Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Pulmonary injury ensues after exposure to 1,1-dichloroethylene (DCE), a chemical that is commonly used in the plastics industry and is a prevalent environmental contaminant (1). Cytotoxicity is manifested selectively in bronchiolar Clara cells, and is linked to metabolic activation of DCE to reactive intermediates that bind covalently to tissue macromolecules, including microsomal proteins (2). Magnitudes of covalent binding of DCE to lung proteins are associated with glutathione (GSH) content, in that the amounts of binding increased as GSH levels decreased (5, 6). Moreover, binding to lung microsomes was significantly inhibited when incubations with DCE were performed in the presence of GSH (3), and was increased in mice pretreated with diethylmaleate to deplete GSH (7). Furthermore, studies with isolated lung cells showed that binding levels were highest in fractions enriched in Clara cells, and were significantly higher than those in alveolar type II cells or the cell digest comprising nonseparated lung cells (8). Histochemical staining for GSH revealed that Clara cells contained abundant GSH, and the cellular GSH content in this cell type was markedly decreased following DCE exposure (5, 6). These findings strongly supported GSH conjugation of DCE metabolites as a protective mechanism against DCE-induced cytotoxicity.

It was of interest to identify the DCE metabolites that formed conjugates with GSH. The primary metabolites formed from DCE metabolism have been identified in rat hepatic microsomal incubations as DCE-epoxide, 2,2-dichloroacetaldehyde, and 2-chloroacetyl chloride (9). We have previously investigated the formation of the three primary DCE metabolites in murine liver microsomal incubations by identifying these intermediates as their GSH conjugates and/or their hydrolyzed products (13). The scheme of the proposed pathway of DCE metabolism is illustrated in Figure 1. The GSH conjugates 2-(S-glutathionyl)acetyl glutathione ([B]) and 2-S-glutathionyl acetate ([C]) were the major products formed and are believed to be derived from the DCE-epoxide. The acetal, the hydrolysis product of 2,2-dichloroacetaldehyde, and S-(2-chloroacetyl)-glutathione ([D]), the GSH-conjugated product of 2-chloroacetyl chloride, were detected, but the levels were minimal compared with those obtained for the DCE-epoxide-derived conjugates [B] and [C]. S-(2,2-Dichloro-1-hydroxy) ethyl glutathione ([A]), the GSH conjugate formed from reaction of 2,2-dichloroacetaldehyde with GSH (11), was not detected in our microsomal incubations (13). We have recently investigated the metabolites formed from DCE in murine lung microsomal incubations, and confirmed that the acetal and conjugates [B] and [C] were also the major byproducts of DCE metabolism (14). These findings suggested that the DCE-epoxide is the major metabolite generated by lung microsomes, that it appears to conjugate efficiently with GSH, and that it is probably responsible for the depletion of GSH observed in the lung after DCE treatment (5, 6). Furthermore, we have identified CYP2E1 as the major cytochrome P450 isozyme that catalyzes the formation of reactive intermediates, including the epoxide, from DCE in the lung (15). Of relevance in this context is the predominant localization of CYP2E1 in Clara cells (16), suggesting that DCE metabolism may occur primarily in this cell population.

View larger version (11K): [in this window] [in a new window]   Figure 1.   Scheme of the proposed pathway of DCE metabolism. [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.

The selective Clara cell damage induced by DCE, the high levels of DCE binding detected in the Clara cell population, and the localization of CYP2E1 in this cell type led us to hypothesize that metabolic activation occurs in situ in the target Clara cells. To test this hypothesis, we reasoned that an immunohistochemical approach was highly desirable. As an initial step, we synthesized conjugate [C], and prepared for immunization a hapten consisting of conjugate [C] coupled to bovine serum albumin (BSA). A polyclonal antibody to the hapten was developed, and data from our recent studies established its specificity for recognition of [C], a conjugate that is formed by reaction of the DCE-epoxide with the cysteine -SH group of GSH (17). In the investigation reported here, we used this antibody in immunohistochemical studies to identify the cellular location of conjugate [C], and hence of the DCE-epoxide, within lung cells. To establish that our antibody recognized sites of reactivity of the epoxide with protein -SH groups, we performed immunoblotting with a synthesized conjugate consisting of the epoxide coupled to L-cysteine and BSA. We further confirmed the in vivo formation of the epoxide by identifying the GSH conjugates [B] and [C] in lung cytosol, and determined their production under conditions in which sulfydryl and CYP2E1 levels were inhibited by buthionine sulfoximine (BSO) and diallyl sulfone (DASO₂), respectively.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

Chemicals and reagents were purchased from the suppliers named, as follows: DCE (Aldrich Chemical Co., Montréal, PQ, Canada); [U-¹⁴C]DCE (specific activity: 1.66 Ci/ mol; Amersham Corp., Arlington Heights, IL); DASO₂ (Parish Chemical Co., Orem, UT); biotinylated goat antirabbit IgG, avidin-biotin complex (Vector Laboratories, Inc., Burlingame, CA); and L-cysteine (Sigma Chemical Co., St. Louis, MO). The CYP2E1 antibody was obtained from Oxford Biomedical Research, Inc. (Oxford, MI). Goat antirabbit immunoglobulin G (IgG) conjugated to alkaline phosphatase was obtained from Zymed Laboratories (San Francisco, CA). An IgG purification kit (Immunopure [A/G]) was purchased from Pierce Chemical Co. (Rockford, IL). All other chemicals were of reagent grade and were obtained from standard suppliers.

Treatment of Animals

Female CD-1 mice weighing 25 to 28 g were purchased from Charles River Canada (St. Constant, PQ, Canada) and were maintained on a 12-h light/dark cycle. They were freely provided with food (Purina Rodent Chow; Ralston Purina, International, Strathroy, ON, Canada) and drinking water and were acclimatized to laboratory conditions for at least 5 d before being assigned to an experimental group. For detection of DCE metabolites in cytosol in dose-response experiments, mice were treated with DCE (25, 75, 125, 175, and 225 mg/kg, 40 µCi [¹⁴C]DCE), and were killed 1 h later. Mice were pretreated with DASO₂ or BSO to inhibit CYP2E1 and sulfhydryl levels, respectively. DASO₂ (100 mg/kg) was dissolved in water and given to mice by oral gavage; the mice were treated intraperitoneally with DCE (225 mg/kg) 4 h later. The dose- and time-dependent effects of BSO pretreatment on lung sulfhydryl content were initially determined. BSO was dissolved in saline with the aid of 1.0 N NaOH, pH 8.5. In the dose-response studies, mice were treated intraperitoneally with BSO at dosages ranging from 0.5 to 3.0 g/kg. In the time-course experiments, mice were treated intraperitoneally with BSO (1.5 g/kg) and were killed 1, 2, 4, 10, 15, and 24 h later. In subsequent studies, mice were pretreated with BSO, and 4 h later were treated with DCE (225 mg/kg) and killed 1 h after DCE exposure. These mice were killed by cervical dislocation. For immunohistochemical studies, mice were treated with the following protocols: (1) DCE; (2) BSO and DCE; and (3) DASO₂ and DCE. DCE was administered 4 h after treatment with BSO or DASO₂, and mice were killed 1 h after DCE treatment. Control mice were treated with equivalent volumes of the appropriate vehicle and were killed at times corresponding to those in the experimental groups.

Preparation of Cytosol

Lungs of mice were excised, rinsed, blotted, and weighed. For preparation of cytosolic fractions, lung tissue from 10 mice that were treated with [¹⁴C]DCE were pooled and homogenized (1 ml buffer per gram) in cold phosphate-buffered KCl (140 mM KCl, 100 mM K₂HPO₄, 1.5 mM ethylenediamine tetraacetic acid [EDTA], pH 7.4) with a tissue homogenizer. The homogenate was centrifuged at 9,000 × g at 4°C for 40 min, after which the supernatant was obtained and centrifuged at 105,000 × g at 4°C for 40 min. Aliquots of the supernatant were dispensed into Eppendorf tubes, rapidly frozen in liquid nitrogen, and stored at 70^oC. Protein concentrations were determined with the Bradford protein assay (18).

Metabolite Identification

Metabolites of DCE were analyzed and identified through procedures described in our previous studies (13, 14), with modification. Proteins in samples of cytosol (30 mg/ml) were precipitated with 70% perchloric acid (final concentration, 5%), and centrifuged for about 5 min; 100 µl of the supernate were analyzed by high-pressure liquid chromatography (HPLC) using a reverse-phase C-18 column (5 µm, 4.6 × 250 mm; Microsorb-MV; Rainin Instruments Co., Inc., Woburn, MA). The isocratic mobile phase was 0.2% H₃PO₄ with a flow rate of 1.0 ml/min. The column effluent was monitored at 200 nm. For detection of the DCE metabolites, 250-µl aliquots of the column effluent were collected every 15 s for each sample, and levels of radioactivity were determined through liquid scintillation spectroscopy. Levels of metabolites were estimated by summing the amounts of radioactivity associated with each peak and converting the data to nanomolar amounts, using the specific activity of the [¹⁴C]DCE. The HPLC experiments were performed on a Beckman System Gold Nouveau (Beckman Instruments, Mountain View, CA) equipped with a Model 168 diode array UV detector.

Measurement of Sulfhydryl Content

Lungs from two mice were pooled for each sample determination for sulfydryl levels. Tissues were homogenized in 0.1 M phosphate buffer, pH 7.4, and proteins in a 500-µl aliquot of homogenate were precipitated by addition of an equal volume of 4% sulfosalicylic acid. After thorough mixing and centrifugation, 500 µl of the supernate samples were analyzed for concentrations of acid-soluble sulfhydryls according to the method of Ellman (19). Protein concentrations were determined by the method of Lowry and associates (20).

Synthesis of DCE-epoxide-L-Cysteine- BSA Conjugate

The DCE-epoxide was synthesized by oxidation of DCE with m-chloroperbenzoic acid as described in our previous studies (13). Briefly, 310 µl (3.88 mmol) of DCE and 0.25 g (5.8 mmol) of m-chloroperbenzoic acid were dissolved in 5 ml of dry acetonitrile and stirred for 60 min in a water-bath set at 60°C. This solution was combined with 20 ml of an aqueous solution of L-cysteine (0.1 mg/ml), and allowed to react at room temperature for 24 h. The resulting solution was then dried in vacuo and extracted with 50% diethylether in water. The aqueous layer was extracted a second time with the diethylether and dried down.

The DCE-epoxide-cysteine conjugate was coupled to BSA using glutaraldehyde as the chemical crosslinking agent. The chemical structure of the synthesized conjugate is depicted in Figure 2. The conjugate (0.3 mg) was dissolved in 1 ml phosphate-buffered saline (PBS) and combined with 15 mg of BSA. Glutaraldehyde (0.2%, 1 ml) was added slowly to the solution over a period of 0.5 h, and allowed to react for 1 h with constant stirring. Sodium borohydride (10 mg/ml, 0.5 ml) was then added and stirred for 15 min. The solution was subsequently dispensed into dialysis tubing and dialyzed overnight against PBS (500 ml), with four changes of buffer. Aliquots of the epoxide- cysteine-glutaraldehyde-BSA conjugate were stored at 20°C. L-Cysteine was also coupled to BSA with glutaraldehyde and the product was used as a control conjugate. The glycine-glutaraldehyde-BSA conjugate was used to reduce nonspecific binding of the antibody to antigenic sites, and was prepared as previously described (17). Protein concentrations were determined with the Bradford protein assay (18).

View larger version (10K): [in this window] [in a new window]   Figure 2.   Chemical structure of the synthesized DCE-epoxide-cysteine-glutaraldehyde-BSA conjugate. Details of the synthesis are described in MATERIALS AND METHODS.

Synthesis and Purification of Conjugate [C]

Conjugate [C] was synthesized and purified according to methods described in our previous studies (17), with slight modifications. Briefly, conjugate [C] was synthesized by combining GSH (307 mg) with chloroacetic acid (94 mg) in 10 ml of 100 mM potassium phosphate buffer, pH 7.4, yielding a final equimolar concentration of 100 mM. The solution was heated at 50°C for 2 h and then cooled overnight at room temperature. The solution was adjusted to pH 2.0 with 6 N HCl and was then extracted with diethylether (10 ml). Conjugate [C] was isolated by passage through a C-18 extraction cartridge and elution with methanol. The samples were dried down, reconstituted with water, and analyzed via HPLC with a reverse-phase C-18 column (5 µm, 4.6 × 250 mm; Phenomenex, Torrance, CA). The mobile phase was 0.1% trifluoroacetic acid in H₂O and the flow rate was 1 ml/min. The peak for conjugate [C] was identified by an elution time similar to that identified for the authentic standard. The eluent corresponding to the peak for conjugate [C] was collected from approximately 40 HPLC injections, pooled, concentrated in vacuo, and stored at 20°C. The identity and purity of the sample were confirmed by proton nuclear magnetic resonance (¹H-NMR) analysis and electrospray mass spectroscopy, as described in our previous studies (17).

Antibody Production and Purification

Rabbits were immunized with a hapten consisting of chemically synthesized conjugate [C] coupled to BSA as the carrier protein, with glutaraldehyde as the chemical cross-linking agent, prepared as described previously (17). The properties of the resulting rabbit polyclonal antibodies have been characterized, and the antibodies have been confirmed to be effective in recognizing conjugate [C], but had minimal reactivity with the carrier protein or the cross-linking agent. The antiserum was affinity-purified with a protein A/G column, and the IgG fraction obtained was used for the immunohistochemical experiments. Protein concentrations were determined with the Bradford protein assay (18).

Protein Immunoblotting

Samples of synthesized conjugates (DCE-epoxide-cysteine- glutaraldehyde-BSA, cysteine-glutaraldehyde-BSA) and BSA were applied to the wells of a slot-blotting apparatus and onto a nitrocellulose membrane (Hoefer Slot Blot PR 648 Apparatus; Hoefer Scientific Instruments, San Francisco, CA). The samples contained concentrations of protein ranging from 10-90 µg. The protein blots were prepared as described in our previous studies (17). Briefly, the membrane was rinsed with PBS and blocked for 2 h with 3% BSA diluted in PBS-Tween 20. After further washing, the membrane was reacted overnight with the conjugate [C] IgG (708 ng/ml) diluted in PBS-Tween 20 containing 1% BSA and glycine-glutaraldehyde-BSA (1 mg/ml). The membrane was then reacted with a secondary antibody (goat antirabbit IgG conjugated to alkaline phosphatase, 1:1,000) diluted in PBS-Tween 20 containing 1% BSA. The color reaction was developed with 5-bromo-4-chloro-3-indolylphosphate and p-nitroblue tetrazolium chloride.

Immunohistochemical Staining for Conjugate [C]

Lung tissue was fixed with 4% paraformaldehyde in 0.1 M Sorensen's buffer (12.0 mM NaH₂PO₄, 69.0 mM Na₂HPO₄, pH 7.4) by vascular perfusion and tracheal instillation according to the method described in our previous studies (16). Paraffin-embedded tissue sections were adhered to gelatin-coated slides and subjected to immunohistochemical staining for conjugate [C]. Labeling of conjugate [C] was done with the avidin-biotin complex technique, using procedures described previously (16). The tissue sections were deparaffinized, cleared, and hydrated. After rinsing in PBS, the sections were incubated in 5% normal goat serum and 3% BSA. After further rinsing in PBS, the sections were incubated with the conjugate [C] antibody for 60 min. The antibody was diluted in PBS containing 1% normal goat serum and 0.1% of the glycine-glutaraldehyde-BSA conjugate. Inclusion of the glycine-glutaraldehyde-BSA conjugate in the incubations effectively blocked nonspecific antibody binding. The sections were rinsed thoroughly to remove unbound antibodies, and were reacted with biotinylated goat antirabbit IgG for 10 min. Endogenous peroxidase activity was blocked by incubating tissue sections for 30 min with 1% hydrogen peroxide in nanopure water. Sections were then reacted with streptavidin conjugated to horseradish peroxidase for 10 min, and the immunoperoxidase color reaction was developed by incubation in PBS containing 0.05% 3,3'-diaminobenzidine and 0.01% hydrogen peroxide. The sections were then rinsed in running tap water for 5 min. After incubation for 5 min in 0.15 M sodium chloride containing 0.5% copper sulfate, the sections were dehydrated, cleared, and mounted. Controls for the specificity of the immunohistochemical reactions included incubations performed in the presence of a nonspecific antibody or incubations in which the specific antibody was omitted.

Statistical Analysis

Data are expressed as mean ± standard deviation (SD). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Tukey's test for pairwise multiple comparisons to identify significant differences between treatment groups. The level of significance was set at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Detection of the DCE-Epoxide in Lung Cytosol

The formation of DCE metabolites was examined in cytosolic fractions isolated from the lungs of mice treated in vivo with DCE. HPLC analysis of cytosolic samples showed that the acetal, the hydrate of 2,2-dichloroacetaldehyde, and the epoxide-derived GSH conjugates [B] and [C] were detectable. A representative chromatogram of the metabolites is shown in Figure 3. Conjugate [C] was the major species formed, with lower levels for conjugate [B]; in contrast, minimal levels of the acetal were detected as compared with those obtained for the epoxide. Peaks eluting from the column between 2 and 4 min were also observed; glycolic acid and formaldehyde, which are decomposition products of the DCE-epoxide, elute in this region (14). Time-course experiments revealed that conjugate [C] was formed rapidly and was present at detectable levels (0.41 ± 0.06 nmol/mg protein) at 15 min after DCE (125 mg/kg) treatment. The amounts of conjugate [C] found in the cytosol increased at 60 min and declined thereafter, but were still detectable at 2 h after DCE treatment. However, formation of conjugate [B] was low at 15 to 60 min after DCE (0.04-0.18 ± 0.02 nmol/mg protein). Dose-response experiments showed that formation of conjugate [C] was concentration-dependent and increased progressively with exposure to concentrations of DCE ranging from 25 to 225 mg/kg (Figure 4). Formation of conjugate [B] was also dose-dependent, but the increases detected at doses greater than 75 mg/kg were low (Figure 4). As expected, the amounts of DCE-epoxide formed in the cytosol, as estimated from the total amounts of GSH conjugates generated, also increased progressively with doses of DCE administered.

View larger version (13K): [in this window] [in a new window]   Figure 3.   Chromatograms of GSH conjugates [B] and [C] detected in lung cytosol of mice treated with [14C]DCE (40 Ci, 125 mg/kg) alone (A), or in conjunction with BSO (B) or DASO2 (C ). Cytosolic fractions were isolated from lung tissue pooled from 10 mice. Proteins were precipitated with 70% perchloric acid, and 100 µl of the supernate was analyzed with reverse-phase HPLC. Aliquots of the column effluent were collected and levels of radioactivity were determined through liquid scintillation spectroscopy.

View larger version (19K): [in this window] [in a new window]   Figure 4.   Dose-dependent formation of GSH conjugates [B] and [C] in lung cytosol from mice treated with doses of DCE ranging from 25 to 225 mg/kg. Cytosolic proteins were precipitated with perchloric acid, and 100 µl of the supernate was analyzed with reverse-phase HPLC. Aliquots of the column effluent were collected and levels of radioactivity were determined through liquid scintillation spectroscopy. Data are expressed as mean ± SD.

The formation of the DCE-epoxide was also determined in cytosol isolated from mice pretreated with BSO. BSO is a chemical that inhibits the synthesis of GSH by inhibiting -glutamylcysteine synthetase, an enzyme that has a key role in synthesis and maintenance of cellular GSH (21, 22). In preliminary experiments, time-course and dose- response studies were done to evaluate BSO-induced alterations in lung sulfhydryl content. On the basis of data obtained from these experiments, a time-point of 4 h and a dose of 1.5 g/kg BSO were selected. This dose was within the range of concentrations used in previous studies (22). Maximal loss of sulfhydryl content was estimated to be about 40% of the control level (20.2 nmol/mg protein). Preliminary experiments also confirmed that BSO did not cause alterations in p-nitrophenol hydroxylation, an enzyme activity associated with CYP2E1, the P450 enzyme catalyzing the oxidation of DCE (15, 23).

In mice treated with a dose of 125 mg/kg DCE, the amounts of conjugate [C] found in lung cytosol were significantly higher (108%) than those detected for conjugate [B] (Figure 5, top). Conjugates [B] and [C] were also detected in cytosolic fractions from mice treated with BSO and DCE, but the levels were significantly reduced, and both conjugates comprised only a small fraction of the amounts detected in mice treated with DCE alone. Moreover, conjugate [B] was formed at significantly lower levels than conjugate [C]. Epoxide formation in the cytosol, as estimated from the total levels of conjugates [B] and [C], was inhibited by about 70% after treatment with both BSO and DCE, as compared with the amount produced by treatment with DCE alone (Figure 5, bottom). Mice were also give DASO₂ and were subsequently treated with DCE (Figure 5, top). As was found with BSO, DASO₂ produced marked inhibition of formation of conjugates [B] and [C] in the cytosol as compared with levels of both conjugates detected in mice treated with DCE alone. The amount of conjugate [B] formed was significantly lower than that of conjugate [C]. Epoxide formation in lung cytosol of mice treated with both DASO₂ and DCE comprised only a small amount (14%) of the level seen in mice treated with DCE alone (Figure 5, bottom). Hence, epoxide levels differed for the two pretreatments, and were significantly lower for DASO₂ than for BSO.

View larger version (14K): [in this window] [in a new window]   Figure 5.   Measurement of GSH conjugates [B] and [C] (top panel ) and the DCE-epoxide (bottom panel ) in lung cytosol (conjugates [B] and [C]) isolated from mice treated with [14C]DCE (125 mg/kg) alone, with DCE and BSO, or with DASO2. Cytosolic proteins were precipitated, and 100 µl of the supernate was analyzed with reverse-phase HPLC. Aliquots of the column effluent were collected and levels of radioactivity were determined through liquid scintillation spectroscopy. Data are expressed as mean ± SD, and were analyzed by one-way ANOVA and Tukey's test (*significantly different from conjugate [B] in mice subjected to the same treatment [P < 0.001]; **significantly different from conjugate [B] in mice treated with DCE alone [P < 0.001]; +significantly different from conjugate [C] in mice treated with DCE alone [P < 0.001]; ++significantly different from conjugate [C] in mice treated with DCE alone or with BSO/DCE [P < 0.001]). Levels of DCE-epoxide formed in lung cytosol were estimated by summing the levels of radioactivity for the conjugate [B] and conjugate [C] peaks (*significantly different from mice treated with DCE alone [P < 0.001]; **significantly different from mice treated with DCE or BSO/DCE [P < 0.001]).

Protein Immunoblotting

Results of the slot blots revealed that the conjugate [C] antibody recognized a conjugate of DCE-epoxide-cysteine- BSA (Figure 6). The immunoreactivity was concentration-dependent at protein contents ranging from 10-90 µg, and was minimal for reactions conducted with BSA or with the cysteine-glutaraldehyde-BSA conjugate.

View larger version (47K): [in this window] [in a new window]   Figure 6.   Protein immunoblotting for the chemically synthesized conjugate DCE-epoxide-L-cysteine-glutaraldehyde-BSA or controls (BSA, L-cysteine-glutaraldehyde-BSA). Samples were applied to the wells of a slot-blotting apparatus; Lanes 2-6 were loaded with 10, 20, 30, 50, 70, and 90 µg of the epoxide-cysteine- glutaraldehyde-BSA conjugate, respectively. Lanes 1 and 7 were loaded with BSA (20 µg) and L-cysteine-glutaraldehyde-BSA (20 µg). The top and bottom rows are duplicates of the same samples. The membrane was reacted with the anticonjugate [C] IgG antibody (708 ng/ml) diluted in PBS-Tween 20 containing 1% BSA and the conjugate glycine-glutaraldehyde-BSA. The membrane was subsequently reacted with a goat antirabbit IgG conjugated to alkaline phosphatase.

Immunohistochemical Localization of Conjugate [C]

Immunohistochemical studies were performed on lung tissues from groups of mice that were treated with DCE alone or that were pretreated with BSO or DASO₂ and subsequently treated with DCE. The results of these studies are illustrated in Figure 7. In tissue sections from lungs of mice treated with DCE (125 mg/kg), the bronchiolar epithelium exhibited staining for conjugate [C], and this staining was most prominent in the apical portions of the Clara cells (Figure 7a). Staining for conjugate [C] increased with exposure to increasing dosages of DCE, and was proportional to the amounts of DCE given. Staining was most intense in tissue sections from mice treated with 225 mg/kg, the highest DCE dose administered in this study (Figure 7c). Staining with this high dose was not confined to the apices of the Clara cells, but appeared to encompass a greater portion of the cellular area. No specific staining was seen in lung sections from untreated mice (Figure 7d), sections reacted with a nonspecific antibody, or sections that were not incubated with a primary antibody. In lung sections from mice treated with DCE in conjunction with BSO, staining for conjugate [C] in the bronchiolar epithelium was decreased slightly; the staining was seen in the Clara cells, and was decreased in comparison with the levels found in nonpretreated mice given the same DCE dose (Figures 7c and 7e). The combined treatment with DASO₂ and DCE also produced diminished staining for conjugate [C] in the bronchiolar epithelium and in Clara cells (Figure 7f). This staining was markedly weaker than that seen in lung sections from mice treated with both BSO and DCE, and was almost comparable to the staining observed in the controls.

View larger version (147K): [in this window] [in a new window]   Figure 7.   Immunohistochemical detection of conjugate [C] in bronchiolar epithelium of murine lung. Nonciliated Clara cells are labeled (arrows). In mice treated with DCE alone, the doses used were 125 mg/ kg (a), 175 mg/kg (b), and 225 mg/kg (c). Mice were also pretreated with BSO (e) and DASO2 (  f  ) before treatment with DCE (225 mg/kg). Controls consisted of reactions conducted in the absence of the specific antibody (d). Immunohistochemical staining was achieved with a rabbit anticonjugate [C] IgG antibody and the avidin-biotin complex procedure. Bar = 50 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In previous studies we have investigated the metabolism of DCE in lung and liver microsomal incubations, and identified the major products formed as the DCE-epoxide- derived GSH conjugates [B] and [C], and as the acetal, the hydrate of 2,2-dichloroacetaldehyde (13, 14). In the present study, significantly higher levels of conjugate [C] than of conjugate [B] were detected in lung cytosol isolated from mice treated in vivo with DCE, and this result was consistently obtained with all the doses of DCE investigated (Figure 4). These results differed from those obtained in our previous in vitro studies, in which substantially higher levels of conjugate [B] than of conjugate [C] were detected in both lung and liver microsomal incubations (13, 14). These findings are in agreement with those reported in previous studies, in which the amounts of conjugate [C] detected in isolated hepatocytes were approximately threefold higher than those of conjugate [B] (11). The reverse situation was found in rat liver microsomal incubations, where conjugate [B] was the major conjugate formed. The higher levels of conjugate [B] than of conjugate [C] detected in our in vitro experiments may be ascribed to an excess of GSH used in our incubations to capture the epoxide. This assumption is consistent with the concept that excess levels of GSH present in the microsomal incubations favor the formation of conjugate [B], whereas in the in vivo situation there is more limited availability of GSH for conjugation reactions, and hence the conditions favor the formation of conjugate [C]. This premise is supported by data from our dose-response experiments, in which the highest levels of conjugate [B] relative to those of conjugate [C] were formed at the lower doses of DCE (Figure 4). The amount of conjugate [B] formed at 75 mg/kg of DCE was about 600% greater than that generated at the 25 mg/kg dose; the increases at the higher doses were low, and were only about 20% for DCE doses of 125 to 225 mg/kg (Figure 4). This phenomenon may be related to decreasing amounts of GSH available for conjugation, as a result of its consumption through reaction with the DCE metabolites (5, 6). However, the reduced access to GSH was not sufficient to restrict the production of conjugate [C], and this is probably associated with the mechanism by which the GSH conjugates are generated from the DCE-epoxide. 2-S-Glutathionylacetyl chloride, the intermediate formed from initial reaction of GSH with the epoxide, either undergoes hydrolysis to form conjugate [C] or reacts with a second molecule of GSH to form conjugate [B]. Once formed, conjugate [B] can also hydrolyze to conjugate [C] under aqueous conditions (11). This finding raises the question of whether the quantities of conjugate [C] detected in vivo in the lung are a result of hydrolysis of conjugate [B] to conjugate [C]. Data from our time-course studies showed that the amounts of conjugate [B] (0.04-0.18 nmol/mg protein) detected from 15 min to 1.0 h in mice treated with 125 mg/kg of DCE amounted to only a small portion of the levels detected for conjugate [C]. The quantity of conjugate [C] detected at 15 min after DCE was about tenfold higher than that identified for conjugate [B]. Furthermore, previous studies showed that hydrolysis of conjugate [B] to conjugate [C] occurred at pH 8.0 over a period of 48 h (11). At pH 7.5, the half-life of conjugate [B] was estimated at about 3 h (11), suggesting that a high rate of degradation of conjugate [B] was not a major contributing factor in the lower levels of conjugate [B] relative to conjugate [C] detected in our experiments performed at 1 h after DCE treatment. These findings indicated that it is unlikely that the hydrolysis of conjugate [B] contributes significantly to the levels of conjugate [C] detected in vivo under physiologic conditions. Thus, our results showed that the specific conjugates generated from DCE in vivo differed qualitatively and quantitatively from those formed in vitro, and that this may have been influenced in part by the size of the GSH pool available for conjugation reactions. Other factors, such as the amounts of epoxide formed or as yet unidentified, may also account for the difference in the relative amounts of conjugates [B] and [C] formed in both of these systems.

It is of interest to compare the results of BSO pretreatment with those obtained in experiments in which mice were pretreated in vivo with DASO₂, an efficacious inhibitor of lung CYP2E1 (24). In our previous studies, treatment of mice with DASO₂ resulted in an 80% reduction in p-nitrophenol hydroxylation, a CYP2E1-selective enzyme catalytic activity (24). Since the oxidation of DCE is catalyzed predominantly by CYP2E1 to yield the DCE-epoxide (13, 23), this treatment regimen is expected to inhibit DCE metabolism, and hence to decrease the production of GSH conjugates, owing to restricted synthesis of the epoxide. Under these conditions, in which GSH is probably not a limiting factor, the total amount of conjugates formed should reflect the quantity of the epoxide generated. As expected, epoxide formation was markedly depressed, and comprised only about 14% of the level identified in mice treated with DCE alone (Figure 5). These results indicated that the formation of GSH conjugates can be inhibited more efficiently by blocking the activation pathway, and is less efficiently achieved by lowering sulfydryl levels and hence inhibiting the detoxication pathway. This may be ascribed to the ability to achieve a high level of CYP2E1 inhibition, whereas the BSO maneuver was not as effective at depressing lung sulfydryl levels, at least to a magnitude comparable to that produced by DASO₂.

Several investigators, including ourselves, have postulated that selective damage to Clara cells induced by a variety of chemicals, including DCE (8), naphthalene (25, 26), 4-ipomeanol (27), and 1-nitronaphthalene (28, 29), is mediated by reactive intermediates generated in situ within this cell type. This concept arose from studies showing that production of reactive metabolites was associated with Clara cell cytotoxicities (24, 27). Moreover, the Clara cell lesion correlated with preferential covalent binding of metabolites to this cell type (8, 30), and coincided with localization of high concentrations of P450 enzymes in Clara cells (31, 32). For example, Clara cell damage induced by naphthalene is mediated by oxidative metabolism via CYP2F2 and CYP2B, both of which are localized in Clara cells (33). On the other hand, DCE is metabolically activated by CYP2E1, a P450 enzyme that also resides primarily in Clara cells (16). Inhibition of CYP2E1 by DASO₂ caused a significant reduction in formation of the DCE-derived conjugates [B] and [C], and hence in formation of the epoxide, and protected the Clara cells from DCE-induced injury (24). Taken together, these findings have provided evidence, albeit indirect, to support in situ formation of DCE metabolites within the target Clara cells.

A primary objective of this study was to obtain more direct evidence that the in situ formation of a reactive metabolite within Clara cells is associated with the susceptibility of this cell population to chemically induced damage. We have used the DCE-induced model of Clara cell cytotoxicity to address this objective (1). In this model, the ultimate toxic metabolite is believed to be the DCE-epoxide (13, 14). It is readily trapped by GSH, and the studies described herein have shown that conjugation of the thiol nucleophile with the epoxide gives rise in vivo to the major GSH conjugate [C]. We developed a polyclonal antibody directed against conjugate [C], which was synthesized by reaction of the DCE-epoxide with the SH group of the cysteine of GSH. We confirmed by protein immunoblotting that this antibody recognizes an antigen consisting of the epoxide-cysteine-glutaraldehyde-BSA conjugate (Figure 6). On the basis of these findings, we predicted that this antibody could be used as a probe to identify tissue protein sites of binding of the electrophilic epoxide to cysteinyl thiols, which are major nucleophilic centers on proteins. Results of our immunohistochemical studies showed that conjugate [C] was localized in the bronchiolar Clara cells (Figure 7). Staining was mainly concentrated in the apical cytoplasm, a region that contains abundant networks of smooth endoplasmic reticulum, where P450 enzymes including CYP2E1 reside (16). These findings suggested that conjugation of the DCE-epoxide occurred at the site of its formation within the Clara cells. The level of staining for conjugate [C] followed a dose-dependent pattern, and was most pronounced at the highest dose of DCE (225 mg/kg) given to the mice (Figures 7a-7c). These observations are in agreement with the levels of conjugate [C] identified in the lung cytosol for the same DCE doses (Figure 4). Staining in lung sections from mice pretreated with BSO was reduced as compared with the level seen in mice treated with the same DCE dose (225 mg/kg; Figures 6c and 6e). However, the lowest levels of staining were observed in lung sections from mice pretreated with DASO₂ (Figure 7f). Both of these pretreatments produced staining levels that were consistent with the relative amounts of conjugate [C] detected in the cytosol (Figure 5). These results suggested that the GSH conjugates detected in lung cytosol reflected the amounts formed primarily in the Clara cells, and indicated that the DCE-epoxide is generated and conjugated in this cell type. In conclusion, findings derived from application of a novel immunohistochemical approach have provided the most direct evidence to date for the proposal that generation of the epoxide in situ within Clara cells mediates their particular susceptibility to cytotoxicity induced by DCE.