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BIOACTIVATION OF 1,1-DICHLOROETHYLENE TO ITS EPOXIDE BY CYP2E1 AND CYP2F ENZYMES

Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada (A.C.S., K.S.C., P.-G.F.); Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California (R.M.B.); Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (B.I.G.); and Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah (C.A.R., G.S.Y., D.L.L.)

(Received December 3, 2003; accepted June 4, 2004)

	Abstract

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1,1-Dichloroethylene (DCE) exposure to mice elicits lung toxicity that selectively targets bronchiolar Clara cells. The toxicity is mediated by DCE metabolites formed via cytochrome P450 metabolism. The primary metabolites formed are DCE epoxide, 2,2-dichloroacetaldehyde, and 2-chloroacetyl chloride. The major metabolite detected is 2-S-glutathionyl acetate [C], a putative conjugate of DCE epoxide with glutathione. In this investigation, studies were undertaken to test the hypothesis that CYP2E1 and CYP2F2 are involved in bioactivation of DCE to the epoxide in murine lung. We have developed a method using liquid chromatography/mass spectrometry (LC/MS) to evaluate the kinetics of the rates of production of conjugate [C] by recombinant CYP2E1 and CYP2F enzymes and lung microsomes. Concentration-dependent formation of conjugate [C] was found in incubations of DCE with recombinant CYP2E1 and CYP2F enzymes and lung microsomes from CD-1, wild-type (mixed 129/Sv and C57BL), and CYP2E1-null mice. Recombinant rat CYP2E1 exhibited greater affinity and catalytic efficiency for DCE metabolism than did recombinant human CYP2E1, mouse CYP2F2, goat CYP2F3 or rat CYP2F4. In the lung microsomal incubations, the rates of conjugate [C] production were higher in CD-1 mice than in either wild-type or CYP2E1-null mice; the level of [C] in CYP2E1-null mice was about 66% of that in wild-type mice. These results demonstrated that LC/MS analysis is a suitable method for detection and quantitation of conjugate [C], and that CYP2E1 and CYP2F2 catalyze the bioactivation of DCE to the epoxide in murine lung. The results also demonstrated that CYP2E1 is the high-affinity enzyme involved in DCE bioactivation.

Previous studies in rat liver have identified the primary metabolites formed from DCE in microsomal incubations as the epoxide, 2,2-dichloroacetaldehyde, and 2-chloroacetyl chloride (Fig. 1) (Costa and Ivanetich, 1982; Liebler and Guengerich, 1983; Liebler et al., 1985). The major products formed in murine lung and liver microsomal incubations containing glutathione (GSH) were the conjugates 2-(S-glutathionyl) acetyl glutathione [B] and 2-S-glutathionyl acetate [C] (Fig. 1) (Dowsley et al., 1995, 1996). These conjugates are the products of conjugation of GSH with DCE epoxide (Dowsley et al., 1995, 1996). The acetal of 2,2-dichloroacetaldehyde has also been detected in the microsomal incubations. However, S-(2,2-dichloro-1-hydroxy)ethyl glutathione [A], the product of GSH conjugation with 2,2-dichloroacetaldehyde, was not detected (Dowsley et al., 1995). S-(2-Chloroacetyl)glutathione [D] and chloroacetic acid, the GSH-conjugated and hydrolysis products of 2-chloroacetyl chloride, respectively, were also formed in the microsomal incubations but at minimal levels (Dowsley et al., 1995). Hence, the DCE epoxide was the major metabolite produced in vitro, and was monitored in this study as its GSH conjugate [C]. Subsequent studies confirmed that DCE epoxide was also generated in vivo, and its products were detected in lung and liver cytosol and in bile of DCE-treated mice (Forkert, 1999a,b).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Proposed scheme for 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.

In view of the finding that cytochrome P450-dependent bioactivation of DCE is a mechanism through which lung toxicity is mediated, it was of interest to identify the P450 enzymes responsible for DCE oxidation. Previous studies showed selective loss of CYP2E1 catalytic activity and protein content in lung microsomes incubated with DCE (Lee and Forkert, 1995). More importantly, formation of DCE epoxide and 2,2-dichloroacetaldehyde was inhibited by about 50% in lung microsomes preincubated with a CYP2E1-inhibitory antibody before incubation with DCE (Dowsley et al., 1996). There was also a 50% inhibition of formation of DCE epoxide and 2,2-dichloroacetaldehyde in mice treated with diallyl sulfone, a CYP2E1 inhibitor (Forkert et al., 1996). These findings supported the role of CYP2E1 in DCE bioactivation, but suggested that an additional P450 enzyme may also be involved.

Cytochrome P450 enzymes, such as CYP2E1, are predominantly expressed in liver, with lower levels in other tissues such as the lung. The CYP2F subfamily is distinct in that it has selective expression in the lung with little expression in the liver (Hakkola et al., 1994). This subfamily is also distinct for its lack of gene diversity, with only a single member expressed in each of the species studied: human (2F1) (Nhamburo et al., 1989, 1990; Raunio et al., 1998), mouse (2F2) (Ritter et al., 1991), and goat (2F3) (Wang et al., 1998). The sequence similarities of CYP2F subfamily members among different species are high, with identities of 80 to 84% at both the nucleic and amino acid levels (Chen et al., 2002). Like CYP2E1, CYP2F2 resides predominantly in Clara cells of murine lung (Buckpitt et al., 1995; Forkert, 1995). Members of the CYP2F subfamily have been reported to bioactivate naphthalene (Nagata et al., 1990; Ritter et al., 1991; Lanza et al., 1999), styrene (Carlson, 1997), and 3-methylindole (Wang et al., 1998; Lanza et al., 1999; Lanza and Yost, 2001). Similar to these compounds, DCE is a low molecular weight chemical identified in preliminary studies as a potential substrate for CYP2F. Moreover, localization of this P450 in the Clara cells, coupled with the cytotoxic lesions induced by DCE in this cell type, supported a role for CYP2F2 in DCE bioactivation.

In the present study, we have established LC/MS methodology for detection of conjugate [C] and tested the hypothesis that DCE is bioactivated by CYP2E1 and CYP2F enzymes. The results showed that conjugate [C] was formed from DCE in lung tissues of CD-1, wild-type (mixed 129/Sv and C57BL), and CYP2E1-null mice, and in incubations containing recombinant CYP2E1 (rCYP2E1) (rat and human), recombinant CYP2F2 (rCYP2F2) (mouse), recombinant CYP2F3 (rCYP2F3) (goat), and recombinant CYP2F4 (rCYP2F4) (rat). The results further showed that DCE metabolism occurred with different affinities and catalytic efficiencies, suggesting that the toxicities elicited by DCE may be manifested at different severities in various species.

	Materials and Methods

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Chemicals and Reagents. Chemicals were purchased from suppliers as follows: 1,1-dichloroethylene (>99% purity) and glutathione, Aldrich Chemical Co. (Montreal, Québec, Canada); glucose 6-phosphate, glucose-6-phosphate dehydrogenase, NADP⁺, NADPH, S-ethyl glutathione, -aminolevulinic acid, lysozyme, magnesium acetate, dithiothreitol, Lauria Bertani (LB) broth dry mix, Terrific Broth dry mix, and phenylmethylsulfonyl fluoride, Sigma-Aldrich (St. Louis, MO); leupeptin, bestatin, and aprotinin, Invitrogen (Carlsbad, CA); ampicillin, Roche Diagnostics (Indianapolis, IN); isopropyl-thiogalactopyranoside (IPTG), Fisher Scientific (Pittsburgh, PA); recombinant rat CYP2E1- and human CYP2F1-expressed ß-lymphoblastoid microsomes, BD Gentest (Woburn, MA); and recombinant human CYP2E1, BD Biosciences Discovery Labware (Bedford, MA). All other chemicals and reagents were purchased from standard suppliers.

Treatment of Animals. Female CD-1 mice, weighing 20 to 25 g, were purchased from Charles River Canada (St. Constant, Quebec, Canada), maintained on a 12-h light/dark cycle, and given food (Mouse Diet 5015; PMI Nutrition International, Inc., Brentwood, MO) and water ad libitum. They were acclimatized to laboratory conditions for 1 week after arrival before being assigned to an experimental group. Lung microsomes were prepared from mice sacrificed by cervical dislocation. Female wild-type and CYP2E1-null mice were obtained from a colony (mixed 129/Sv and C57BL) developed (Lee et al., 1996) at the National Cancer Institute (Bethesda, MD) and were re-derived and bred at Charles River Laboratories, Inc. (Wilmington, MA). Female mice weighing 22 to 35 g (2-3 months old) were quarantined at the National Institute of Environmental Health Sciences (Research Triangle Park, NC) for 1 week before use in temperature- and humidity-controlled rooms with a 12-h light/dark cycle. NIH 31 rodent chow diet and tap water were provided ad libitum. All animal care and procedures were performed according to the National Institutes of Health guidelines (NIH, 1985). Mice were euthanized using CO₂/O₂ and the lungs were removed and stored at -70°C until used.

Preparation of Microsomes. Microsomes were prepared according to procedures used in our previous studies (Forkert et al., 1987), with minor modifications. Lung tissues from 30 mice were pooled for each lung microsomal sample. Microsomes were prepared by mincing the lung tissue with a razor blade while on ice. The minced tissue was then homogenized with a ratio of 1 g of tissue to 4 ml of cold phosphate-buffered KCl (1.15% KCl, 100 mM K₂HPO₄, 1.5 mM EDTA, pH 7.4). The homogenate was centrifuged at 12,500g for 23 min at 2°C. The supernatant was then centrifuged at 105,000g for 68 min. The pellet was homogenized using about three strokes of the pestle, and the resultant homogenate was centrifuged at 12,500g for 68 min. The final pellet was homogenized, using a hand-held pestle in a 1.5-ml tube with 400 µl of buffer. Aliquots of lung microsomes were flash frozen in liquid nitrogen and stored at -70°C. The microsomal protein concentration was determined by the Bradford method, using bovine serum albumin as the standard (Bradford, 1976).

Cloning and Expression of CYP2F3. The full-length cDNA of CYP2F3 was cloned into a pCW bacterial expression vector. This vector, a generous gift from Dr. F. P. Guengerich, allows for coexpression of both cytochrome P450 genes and cytochrome P450 oxidoreductase. The 2F3/oxidoreductase construct was transformed into competent DH5 bacterial cells. The transformation mixture was spread onto LB plates containing 100 µg/ml ampicillin, and approximately 14 h later, colonies were selected. Several of the ampicillin-resistant colonies were selected and screened using a restriction digest test. The positive clones were verified by sequence analysis.

The correct bacterial clones were used to then inoculate 5 ml of LB broth containing 100 µg/ml ampicillin. The culture was placed into a shaker/incubator set at 37°C/200 rpm and was left to grow overnight. This 5-ml culture was then used to inoculate a 500-ml flask of Terrific Broth media containing 100 µg/ml ampicillin. The culture was incubated at 37°C/200 rpm for approximately 2 h when an aliquot of -aminolevulinic acid was added (final concentration 0.5 mM). The culture was incubated for another hour, and then an aliquot of IPTG was added (final concentration 1 mM). Upon induction with IPTG, the rotation speed was decreased to 110 rpm and the temperature was reduced to 30°C. The culture was allowed to incubate for an additional 16 h before the bacterial cells were harvested.

Preparation of Bacterial Membranes. The bacterial membranes were prepared using the method established by Guengerich et al. (1996). The bacterial cell pellets were resuspended in 100 mM tris-acetate buffer (pH 7.6) containing 500 mM sucrose and 0.5 mM EDTA, using approximately 14 ml for each gram wet weight of cells. The suspension was diluted with an equal volume of water containing 0.10 mg/ml lysozyme. The suspension was gently shaken for 30 min to hydrolyze the outer bacterial membrane. The resulting spheroplasts were pelleted at 4000g for 10 min and then resuspended in 100 mM potassium phosphate buffer (pH 7.4) containing 6 mM magnesium acetate, 20% glycerol (v/v), and 0.10 mM dithiothreitol, using approximately 2 ml for each gram wet weight. Protease inhibitors were then added (1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, 10 µM bestatin, and 0.04 U/ml aprotinin). The cells were lysed with two 20-s sonicator bursts (Branson Sonic Power; Branson Ultrasonics Corp., Danbury, CT) at 70% power. The resulting lysate was subjected to centrifugation at 10,000g for 10 min, and the resulting supernatant was then spun at 100,000g for 60 min. The membrane pellet was resuspended in 50 mM tris-acetate buffer (pH 7.6) containing 0.25 mM EDTA and 250 mM sucrose, and was stored at -70°C until further use.

Synthesis and Characterization of Conjugate [C]. Conjugate [C] was synthesized by combining 0.181 g of chloroacetic acid and 0.589 g of GSH in 10 ml of 100 mM phosphate buffer, pH 7.4 (Dowsley et al., 1995). The reaction was initiated by adjusting the pH to 7.4 with 0.5 N NaOH and then incubated for 2 h at 50°C. The chemical mixture was then reacted overnight at room temperature. The following day, 6 N HCl was added to adjust the pH to 2. Excess chloroacetic acid was extracted using ether. The aqueous phase containing conjugate [C] was removed, and ether remaining in the sample was evaporated. The product was purified by HPLC (Primesphere C-18 column, 5 µm, 250 x 10.00 mm; Phenomenex, Torrance, CA) using 0.1% trifluoroacetic acid in H₂O as the mobile phase and an injection volume of 1 ml (500 µl of 0.1% trifluoroacetic acid and 500 µl of the aqueous sample). The peak corresponding to that for conjugate [C] was collected, lyophilized, and weighed. The identity of the product was confirmed by ¹H NMR analysis. Conjugate [C] was used as a standard for LC/MS analysis. S-Ethyl glutathione (S-Et-GSH) was used as the internal standard.

Preparation of Analytical Standards. The analytical standards for conjugate [C] and the internal standard, S-Et-GSH, were prepared by dissolving 10 mg of each compound in 1 ml of H₂O. Stock standards were prepared by serial dilution. All standards were stored at -20°C and protected from light for the duration of the study. Quality control samples, used to validate the assay and to verify the quantitative accuracy of the assayed unknown samples, were prepared at concentrations of 50 ng/ml, 1 µg/ml, and 20 µg/ml in H₂O.

Quantitative Analysis of Conjugate [C] by LC/MS. Samples were thawed, vortexed, diluted 10-fold in H₂O containing 5 µg/ml S-Et-GSH, vortexed again, and analyzed by LC/MS. Liquid chromatographic separation of conjugate [C] and the internal standard (S-Et-GSH) was achieved using an Inertsil ODS-3 (100 x 3.0 mm, 3-µm particle size) reversed-phase HPLC column (MetaChem Technologies, Torrance, CA). A stepwise gradient of 0.1% (v/v) formic acid in dH₂O and methanol was used for separation. The column was equilibrated with 0.1% (v/v) formic acid at 25°C and a flow rate of 0.25 ml/min. The mobile phase was maintained at this composition for 3.75 min. A linear increase in the concentration of methanol to 25% followed from 3.75 to 4.00 min. The concentration of methanol was maintained at 25% for 3.90 min and returned to its initial conditions of 0.1% (v/v) formic acid between 7.90 and 8.00 min. The duration of the assay was 10.50 min with a 3.50-min time delay between samples to ensure proper re-equilibration of the LC column. The autosampler injection volume was set at 20 µl and was maintained at room temperature. Under these conditions, conjugate [C] eluted at 4.7 min, whereas S-Et-GSH eluted at 8.3 min.

The mass spectrometer was equipped with an electrospray ionization source and was operated in selected-ion monitoring mode. The [M + H]⁺ ions produced from conjugate [C] and S-Et-GSH were m/z 366 and m/z 336, respectively. However, the [M - Glu]⁺ ion of conjugate [C] at m/z 237 and the [M - Glu, -H₂O]⁺ ion of S-Et-GSH at m/z 190 were used for quantitative analysis to decrease the background MS signals from contaminating molecules. The mass spectrometer conditions were optimized for detection of conjugate [C] and S-Et-GSH and were as follows: fragmenter, 100 V; capillary voltage, 2500 V; gas temperature (nitrogen), 350°C at 10 l/min; and nebulizer pressure, 25 psig.

Integration and quantification of the chromatographic peaks were performed using the HP Chemstation software package (revision A.06.03) (Agilent Technologies, Palo Alto, CA). Determination of the concentration of conjugate [C] in the samples was achieved using a standard curve (0-25 µg/ml). The standard curve was fit with a quadratic equation weighted 1/Y², where y was the peak area ratio of analyte to internal standard and x was the concentration of analyte. Calibration curves constructed in this manner exhibited a correlation coefficient (R²) of >0.998.

Formation of Conjugate [C] in Microsomal Incubations. Microsomal incubations were performed in a total volume of 170 to 250 µl with a protein concentration of 5 mg/ml. The incubation mixtures consisted of microsomes, 0.1 M phosphate buffer, pH 7.4, and 1 mM NADPH. Lung microsomes were preincubated with NADPH for 3 min at 25°C; GSH and DCE were then added and allowed to react with gentle shaking for 30 min at 25°C. The reactions were terminated by cooling the samples on ice. The microsomal proteins were precipitated by centrifuging the samples for 30 min at 100,000g in a Beckman Airfuge (Beckman Coulter, Fullerton, CA) using an A115 rotor. The supernatant was subjected to LC/MS analysis. To study the effect of substrate concentrations, microsomes were incubated with DCE in concentrations ranging from 0.25 mM to 6.0 mM. For time course studies, formation of conjugate [C] was determined from 2 to 60 min after reaction with DCE (2 mM).

Formation of Conjugate [C] by Recombinant CYP2F and Recombinant CYP2E1 Enzymes. Formation of conjugate [C] by rCYP2F3 was determined in bacterial cell membrane fractions containing 25 pmol of CYP2F3 (goat) expressed in DH5 bacterial cells. Conjugate [C] formation by rCYP2E1 was determined in incubations containing 25 pmol of rat or human CYP2E1. Incubations containing rCYP2F3 or rCYP2E1, DCE, 2 mM NADPH, and 2 mM GSH were carried out for 30 min.

Rates of formation of conjugate [C] by rCYP2F2 and rCYP2F4, the rat CYP2F isoform cloned recently (R. M. Baldwin, M. A. Shultz, and A. R. Buckpitt, manuscript submitted for publication) were determined using insect cell lysates and procedures as described (Shultz et al., 1999), with minor modifications. Reaction mixtures containing 15 pmol of rCYP2F2 or rCYP2F4, NADPH-cytochrome P450 reductase (40 units/pmol P450) and 2 mM 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate in 0.1 M Na₂HPO₄, pH 7.4, were preincubated at 4°C for 2 h. NADPH-cytochrome P450 oxidoreductase was purified from mouse liver and specific activity determined using procedures described previously (Shultz et al., 2001). After the preincubation, an NADPH-regenerating system (2 U of glucose-6-phosphate dehydrogenase, 15 mM glucose 6-phosphate, 2 mM NADP, and 1 mM MgCl₂) and GSH (10 mM) were added; an incubation volume of 250 µl was used. Concentrations of DCE in methanol (2.5 µl) ranging from 0.5 to 5.0 mM were then added and the incubations were carried out at 25°C for 10 min in a shaking water bath. After centrifugation to remove the proteins, the supernatants were stored at -80°C until analysis by LC/MS as described.

Instrumentation. Quantitative analysis of conjugate [C] and S-Et-GSH was performed using a Hewlett-Packard Series 1100 LC-MSD system (Agilent Technologies, Palo Alto, CA). HPLC experiments were performed using a Beckman System Gold Programmed Solvent Model with a Beckman Diode Array Detector Module (Beckman Coulter).

Statistical Analysis. Data are expressed as mean ± S.D. and were analyzed using the Student's t test to identify significant differences between experimental groups (p < 0.05). The apparent K_m and V_max values for the rate of conjugate [C] formation were determined by Michaelis-Menten kinetics using GraphPad Prism Version 4 (GraphPad Software, Inc., San Diego, CA).

	Results

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Synthesis of Conjugate [C]. The synthesized conjugate [C] eluted at 13.4 min on the Primesphere C-18 HPLC column. The identity of conjugate [C] was confirmed by spectroscopic data: ¹H NMR (D₂O): 2.21 (m, 2H, Glu ß), 2.58 (m, 2H, Glu ), 3.11 (dd, 1H, J = 14.1, 5.0, Cys ), 3.39 (d, 1H, J = 15.8, -CH₂COOD), 3.99 (s, 2H, Gly), 4.03 (t, 1H, J = 6.6, Glu ), 4.59 (dd, 1H, J = 8.6, 5.0, Cys ß). These data are consistent with those reported previously for conjugate [C] (Dowsley et al., 1995).

LC/MS Detection of Conjugate [C] in Lung Microsomal Incubations. Selected ion monitoring profiles and spectra for conjugate [C] and the internal standard, S-Et-GSH, are displayed in Fig. 2. The major fragment ion of S-Et-GSH was m/z 190 and was detected at 8.3 min, using the Inertsil ODS-3 column. The ion fragment of conjugate [C] used in analysis had a mass of 237 and eluted at 4.7 min. Selected ion monitoring profiles and spectra depicting the products of the lung microsomal incubations are shown in Fig. 3. The data illustrated in Fig. 3 (a-d) demonstrate increasing conjugate [C] levels with increasing concentrations of DCE (0.5-4.0 mM) used in the lung microsomal incubations. In the controls in which NADPH was omitted from the incubations, conjugate [C] was detected only at background levels (Fig. 3, a^*).

View larger version (23K): [in this window] [in a new window]   FIG. 2. A, mass spectra generated by scanning LC/MS analysis of a 5 µg/ml standard containing conjugate [C] and S-Et-GSH. MS spectra were collected using LC/MS conditions described under Materials and Methods, except that the fragmenter voltage was 75 V and the mass spectrometer was set to scan for ions between m/z 50 and m/z 375. The [M + H]+ ions for conjugate [C] and S-Et-GSH were m/z 366 and 336, respectively. The [M - Glu]+ ion of conjugate [C] (m/z 237) and the [M - Glu, -H2O]+ ion for S-Et-GSH (m/z 190) were used for the quantitative analysis of these compounds. B, mass spectra generated from the analysis of a 5 µg/ml standard containing conjugate [C] and S-Et-GSH using selected ion monitoring LC/MS, as described under Materials and Methods.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Selected ion monitoring profiles generated from the analysis of conjugate [C] (top panel) and the internal standard, S-Et-GSH (bottom panel), from lung microsomal incubations containing various concentrations of DCE with or without NADPH. The concentrations of DCE used in the incubations were 4, 2, 1, or 0.5 mM and are labeled as a, b, c, and d, respectively. a*, 4 mM DCE without NADPH.

Formation of Conjugate [C] by Recombinant CYP2E1 and Recombinant CYP2F Enzymes. In incubations of DCE with the recombinant P450 enzymes, rat and human CYP2E1, CYP2F3, and CYP2F4, production of conjugate [C] was highly correlated with substrate concentrations, with R² values that were equal to or higher than 0.90, whereas the correlation for rCYP2F2 was positive but less robust (R² = 0.52) (Figs. 4 and 5). The kinetics of DCE metabolism in incubations of human and rat rCYP2E1 and rCYP2F isoforms (CYP2F2, CYP2F3, CYP2F4) are shown in Figs. 4 and 5. Kinetic analyses of rCYP2E1 showed that the K_m for rat rCYP2E1 (74 µM) was about 10-fold lower than the K_m for human rCYP2E1 (725 µM) (Fig. 4). The apparent V_max/K_m ratio for rat rCYP2E1 (16 x 10^-3) was 8-fold higher than the ratio for human rCYP2E1 (2 x 10^-3). The K_m for DCE metabolism by rCYP2F2 (254 µM) was about 2- and 22-fold lower than the K_m values for rCYP2F3 (533 µM) and rCYP2F4 (5.6 mM), respectively (Fig. 5). Of the rCYP2F enzymes, the apparent V_max/K_m ratios for rCYP2F2 (11 x 10^-3) and rCYP2F4 (13 x 10^-3) were similar, and were both higher than the ratio for rCYP2F3 (7 x 10^-4).

View larger version (17K): [in this window] [in a new window]   FIG. 4. Kinetic analyses of the rates of formation of conjugate [C] by recombinant rat CYP2E1 (A) and recombinant human CYP2E1 (B). Incubations (N = 3-4) contained 25 pmol of recombinant rat or human CYP2E1, NADPH, GSH, and DCE (0.05-1.0 mM). Conjugate [C] was not formed in incubations in which DCE or NADPH was omitted.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Kinetic analyses of the rates of formation of conjugate [C] in incubations of recombinant CYP2F2 (A), recombinant CYP2F3 (B), or recombinant CYP2F4 (C) with DCE. Reaction mixtures contained 15 pmol of CYP2F2 or CYP2F4, NADPH-cytochrome P450 reductase, NADPH-generating system, GSH, and DCE (5 µM to 5.0 mM). Conjugate [C] was not formed in incubations in which DCE or NADPH was omitted.

Formation of Conjugate [C] in Incubations of Lung Microsomes from CD-1, Wild-Type, and CYP2E1-Null Mice. As expected, experiments using lung and liver microsomes and Western blot analysis confirmed the presence and absence of the CYP2E1 protein in the wild-type and CYP2E1-null mice, respectively (data not shown). Formation of conjugate [C] from DCE was detected in incubations containing NADPH and lung microsomes from CD-1, CYP2E1-null, and wild-type mice (Fig. 6), and was not detected when NADPH was omitted. At a DCE concentration of 4 mM, the levels of conjugate [C] produced in CD-1 mice were significantly higher than in wild-type and CYP2E1-null mice; the levels in wild-type and CYP2E1-null mice amounted to about 43 and 29% of the level in CD-1 mice. The amount of [C] produced in CYP2E1-null mice was about 66% of the level in wild-type mice, but the difference was not statistically significant. Formation of [C] in the microsomal incubations in CD-1, wild-type, and CYP2E1-null mice was time-dependent (data not shown). The rates of formation of conjugate [C] in all three murine strains were highly correlated with DCE concentrations ranging from 0.25 to 6 mM and yielded R² values equal to or higher than 0.942. Maximal levels of [C] were produced at 4 mM DCE in both CD-1 and wild-type mice (Fig. 6, A and B). In CYP2E1-null mice, a peak level was detected at 6 mM DCE, and saturation was not observed at the concentrations used in this study (Fig. 6C).

View larger version (14K): [in this window] [in a new window]   FIG. 6. Kinetic analyses of the rates of formation of conjugate [C] in incubations of DCE with lung microsomes from CD-1 (A), wild-type (B), and CYP2E1-null (C) mice. Lung microsomes (5 mg of protein/ml) were incubated with DCE (0.25-6 mM), NADPH, and GSH in a final volume of 250 µl. Incubations were performed at 25°C for 30 min. Conjugate [C] formation was determined by LC/MS analysis. Conjugate [C] was not formed in incubations in which DCE or NADPH was omitted.

Kinetic analysis of the rates of production of conjugate [C] in the microsomal incubations showed that the K_m for DCE metabolism in CD-1 mice was 3- and 2.4-fold lower than the values in wild-type and CYP2E1-null mice, respectively (Fig. 6). The apparent V_max/K_m ratio (1.0) in CD-1 mice was greater than the ratios in wild-type (0.19) and CYP2E1-null (0.17) mice. Hence, the catalytic efficiency for DCE metabolism was greater in lung microsomes from CD-1 mice than in either the wild-type or CYP2E1-null mice. It is not known what levels of CYP2E1 and CYP2F2 were present in these lung microsomes, and hence the kinetic parameters for the rates of conjugate [C] formation were not directly comparable with those for the recombinant P450 enzymes. However, the amounts of cytochrome P450 content in lung microsomes from CD-1 (220 ± 81 pmol/mg protein), wild-type (210 ± 85 pmol/mg protein), and CYP2E1-null (164 ± 50 pmol/mg protein) mice were not significantly different from one another.

	Discussion

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In previous studies, we have used GSH to trap the DCE epoxide and identified it as its GSH conjugate (Dowsley et al., 1995). This strategy was facilitated by the use of [¹⁴C]DCE and HPLC analysis to detect the DCE metabolites formed. In in vitro studies, lung and liver microsomes were incubated with [¹⁴C]DCE, and identification of DCE metabolites was achieved by HPLC analysis and collection of each metabolite with retention times corresponding to the synthesized standards (Dowsley et al., 1999). Metabolite production was estimated from the radioactivity of the collected HPLC effluents (Dowsley et al., 1999). Estimation of the quantities of DCE metabolites formed by using [¹⁴C]DCE and HPLC analysis has proven to be a reliable method of detection. However, the cost of custom synthesis of [¹⁴C]DCE has become prohibitive, and the radiolabeled compound is additionally a potential hazard in the laboratory. An objective of this study was to develop a sensitive, selective, and reliable method for the detection and quantification of GSH conjugates of reactive DCE metabolites that do not require radiolabeled DCE. We have established methodology using LC/MS for detection and quantitation of conjugate [C], and this technique has eliminated the need for radio-labeled DCE. Our results showed that the major fragment ion of conjugate [C], [M - Glu]⁺, had a mass-to-charge ratio of 237 and eluted at 4.7 min, whereas that of the internal control, S-Et-GSH, [M-Glu, -H₂O]⁺, had a mass of 190 and eluted at 8.3 min (Fig. 2). Using this methodology, the kinetics of DCE metabolism was evaluated. The data presented in this report validate the application of LC/MS for the detection and quantitation of conjugate [C] by P450-dependent oxidation of DCE.

Participation of a specific P450 in the metabolism of a particular chemical is related, in part, to the metabolic activity of the P450 enzyme and its relative abundance in the target cell. In comparison to its content in the liver, CYP2E1 has been shown to be expressed at relatively low levels in the lung. However, this P450 has also been shown to be highly concentrated in the Clara cells and, as a result, its relative concentration at the cellular level is substantial (Forkert, 1995). Considerable evidence has accrued to support the involvement of CYP2E1 in the metabolism of DCE in lung and liver (Dowsley et al., 1995, 1996, 1999; Lee and Forkert, 1994, 1995; Forkert et al., 1996, 1999a,b; for review, see Forkert, 2001). However, more direct evidence was required to definitively affirm the role of CYP2E1 in mediating DCE bioactivation to metabolites such as the epoxide. In this study, we incubated recombinant rat and human rCYP2E1 with DCE and measured the rates of conjugate [C] production. Our results showed that both rat and human rCYP2E1 metabolized DCE, albeit with different affinities and efficiencies. The K_m (725 µM) for DCE metabolism by human rCYP2E1 was about 10-fold higher than the K_m (74 µM) for rat rCYP2E1 (Fig. 4). The V_max/K_m ratio (16 x 10^-3) for rat rCYP2E1 was 8-fold the ratio (2 x 10^-3) for human rCYP2E1. Hence, rat rCYP2E1 exhibited greater affinity for DCE than did the human isoform, as measured using the production of conjugate [C] from DCE as an endpoint. Moreover, the catalytic efficiency of rat rCYP2E1 for DCE metabolism was higher than that of human rCYP2E1. Relevant in this context were findings from comparative studies showing that the mean rate of formation of the DCE epoxide in murine lung microsomal incubations was about 2-fold higher than the level in human lung microsomal incubations (Dowsley et al., 1999). In summary, these studies provided data to unequivocally demonstrate the capabilities of CYP2E1 to bioactivate DCE to the epoxide.

We have also undertaken studies herein to address the involvement of other P450 enzymes, in addition to CYP2E1, in the bioactivation of DCE in lung tissues. Analyses of the results of incubations of DCE with several rCYP2F enzymes showed that conjugate [C] was generated by rCYP2F2, rCYP2F3, and rCYP2F4 (Fig. 5). The quantities of [C] formed in incubations with rCYP2F2 (R² = 0.52), rCYP2F3 (R² = 0.93), and rCYP2F4 (R² = 0.90) correlated with DCE concentrations. Kinetic analyses revealed that the K_m for DCE metabolism for rCYP2F2 (254 µM) was lower than for either rCYP2F3 (533 µM) or rCYP2F4 (5.6 mM), indicating that rCYP2F2 exhibited comparatively greater affinity for DCE than either rCYP2F3 or rCYP2F4. The V_max/K_m ratios for rCYP2F2 (11 x 10^-3) and rCYP2F4 (13 x 10^-3) were similar and were both higher than the ratio for rCYP2F3 (7 x 10^-4). These data were intriguing because rCYP2F4, which had a high K_m value, also had high catalytic efficiency for DCE metabolism and did not reach saturation at 3 mM DCE (Fig. 5C). On the other hand, rCYP2F2 had a lower K_m and reached saturation at concentrations greater than 1 mM DCE (Fig. 5A). On the basis of these findings, we postulated that rCYP2F2 catalyzed the oxidation of DCE with high affinity and was at the same time more sensitive to inactivation through catalytic production of the reactive epoxide, thus resulting in lower production of conjugate [C]. This phenomenon was also observed with rat and human rCYP2E1 (Fig. 4). These findings were consistent with those of recent studies showing the rapid loss of CYP2F2- and CYP2E1-immunoreactive protein after treatment of mice with DCE (Simmonds et al., 2004), presumably the result of enzyme adduction, inactivation, and subsequent degradation. Evaluation of the kinetic data for the rCYP2F and rCYP2E1 enzymes indicated that the relative catalytic efficiencies for DCE metabolism were greater for rat rCYP2E1 than for human rCYP2E1, rCYP2F2, CYP2F3, and rCYP2F4 (Figs. 4 and 5). Nonetheless, these findings supported the idea that bioactivation of DCE in murine lung can be catalyzed by both CYP2E1 and CYP2F2.

In this investigation, we have also performed studies to further characterize the involvement of CYP2F2 in DCE bioactivation using lung microsomes from wild-type (mixed 129/Sv and C57BL) and CYP2E1-null and outbred CD-1 mice. Conjugate [C] was generated by lung microsomes isolated from all three strains of mice, albeit at differing levels (Fig. 6). The rates of formation of conjugate [C] in CD-1 mice (Fig. 6A) were higher than in wild-type mice (Fig. 6B) using the same DCE concentration (4 mM). This finding was consistent with data from previous studies showing significantly higher levels of lung CYP2E1 in CD-1 than in C57BL/6 mice (Forkert et al., 2001). Formation of conjugate [C] in microsomes from CYP2E1-null mice represented about 66% of the level in microsomes from wild-type mice (Fig. 6), indicating that a P450 other than CYP2E1 accounted for DCE bioactivation. In view of the in vitro data presented in this report using recombinant P450 enzymes to generate conjugate [C], we propose that the residual metabolism of DCE in CYP2E1-null mice was the result of CYP2F2 catalysis. This assumption was supported by data from recent studies showing that when mice were pretreated with 5-phenyl-1-pentyne, both CYP2E1 and CYP2F were inhibited, leading to the complete abrogation of conjugate [C] production (Simmonds et al., 2004). Kinetic analyses revealed that the affinity for DCE metabolism in lung microsomes from CD-1 mice were higher than in microsomes from wild-type and CYP2E1-null mice (Fig. 6). The catalytic efficiencies for production of conjugate [C] in wild-type and CYP2E1-null mice were similar and were both lower than in CD-1 mice. These differences in catalytic efficiencies may be ascribed to the relative levels of CYP2E1 present in the lungs of the three murine strains as well as the overall contribution of CYP2F2, a lower-affinity enzyme, in the production of conjugate [C] by microsomes.

Taken together, the results of this study showed that LC/MS was a suitable method for quantitative analysis of conjugate [C] formation, and that both CYP2E1 and CYP2F family enzymes catalyzed the bioactivation of DCE to the epoxide in lung tissue. Our results also demonstrated for the first time that CYP2E1 represents the principal high-affinity enzyme involved in the bioactivation of DCE in murine lung.

	Footnotes

 
This study was supported by Grant MOP 11706 from the Canadian Institutes of Health Research (to P.G.F.) and United States Public Health Service Grants HL13645 and HL60143 from the National Heart, Lung, and Blood Institute (to G.S.Y.). The expression of CYP2F2 and CYP2F4 was conducted with support from the National Institute of Environmental Health Sciences Grant ES 08408 (to A.R.B.).

ABBREVIATIONS: DCE, 1,1-dichloroethylene; S-Et-GSH, S-ethyl glutathione; GSH, glutathione; [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; HPLC, high performance liquid chromatography; IPTG, isopropyl-thiogalactopyranoside; LC/MS, liquid chromatography/mass spectrometry; P450, cytochrome P450; LB broth, Lauria Bertani broth; r, recombinant.

Address correspondence to: Dr. Poh-Gek Forkert, Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6. E-mail: forkertp{at}post.queensu.ca

	References

Top Abstract Materials and Methods Results Discussion References

 


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