl)glutathione.

Investigations of the isozyme-selective metabolism of DCE have produced results indicating that CYP2E1 is the major P450 enzyme involved in the oxidation of DCE to reactive intermediates including the DCE epoxide and 2,2-dichloroacetaldehyde. These metabolites, which were formed in lung microsomal incubations, were significantly inhibited when the microsomes were preincubated with inhibitory CYP2E1 monoclonal antibodies (Dowsley et al., 1996). Importantly, pretreatment of mice with diallyl sulfone (DASO₂) significantly inhibited the CYP2E1 enzyme, decreased the generation of 2,2-dichloroacetaldehyde and the epoxide, and protected from DCE-induced bronchiolar Clara cell cytotoxicity (Forkert et al., 1996b). Moreover, other studies have identified differing quantities of CYP2E1 in the lungs of female and male mice (Forkert et al., 1996a). Levels of CYP2E1-dependent p-nitrophenol (PNP) hydroxylase activity, were more than 50% higher in female than in male mice, and these amounts correlated with formation of DCE epoxide that was about 2-fold higher in the former than in the latter. These findings are consistent with involvement of CYP2E1 in the bioactivation of DCE.

The selective Clara cell damage evoked by DCE and the high level of covalent binding of DCE metabolites to the Clara cells suggested that bioactivation occurs in situ within this cell type (Forkert et al., 1990). This assertion is consistent with findings from previous studies showing that the CYP2E1 protein and mRNA are localized preferentially within the Clara cells (Forkert, 1995). Significantly, results from immunohistochemical studies indicated that the epoxide is formed and localized within the Clara cells (Forkert, 1999). Furthermore, pretreatment of mice with DASO₂ inhibited formation of the epoxide, and markedly decreased the level of immunoreactivity in the bronchiolar epithelium and Clara cells. These findings supported the premise that the cell-selective cytotoxicity ensuing after DCE exposure is mediated by in situ metabolism of DCE to reactive intermediates including the epoxide within the Clara cells. These data further supported the contention that the DCE epoxide is the reactive species responsible for the Clara cell damage.

In preliminary studies, we have serendipitously identified strain-related differences in CYP2E1 levels in A/J and C57BL/6 mice. Here we have tested the hypothesis that DCE metabolism differs in these strains of mice, leading to differences in the severity of DCE-induced lung cytotoxicity. These studies were also performed in CD-1 mice, an outbred strain that is more economical and that is commonly used in experimental studies. To identify and to evaluate these differences in A/J, CD-1, and C57BL/6 mice, we have determined lung CYP2E1-dependent PNP hydroxylase activity, measured covalent binding of [¹⁴C]DCE to lung proteins, and estimated formation in vivo of conjugate [C]. We have also determined levels of GSH in the lungs of the three strains of mice under control conditions and after treatment with DCE. In addition, we have determined the lung distribution of formation of the DCE epoxide and evaluated the histopathological alterations ensuing from DCE exposure.

	Materials and Methods

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Chemicals and Reagents. Chemicals and reagents were obtained from suppliers as follows: 1,1-dichloroethylene (DCE) (>99% purity), phosphoric acid (85%, v/v), and GSH (Aldrich Chemical Co., Montreal, Quebec, Canada); Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA); bovine serum albumin (BSA), glucose 6-phosphate, glucose-6-phosphate dehydrogenase, p-nitrophenol, 4-nitrocatechol, glutaraldehyde (50% aqueous), paraformaldehyde, hydrogen peroxide (30%, v/v), 3,3'-diaminobenzidine tetrahydrochloride, -glutamyl glutamate, NADP⁺, and NADPH (Sigma Chemical Co., St. Louis, MO); [¹⁴C]DCE (99% pure by gas liquid chromatography, specific activity 11.3 nCi/nmol, (Amersham Pharmacia Biotech, Arlington Heights, IL); Universal scintillation fluid (ICN Chemical Co., Costa Mesa, CA); Spectrapor-3 dialysis tubing, 3500 molecular weight cut-off (Fisher Scientific, Nepean, Ontario, Canada); sodium pentobarbital (Somnotol; MTC Pharmaceuticals, Hamilton, Ontario, Canada); biotinylated goat anti-rabbit IgG, avidin-biotin blocking reagent (Vector Laboratories, Inc., Burlingame, CA); streptavidin-conjugated to horseradish peroxidase (Zymed Laboratories, San Francisco, CA). The DCE epoxide-derived GSH conjugate [C] used as a standard for metabolite identification was synthesized as described (Dowsley et al., 1995). The conjugate glycine-glutaraldehyde-BSA that is used as a blocking reagent in immunohistochemical studies was synthesized as previously described (Forkert et al., 1997). All other chemicals were of reagent grade and were purchased from standard commercial suppliers.

Animal Treatment. Female A/J mice of 20 to 25 g body weight were purchased from Jackson Laboratories (Bar Harbor, ME). Female C57BL/6 and CD-1 mice, also of 20 to 25 g body weight, were purchased from Charles River Canada (St. Constant, Quebec, Canada). Mice were acclimated to laboratory conditions for a period of no less than 7 days and were maintained on a 12-h light/dark cycle. Access to food (Mouse Diet 5015; PMI Nutrition International, Inc., Brentwood, MO) and drinking water was ad libitum.

Preparation of Microsomes. Groups of mice (n = 30) were sacrificed by cervical dislocation; lungs were pooled and homogenized in 4 volumes of cold phosphate-buffered KCl (100 mM K₂HPO₄, 1.15% KCl, 1.15 mM EDTA, pH 7.4). Microsomes were prepared by differential centrifugation as described previously (Forkert, 1995). Microsomal pellets were resuspended in 1 ml of the same buffer, and aliquots were frozen in liquid nitrogen and stored at 70°C. Protein concentrations were determined by the Bradford method (1976) using BSA as the standard.

p-Nitrophenol Hydroxylase Activity. PNP hydroxylase activity was used as a catalytic marker for CYP2E1-dependent activity. Levels of hydroxylase activity were determined by the method of Koop (1986) as described (Forkert et al., 1996b). Reaction mixtures in a total volume of 1 ml contained 100 mM K₂HPO₄ buffer, pH 6.8, 1.5 mM EDTA, 1.0 mM ascorbic acid, 1.5 mg of microsomal protein and an NADPH-generating system (7.5 mM glucose 6-phosphate, 5.0 mM magnesium chloride, 2 U of glucose-6-phosphate dehydrogenase, and 0.4 mM NADP⁺). The reaction mixtures were preincubated for 3 min at 37°C after which 100 mM PNP in dimethyl sulfoxide (2.0 µl) was added, and the reaction continued for 10 min in a shaking water bath. Following termination of the reaction by cooling the samples on ice, proteins were precipitated by addition of perchloric acid (PCA; 70%, 25 µl). Following centrifugation, NaOH (50 µl, 0.9 M) was added to an aliquot of the supernatant, and formation of 4-nitrocatechol was determined spectrally at 546 nm. Levels of 4-nitrocatechol generated were estimated by reference to a standard calibration curve relating absorbance to known amounts of 4-nitrocatechol.

Covalent Binding of [¹⁴C]DCE. Groups of A/J, CD-1, and C57BL/6 mice (n = 5 for each strain) were treated with [¹⁴C]DCE (20 µCi/kg, 125 mg/kg, i.p.) and were sacrificed 1 h after treatment. Lungs were frozen in liquid nitrogen until processed. Covalent binding of DCE metabolites to lung proteins was determined by equilibrium dialysis as described previously (Forkert, 1999). Tissues were homogenized in 5 volumes of cold 0.01 M sodium phosphate buffer, pH 7.0, containing 2% SDS. After addition of 5 volumes of 4% SDS, the samples were boiled for 15 min. Aliquots (1 ml) were transferred to dialysis tubing (molecular weight cut-off 3500 Da) and dialyzed overnight, with stirring, against 500 ml of 0.01 M sodium phosphate buffer, pH 7.0, containing 0.1% SDS. Subsequently, aliquots (250 µl) of the dialysate were reacted overnight with 1 ml of 1.0 N NaOH to solubilize the proteins. Levels of radioactivity in the dialysate and buffer were determined. The difference in the amounts of radioactivity in the dialysate and buffer was regarded as the quantity of covalently bound DCE in the sample. Protein concentrations were determined by the method of Lowry et al. (1951).

Formation of DCE Epoxide Formation of DCE epoxide was determined in lung cytosol 1 h after treatment with [¹⁴C]DCE (40 µCi/kg, 125 mg/kg, i.p.). Lungs from 10 mice were pooled and homogenized in cold phosphate-buffered KCl, pH 7.4, in a volume of 1 ml per gram of lung tissue (n = 3 for A/J, CD-1, and C57BL/6 mice). Lung cytosolic fractions were isolated according to procedures used in our previous studies (Forkert, 1999). Aliquots of cytosol were frozen in liquid nitrogen and stored at 70°C until metabolite determination. Protein concentrations were determined by the method of Bradford (1976). Proteins in the samples (250 µl) were precipitated with 70% (v/v) PCA and removed by centrifugation. The supernatant (100 µl) was subjected to reversed-phase HPLC analysis using a C₁₈ column (5 µm, 4.6 × 250 mm, Microsorb-MV, Rainin Instruments Co., Inc., Woburn, MA). The mobile phase (0.2% phosphoric acid, pH 2.4) was set at 1 ml/min isocratic flow and monitored at 200 nm. Fractions of the column effluent (0.25 ml) were collected, and radioactivity was determined by liquid scintillation spectroscopy. Metabolites were identified by retention times of the synthesized standards, and levels were estimated by summing the radioactivity associated with each peak and converting the data to nanomolar amounts, using the specific activity of [¹⁴C]DCE.

Measurement of Lung Glutathione Content Lung GSH levels were determined in mice treated with DCE (125 mg/kg DCE, i.p.), using procedures described previously (Fariss and Reed, 1987). Mice were sacrificed 1 h after DCE treatment. Lung tissue (50-100 mg) was pulverized in liquid nitrogen and then mixed with 1 ml of 10% (v/v) PCA containing 1 mM bathophenanthroline disulfonic acid. PCA-insoluble material was removed by centrifugation. Aliquots of the supernatant (0.5 ml) were combined with 50 µl of -glutamyl glutamate (1.5 mM in PCA, 0.3%, v/v), which was used as the internal standard. Iodoacetic acid (100 mM in 0.2 mM m-cresol purple, 50 µl) was then added, and the pH was adjusted to 8 or 9 with 0.48 ml of a solution containing 2 M KOH and 2.4 M KHCO₃. Following a 10-min incubation in the dark, 1 ml of Sanger's reagent (1-fluoro-2,4-dinitrobenzene, 1%, v/v, in ethanol) was added, and the mixture was stored overnight at 4°C. After centrifugation, 100 µl of the supernatant was subjected to HPLC analysis using an aminopropyl silica ion-exchange column (5 µm, 4 × 250 mm, SGE International, PTY Ltd., Ringwood, Australia). Mobile phase A (80% methanol in H₂O) was maintained at a flow rate of 0.75 ml/min. Increasing concentrations of mobile phase B (0.5 M sodium acetate in 64% methanol) was added to elute the chromophore derivative. Following injection of the derivatized sample (100 µl), the gradient of the mobile phase was adjusted from 100% to 5% mobile phase A (0 to 95% mobile phase B) over 10 min. The concentrations of the derivatized samples of GSH and -glutamyl glutamate were monitored and estimated at 360 nm.

Immunohistochemical Detection and Localization of Conjugate [C] In the immunohistochemical studies, we have applied a polyclonal antibody raised against a hapten consisting of the chemically synthesized conjugate [C] cross-linked by glutaraldehyde to the carrier protein BSA (Forkert et al., 1997). Previous studies have established that this antibody recognizes [C] as well as sites of binding of the epoxide to cysteine residues of proteins (Forkert, 1999). For the sake of convenience and ease of reporting, we have designated herein the proteins detected by this antibody as conjugate [C], but with the assumption that proteins containing cysteine residues are also detected. Immunohistochemical studies for detection and localization of conjugate [C] were performed in lung tissues from all three strains of mice treated with DCE (125 mg/kg, i.p.) or the vehicle. The immunohistochemical experiments were carried out as described in our previous studies (Forkert, 1999). Briefly, tissues were fixed with 4% paraformaldehyde containing 2% glutaraldehyde in 0.2 M sodium cacodylate, pH 7.3. Immunohistochemical localization of conjugate [C] was performed in paraffin-embedded tissue sections using the avidin-biotin complex technique as described (Forkert, 1999). The distribution of [C] was visualized by development in 3,3'-diaminobenzidine (0.05% in 0.01% hydrogen peroxide in PBS). Tissue sections were then dehydrated, cleared, and mounted.

Histopathology. Lung cytotoxicity in A/J, CD-1, and C57BL/6 mice was assessed at 24 h following DCE treatment (50, 75, 125, and 175 mg/kg, i.p.). Lung tissue was prepared for histopathologic evaluation as previously described (Forkert, 1995), with minor modifications. Lungs were fixed by intratracheal instillation and vascular perfusion through the left ventricle with 4% paraformaldehyde in 0.1 M Sorensen's phosphate buffer (12.0 mM NaH₂PO₄, 69.0 mM Na₂HPO₄), pH 7.4. Tissues were processed and embedded in paraffin, using standard procedures. Lung sections (5 µm) were stained with hematoxylin and eosin.

Statistical Analysis Data are expressed as mean ± S.D. Statistical analysis was performed by one-way or two-way analysis of variance followed by the Tukey test to identify significant differences between experimental groups (p < 0.05).

	Results

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Levels of Lung CYP2E1. Hydroxylation of PNP was used as an index of the catalytic activity of the CYP2E1 enzyme in the lungs of A/J, CD-1, and C57BL/6 mice. Our results showed that hydroxylase activity was highest in A/J mice, intermediate in CD-1 mice, and was lowest in C57BL/6 mice (Fig. 2). The levels in the lungs of A/J mice were about 20 and 40% higher than those in the lungs of CD-1 and C57BL/6 mice, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Covalent binding of [14C]DCE and PNP hydroxylase activity in the lungs of A/J, CD-1, and C57BL/6 mice. Binding levels to lung proteins were determined in homogenates at 1 h after treatment of mice with [14C]DCE (20 µCi, 125 mg/kg, i.p.). Data are expressed as mean ± S.D. (n = 5 mice of each strain). a, significantly different from binding levels in lungs of CD-1 and C57BL/6 mice (p < 0.001); b, significantly different from levels of hydroxylase activity in C57BL/6 mice (p < 0.001); c, significantly different from levels of hydroxylase activity in lungs of CD-1 and C57BL/6 mice (p < 0.001).

Covalent Binding of [¹⁴C]DCE to Lung Proteins. Covalent binding of [¹⁴C]DCE to proteins in lung homogenates was detected in A/J, CD-1, and C57BL/6 mice. Levels of DCE binding to lung proteins in A/J mice were significantly higher than those in CD-1 and C57BL/6 mice (Fig. 2). However, the difference in binding levels between CD-1 and C57BL/6 mice did not achieve statistical significance. Binding in A/J mice was estimated to be about 25 and 35% higher than levels detected in CD-1 and C57BL/6 mice, respectively. Regression analysis of the relationship between PNP hydroxylase activity and covalent binding of [¹⁴C]DCE in the lungs of the three strains of mice showed a highly positive correlation between these two parameters (r² = 0.9555) such that increased hydroxylase activity coincided with enhanced binding levels (Fig. 3). Hence, binding levels were highest in A/J mice, occurred at intermediate levels in CD-1 mice, and were found at lowest levels in C57BL/6 mice.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Regression analysis of the relationship between PNP hydroxylase activity and covalent binding of [14C]DCE in the lungs of A/J, CD-1, and C57BL/6 mice. Data are derived from those in Fig. 2.

Formation of Conjugate [C]. Representative radiochromatograms of conjugate [C] detected in lung cytosol isolated from the lungs of the three strains of mice are depicted in Fig. 4. The peak for [C] eluted at 7.5 min on the column and was similar to the retention time detected for the synthesized standard (not shown). A smaller peak eluted at 2 to 4 min, and this peak has been identified previously as containing glycolic acid and formaldehyde, both of which are believed to be degradation products of DCE epoxide (Dowsley et al., 1995). The amounts of conjugate [C] detected in cytosolic fractions isolated from the lungs of the three strains of mice are summarized in Table 1. Levels in cytosolic samples from CD-1 and A/J mice were comparable, and both were significantly higher than those from C57BL/6 mice. The level in C57BL/6 mice comprised only about 30% of the amounts detected in CD-1 and A/J mice.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Chromatograms of DCE metabolites detected in cytosol isolated from the lungs of A/J (a), CD-1 (b), and C57BL/6 (c) mice. Mice were treated with [14C]DCE (40 µCi, 125 mg/kg, i.p.), and cytosolic fractions were prepared 1 h after treatment. Cytosolic proteins were precipitated with 70% PCA followed by centrifugation, and 100 µl of the supernatant was subjected to reversed-phase HPLC analysis. Aliquots (250 µl) of the column effluent were collected, and levels of radioactivity were determined. Data are expressed as mean ± S.D. [C], 2-S-glutathionyl acetate.

Glutathione Content. The GSH contents in the lungs of A/J, CD-1, and C57BL/6 mice were determined to assess whether GSH levels contributed to potential differences in DCE metabolism in the three murine strains. Levels of GSH were similar in the lungs of all three strains of untreated mice (Fig. 5). Treatment of mice with DCE produced significant decreases in GSH levels, and these were comparable in all murine strains tested, with decreases ranging from 40 to 60% of control levels. Hence, no strain-related differences in GSH levels were observed in either untreated or DCE-treated mice.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Levels of GSH in the lungs of control and DCE-treated A/J, CD-1, and C57BL/6 mice. Mice were treated with DCE (125 mg/kg, i.p.), and GSH contents were determined 1 h after treatment. Data are expressed as mean ± S.D. and were analyzed by two-way analysis of variance and the Tukey test. *, significantly different from controls (p < 0.001).

Detection and Localization of Conjugate [C] in Lung Tissue. Studies using immunohistochemical procedures were performed to detect and to localize conjugate [C] in lung tissues from the three strains of mice. Immunohistochemical experiments revealed no specific staining in the lungs of all control untreated mice (Fig. 6a). In tissue sections from the lungs of DCE-treated mice, staining was found mainly in the bronchiolar epithelium and was localized in the Clara cells. The amounts of immunohistochemical staining for conjugate [C] varied in the lungs of C57BL/6, CD-1 and A/J mice. Staining was highest in the bronchiolar epithelium of lungs from A/J mice and was concentrated in the apical cytoplasms of the Clara cells (Fig. 6c). The levels of staining in the bronchiolar epithelium of CD-1 mice were slightly lower than seen in the lungs of A/J mice (Fig. 6b). However, staining in the lungs of C57BL/6 mice was low and confined to the distal apices of the Clara cells (Fig. 6d).

View larger version (109K): [in this window] [in a new window]   Fig. 6.   Localization and distribution of conjugate [C] and protein adducts in the lungs of A/J, CD-1, and C57BL/6 mice. Mice were untreated or treated with DCE (125 mg/kg), and were sacrificed 1 h after treatment. Immunohistochemical staining was performed using the avidin-biotin complex procedure and a rabbit polyclonal antibody that recognizes conjugate [C] as well as proteins containing cysteine residues. Immunohistochemical staining is localized predominantly in the Clara cells (arrows) and is depicted in representative lung sections from untreated A/J mice (a), and DCE-treated CD-1 (b), A/J (c), and C57BL/6 mice (d). Scale bar = 10 µm.

Histopathology. Histopathologic observations revealed normal morphology in the lungs of all untreated mice, with the usual distribution of ciliated and nonciliated Clara cells (Fig. 7a). In the experimental groups, mice were administered 50, 75, 125, or 175 mg/kg of DCE. Vacuolated Clara cells were apparent 24 h after treatment of A/J and CD-1 mice with 50 mg/kg DCE, whereas this vacuolization was manifested in only the occasional Clara cell in C57BL/6 mice. Vacuolization of Clara cells was more severe after treatment of A/J (Fig. 7c) and CD-1 (Fig. 7b) mice with 75 mg/kg DCE and was more severe than seen in the lungs of C57BL/6 (Fig. 7d) mice. Treatment with 125 and 175 mg/kg produced denudation of airway epithelium in A/J and CD-1 mice, and in many cases, the bronchioles were devoid of an epithelial lining. Treatment of C57BL/6 mice with 125 mg/kg elicited foci of denuded airway epithelium, and an increase of the dose to 175 mg/kg produced marked exfoliation of epithelial cells. Hence, bronchiolar damage was similar in the lungs of A/J and CD-1 mice and was more severe than in the lungs of C57BL/6 mice. Differences in the histopathologic effects elicited by DCE in the three strains of mice were more evident at lower than higher chemical doses.

View larger version (156K): [in this window] [in a new window]   Fig. 7.   Pulmonary bronchioles in the lungs of untreated A/J (a) and DCE-treated CD-1 (b), A/J (c), and C57BL/6 (d) mice. Mice were treated with the vehicle or with DCE (75 mg/kg) and sacrificed 24 h later. Lungs were fixed in 4% paraformaldehyde in 0.1 M Sorensen's phosphate buffer, pH 7.4 and embedded in paraffin. Lung sections were stained with hematoxylin and eosin. Scale bar = 10 µm.

	Discussion

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Our previous studies have produced data establishing a major role for CYP2E1 in DCE metabolism (Lee and Forkert, 1995; Dowsley et al., 1996; Forkert, 1999). Here, we have investigated strain-related differences in CYP2E1 levels in the lungs of A/J, CD-1, and C57BL/6 mice. Our results showed that lung CYP2E1 was present at significantly higher levels in A/J than in either CD-1 or C57BL/6 mice (Fig. 2). In view of the central role of CYP2E1 in DCE metabolism, we reasoned that DCE metabolism is likely to be augmented in A/J mice and diminished in C57BL/6 mice. Our findings showed that covalent binding of [¹⁴C]DCE, which represents in part an indirect index of metabolite formation, was significantly higher in A/J mice than in either CD-1 or C57BL/6 mice (Fig. 2). Regression analysis revealed a highly positive correlation between PNP hydroxylase activity and binding levels in the three strains of mice (Fig. 3), suggesting that the extents of DCE metabolism are linked to levels of lung CYP2E1 available for bioactivation.

Previous studies have confirmed that the metabolites formed from DCE metabolism in vitro and in vivo are the secondary metabolites [B] and [C] (Dowsley et al., 1996; Forkert, 1999). As expected, conjugates [B] and [C] were formed at markedly lower levels under in vivo than in vitro conditions. In this in vivo study, we have found that conjugate [C] was the major metabolite detected in lung cytosol from mice treated with DCE, whereas [B] was formed at negligible levels. These results are in agreement with those reported in our previous in vivo studies (Forkert, 1999), which revealed that conjugate [C] was the major metabolite found in lung cytosol from DCE-treated mice (Fig. 4). Our studies also demonstrated that levels of [C] detected in lung cytosol were significantly higher in A/J and CD-1 mice than in C57BL/6 mice (Fig. 4 and Table 1). These findings indicated that the DCE epoxide is generated to a significantly greater extent in the lungs of A/J and CD-1 mice than in C57BL/6 mice. These discrepancies are likely not due to different magnitudes of conjugation of the epoxide to GSH. Levels of lung GSH under control conditions were similar in all the three strains of mice (Fig. 5). Moreover, treatment with DCE produced significant decreases in GSH levels that were comparable in all three strains of mice. Hence, the quantities of conjugate [C] detected in lung cytosol from the DCE-treated mice most likely represented the amounts of DCE epoxide generated and were not due to differing rates of GSH conjugation. However, the possibility exists that the extent of generation of the GSH conjugates may be influenced also by potential differing rates of DCE distribution to the lungs of the three strains of mice.

Selective damage of Clara cells is induced by exposure to a broad spectrum of chemicals, including trichloroethylene (Forkert et al., 1985), naphthalene (Mahvi et al., 1977), carbon tetrachloride (Boyd et al., 1980), 4-ipomeanol (Boyd, 1977), bromobenzene (Forkert, 1985) and DCE (Forkert and Reynolds, 1982). It has been postulated that this cell-specific lesion is mediated by reactive intermediates produced in situ within this cell type. This concept emanated from studies showing that the cell-specific lesions are associated with preferential covalent binding of metabolites and localization of high concentrations of P450 enzymes within the Clara cells (Boyd, 1977; Serabjit-Singh, 1980; Forkert et al., 1990). Relevant in the context of DCE metabolism are results from previous lung cell isolation studies showing that binding of [¹⁴C]DCE is 4-fold higher in cell fractions enriched in Clara cells than in fractions enriched in alveolar type II cells or in mixed cells from whole lung (Forkert et al., 1990). Also pertinent is the finding that CYP2E1 resides preferentially within the Clara cells (Forkert, 1995). In more recent immunohistochemical studies, conjugate [C] was localized predominantly within the Clara cells and was markedly diminished in lung sections from mice pretreated with DASO₂ to inhibit CYP2E1 (Forkert, 1999). In this investigation, the immunohistochemical experiments produced results confirming preferential staining of the Clara cells in DCE-treated mice (Fig. 6). This staining was highest in lung sections from A/J mice, and was lowest in those from C57BL/6 mice. Intermediate amounts of staining were found in bronchioles from CD-1 mice and were consistent with levels detected in this murine strain in previous studies (Forkert, 1999). These results suggested that the DCE epoxide is formed and conjugated within the Clara cells. It should be reiterated that, since our antibody also detects the DCE epoxide bound to cysteine-containing proteins, the amounts of immunohistochemical staining seen in the Clara cells should represent signals for both these proteins and [C]. Importantly, the findings indicated that the epoxide is formed to the greatest extent in the Clara cells of A/J mice, is formed at intermediate levels in those of CD-1 mice, and is formed at relatively low levels in those of C57BL/6 mice.

Evaluation of the relative severities of Clara cell toxicity in A/J, CD-1, and C57BL/6 mice was facilitated by our dose-response studies using doses of 50 to 175 mg/kg DCE. Histopathologic observations of lung sections from DCE-treated mice revealed that the severities of bronchiolar Clara cell damage was incremental with dose in all strains of mice. However, the onset of Clara cell cytotoxicity differed in the three strains of mice. Vacuolization of Clara cells was evident following treatment of A/J and CD-1 mice with 50 mg/kg DCE. Damage to Clara cells was augmented when the dose was increased to 75 mg/kg DCE, and at doses of 125 to 175 mg/kg, injury was severe, resulting in extensive areas of airway devoid of epithelial lining cells. In C57BL/6 mice, the cytotoxic effects of DCE exposure were less severe, and vacuolated Clara cells were not observed at the low dose of 50 mg/kg DCE. Vacuolization of a few Clara cells was evident following treatment with a DCE dose of 75 mg/kg (Fig. 7d), and epithelial cell sloughing was observed at doses of 125 and 175 mg/kg. Treatment with the highest DCE dose of 175 mg/kg evoked airway lesions that were comparable in all three strains of mice. These results indicated that Clara cell cytotoxicity was relatively more severe in A/J and CD-1 mice than in C57BL/6 mice. These data coincided with the extents of covalent binding of DCE to lung proteins; the levels in A/J mice amounted to about 150% of those detected in C57BL/6 mice (Fig. 2). In this context, previous studies have demonstrated that magnitudes of DCE binding are proportional to the amounts of DCE administered and are associated with severities of Clara cell cytotoxicity (Moussa and Forkert, 1992). Taken together, our results are consistent with these previously reported findings and confirmed the higher levels of DCE metabolism and enhanced susceptibility of A/J mice, relative to those manifested in C57BL/6 mice. The results with CD-1 mice suggested that this outbred murine strain might be classified tentatively as a strain susceptible to cytotoxicity induced through activation of CYP2E1-selective substrates.

It is well established that the liver is highly responsive to the inducing effects of certain chemicals including ethanol, acetone, isopropanol, and pyridine (Coon and Koop, 1987; Kaul and Novak, 1987; Forkert et al., 1994). Severalfold increases in hepatic CYP2E1 are achieved, depending on the inducing chemical and route of exposure (Forkert et al., 1991, 1994). In contrast, the lung is refractory to induction by chemicals that are prototypic CYP2E1 inducers in the liver (Ronis et al., 1996). In our hands, significant increases in the CYP2E1 enzyme were not observed in the lungs of mice treated with acetone, pyridine, ethanol, isoniazid, and cigarette smoke (unpublished data). The results of this study have demonstrated that strain-related differences in CYP2E1 levels manifested in the lungs of A/J, CD-1, and C57BL/6 mice produced amounts of DCE metabolism that are significantly different from one another and was especially evident in A/J and C57BL/6 mice, strains in which CYP2E1 levels are the most discrepant (Fig. 2). These findings suggested that, although the quantity of CYP2E1 in the lungs of A/J mice was only about 40% higher than in C57BL/6 mice, this difference was of sufficient magnitude to produce a significant difference in DCE metabolism in these two strains of mice. This is presumably due to the observation that CYP2E1 is localized primarily in the Clara cells, and hence a 40% increase within a single cell type represented a substantial amount. This assumption is underscored by data showing that Clara cells are more susceptible to DCE-induced cytotoxicity than hepatocytes, even though CYP2E1 content in the lung comprises only a small fraction of the quantities in the liver. These findings indicated that the strains of mice used in this study are appropriate models for investigating metabolism of CYP2E1 substrates. An additional advantage to using these models is the lack of requirement for maneuvers that produce metabolic perturbations in related enzyme systems including GSH and/or other P450 enzymes.