Reaction of Trichloroethylene and Trichloroethylene Oxide with

Trichloroethylene (TCE) has been shown to be toxic to experimental animals and humans. TCE oxide is a reactive electrophile formed during TCE oxidatio...
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Chem. Res. Toxicol. 2001, 14, 451-458

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Reaction of Trichloroethylene and Trichloroethylene Oxide with Cytochrome P450 Enzymes: Inactivation and Sites of Modification Hongliang Cai† and F. Peter Guengerich* Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received October 31, 2000

Trichloroethylene (TCE) has been shown to be toxic to experimental animals and humans. TCE oxide is a reactive electrophile formed during TCE oxidation and rearranges to acylating intermediates [Cai, H., and Guengerich, F. P. (1999) J. Am. Chem. Soc. 121, 11656-11663], which may be related to the toxicity. Mice treated with TCE have been reported to contain N6-dichloroacetylLys residues in P450 2E1, as detected by immunochemical methods. TCE can be oxidized by both P450 2E1 and (rat) 2B1. In this work, direct reaction of TCE oxide with either human P450 2E1, P450 2B1, or NADPH-P450 reductase was shown to lead to enzyme inactivation, and no recovery of the activity of either enzyme occurred, consistent with the view of inactivation reactions with Lys groups and not hydroxyls or Cys. Furthermore, Lys adducts were detected in the reaction of TCE oxide with both P450 2E1 and NADPHP450 reductase, with a larger amount of N6-formylLys observed compared to N6-dichloroacetylLys in both cases. Inactivation of P450 2E1 during NADPH-dependent TCE oxidation was not observed, compared to control experiments. However, inactivation of P450 2B1 during NADPH-dependent TCE oxidation was detected. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of tryptic peptides indicated that the direct reaction of TCE oxide with human P450 2E1 resulted in the modification of peptides containing Lys87 (AVKEALLDYK), Lys251 (VKEHHQSLDPNCPR), and Lys487 (YKLCVIPR), with either a formyl or dichloroacetyl group attached. Lys87 and Lys487 of human P450 2E1 appear to be modified during the oxidation of TCE, using the same approach. The results are considered in the context of comparison of species and P450s.

Introduction (TCE)1

Trichloroethylene is a commodity solvent; production levels in the United States alone have been as high as 275 × 106 kg/year (1). TCE is of concern in regard to several health issues (2). The pharmacology and toxicology of TCE have been reviewed a number of times (2-5). At low exposure levels, TCE is generally considered to be a reasonably safe chemical (6), and TCE had been used as an anesthetic in the past (3, 7). However, the formation of tumors in mice treated with TCE (1, 2) prompted concerns about the toxicology of TCE, along with exposure of large numbers of individuals due to (previous) use of TCE as a solvent in extraction of caffeine from coffee and particularly exposure of workers using TCE vapor in metal degreasing (1, 2). TCE has also been of concern because it is the most common chemical found in waste dump sites (8) and often contaminates drinking water supplies (9). * To whom correspondence should be addressed: Department of Biochemistry, Vanderbilt University School of Medicine, 23rd and Pierce Avenues, 638 Medical Research Building I, Nashville, TN 372320146. Telephone: (615) 322-2261. Fax: (615) 322-3141. E-mail: [email protected]. † Present address: Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research & Development, 2800 Plymouth Rd., Ann Arbor, MI 48105. 1 Abbreviations: TCE, trichloroethylene; DCPIP, 2,4-dichlorophenolindophenol (sodium salt); MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; ESI, electrospray ionization; MOPS, 3-(N-morpholino)propanesulfonic acid; b5, cytochrome b5.

Toxicities of TCE, including carcinogenicity, have been observed at several sites in laboratory animals, including liver (10), lung (11), and kidney (12, 13). Metabolism is generally believed to be involved in the toxicities of TCE, apart from the general anesthetic effects, and the subject has been reviewed extensively (2, 12, 14, 15). The toxicities of TCE in animals have been demonstrated at relatively high doses, and there is considerable concern about extrapolation of these results to humans (16). One approach to estimating human risk has been to extrapolate metabolism from experimental animals to humans using physiologically based pharmacokinetic models (17), including efforts to account for interindividual variability (18). The metabolism of TCE in mammals proceeds by two overlapping pathways, GSH conjugation and oxidation (12, 15). Both pathways can generate reactive electrophiles. The GSH conjugation reaction is very slow with TCE (15). Oxidation of TCE yields TCE oxide. The formation of TCE oxide was proposed in 1977 (19, 20); subsequent work indicated that CO had been misidentified as the product but that the epoxide could be extracted and trapped as a 4-(4-nitrobenzyl)pyridine derivative (21). Protein adducts resulting from TCE are formed in vivo and can also be demonstrated to be produced in P450catalyzed reactions (22). Our own work has shown that the dominant pathway in adduct formation is the rearrangement of TCE oxide to acyl chlorides, which undergo

10.1021/tx0002280 CCC: $20.00 © 2001 American Chemical Society Published on Web 03/09/2001

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reaction with Lys to generate stable N6-formylLys and N6-dichloroacetylLys adducts (23, 24) and unstable esters (25). One concern in the interpretation of in vivo protein binding is the contribution of radiolabel to the protein from metabolites incorporated into amino acids through one-carbon pools (26). However, accelerator mass spectrometry has been used to demonstrate the presence of TCE-derived adducts in mice treated with very low doses of TCE, with a linear dose response to very low levels (27). Although much of the early biochemistry of TCE was demonstrated with the P450 2B subfamily enzyme (rat) P450 2B1 (21, 22), P450 2E1 appears to be a major P450 involved in the metabolism of TCE (28-30). P450 2E1 oxidation of TCE appears to differ somewhat from that by P450 2B1 (24). Pumford and his associates have used antibodies developed against N6-dichloroacetylLys to detect such modifications in circulating proteins of mice treated with TCE (31-34). One of the major proteins detected by this approach is P450 2E1 (33). TCE was also reported to be a mechanism-based inhibitor of mouse P450 2E1 (32). We considered several aspects of the interaction of TCE and TCE oxide with human P450 2E1 and with NADPHP450 reductase, including mechanism-based inactivation, the time course of inhibition of the enzymes in the context of adduct stability (25), and the location of modifications in human P450 2E1.

Experimental Procedures Caution: Care should be exercised in handling TCE oxide, a reactive electrophile. Use adequate skin protection and avoid inhalation. Chemicals. 2,4-Dichlorophenolindophenol sodium salt (DCPIP) was grade I (Sigma Chemical Co., St. Louis, MO). R-Cyano-4-hydroxycinnamic acid and 3-(N-morpholino)propanesulfonic acid (MOPS) were ACS grade (Aldrich Chemical Co., Milwaukee, WI). Phenyl isothiocyanate was sequanal grade (Pierce, Rockland, IL). Solvents for HPLC and HPLC/MS were HPLC grade (EM Science, Gibbstown, NJ). TCE was distilled (atmospheric pressure) to remove inhibitors prior to epoxide synthesis. TCE oxide was synthesized by m-chloroperbenzoic acid treatment of TCE and partially distilled (23). Concentrations of TCE oxide (solution in residual TCE) were estimated colorimetrically using 4-(4-nitrobenzyl)pyridine reagent (21, 23, 35). N5-Formyl-L-ornithine and N5-(dichloroacetyl)-L-ornithine were prepared as described previously (24). Enzymes. Trypsin (sequencing grade, modified) was purchased from Promega (Madison, WI). Recombinant human P450 2E1 was expressed in Escherichia coli and purified as described elsewhere (36, 37). Human P450 2E1 and NADPH-P450 reductase were coexpressed in E. coli, and membranes were prepared and used in some of the experiments (38) (these preparations are termed “P450 2E1/NADPH-P450 reductase membranes”). Rat NADPH-P450 reductase was expressed in E. coli (39) and purified as described previously (40). Rabbit cytochrome b5 (b5) was purified as described elsewhere (41, 42). Spectroscopy. UV/visible spectroscopy was carried out using a Cary 14/OLIS spectrophotometer equipped with a circulating water bath for temperature control (On-Line Instrument Systems, Bogart, GA). HPLC/Electrospray Ionization (ESI)-MS. MS was carried out with a Waters 2690 separations module (Alliance, Waters Corp., Milford, MA) linked with a Finnigan MAT TSQ/SSQ 7000 series mass spectrometer (Finnigan, Sunnyvale, CA). MS studies were performed using the positive ion mode with an electrospray needle voltage of 4.5 kV. N2 was used as the sheath gas (70 psi)

Cai and Guengerich to assist nebulization and as the auxiliary gas (10 psi) to assist with desolvation. The stainless steel capillary was heated to 200 °C, and the electrospray interface and mass spectrometer parameters were optimized to obtain maximum sensitivity. The tube lens and the heated capillary were operated at 75 and 20 V, respectively, and the electron multiplier was set at 2000 V. Matrix-Assisted Laser Desorption Ionization Time-ofFlight (MALDI-TOF) MS. Mass spectra were acquired with a Voyager-Elite instrument (Perseptive Biosystems, Framingham, MA) equipped with a nitrogen laser (λexc ) 337 nm). The accelerating voltage was 20 kV. All spectra were recorded in the positive linear mode. A saturated solution of R-cyano-4hydroxycinnamic acid in a C2H5OH/H2O/HCO2H mixture (45: 45:10), prepared fresh daily, was used as the MALDI-TOF matrix. Samples were prepared by mixing equal volumes of the analyte and matrix solution on the target and letting them dry. Mass calibration was performed by using neurotensin [(M + H+) ) 1672.92] as an external standard. Enzyme Assays. (1) Activity Assays of NADPH-P450 Reductase Treated with TCE Oxide (43). NADPH-P450 reductase (100 µL of a 2.5 µM solution in H2O) was reacted with the indicated amount of TCE oxide (added in 1 µL of CH3CN). An aliquot of the reaction mixture (1 µL) was used for activity assays after reaction for 40 min. A mixture of 0.90 mL of 0.30 M potassium phosphate buffer (pH 7.7), 0.08 mL of either 0.5 mM cytochrome c or 0.1 mM DCPIP, and 1 µL of the enzyme solution was incubated at 30 °C for 5 min to achieve temperature equilibrium. NADPH (10 µL, 10 mM) was added, and the increase in A550 (in the case of cytochrome c) or the decrease in A600 (for DCPIP) was recorded for 4 min. ∆A550 and ∆A600 per minute values were calculated from the initial linear phases. NADPH-cytochrome c reductase activity (nanomoles of cytochrome c reduced per minute per nanomole of reductase) was calculated using an 550 of 21 000 M-1 cm-1 (43-45). In the case of DCPIP assays, the activity (nanomoles of DCPIP reduced per minute per nanomole of reductase) was calculated using an 600 of 21 000 M-1 cm-1 (46). Activity assays of NADPH-P450 reductase reacted with 0.2 mM TCE oxide were also performed at varying times following the procedures described above with both assays. The catalytic activity of unreacted NADPH-P450 reductase was also measured at different times in control experiments. (2) Activity Assays of Purified P450 2E1 Treated with TCE Oxide (47). Purified P450 2E1 (14 pmol in 35 µL of H2O) was reacted with varying amounts of TCE oxide for 30 min and then swept with N2 for 5 min (to remove the residual small amount of TCE) (24) and reconstituted with b5 (0.056 µM), NADPH-P450 reductase (0.084 µM), and l-R-dilauroyl-snglycero-3-phosphocholine (0.84 µM) in 50 mM potassium phosphate buffer (pH 7.4) (total volume of 0.5 mL) for 45 min at room temperature (48). Subsequently, a mixture of 1.0 IU/mL yeast glucose-6-phosphate dehydrogenase, 0.5 mM NADP+, and either 0.50 mM sodium chlorzoxazone or 0.5 mM TCE was added to the reconstituted P450 2E1 mixture (final incubation volume of 0.50 mL) (47). After preincubation for 3 min at 37 °C, 10 mM glucose 6-phosphate was added to start the incubations. In the chlorzoxazone assays, after incubation for 30 min at 37 °C, the reaction was stopped and 6-hydroxychlrozoxazone was analyzed by HPLC (47). In the case of TCE assays, after incubation for 30 min at 37 °C, the reaction was stopped by the addition of 1.0 mL of icecold (CH3CH2)2O. The mixture was mixed using a vortex device, and the layers were separated by a brief centrifugation (3 × 103 × g, 5 min). The organic layer (ether extract) was analyzed directly for chloral using GC (Varian model 3700, Varian, Walnut Creek, CA) with an electron capture detector (24). A standard curve was prepared with authentic chloral hydrate. GC conditions were as follows: initial temperature, 100 °C; temperature gradient, 20 °C/min; final temperature, 200 °C; injector temperature, 250 °C; detector temperature, 340 °C; split

Trichloroethylene-P450 Interaction injection with a ratio of 1:50; SPB-50 capillary column (Supelco, Bellefonte, PA), 0.25 mm × 15 m × 15 m; and 0.5 µm film. In an alternative experiment, at varying times aliquots of reaction mixtures of purified P450 2E1 (70 pmol in 175 µL of H2O) with 3 mM TCE oxide were swept with N2 for 5 min (to remove residual TCE) and reconstituted with the addition of b5 (0.056 µM), NADPH-P450 reductase (0.084 µM), and L-Rdilauroyl-sn-glycero-3-phosphocholine (0.84 µM) in 50 mM potassium phosphate buffer (pH 7.4) (total volume of 0.5 mL) for 45 min at room temperature. Subsequently, assays of P450 2E1 activity were performed following the two procedures described above. At varying times, catalytic activities of unreacted P450 2E1 were also measured in control experiments with both chlorzoxazone and TCE assays. (3) Activity Assays of Purified P450 2B1 Treated with TCE Oxide. Purified rat P450 2B1 (49) (80 pmol in 100 µL of H2O) was reacted with varying amounts of TCE oxide for 30 min and then swept with N2 for 5 min (to remove a small amount of TCE) (24) and reconstituted with the addition of b5 (2.8 µM), NADPH-P450 reductase (3.3 µM), and L-R-dilauroylsn-glycero-3-phosphocholine (33 µM) in 100 mM potassium phosphate buffer (pH 7.4) (total volume of 0.5 mL) (48) at room temperature for 45 min followed by addition of catalase (300 units/mL) (50), superoxide dismutase (80 units/mL) (51), and 50 µM pentoxyresorufin (in 5 µL of Me2SO). The enzyme reaction was initiated at 37 °C with an NADPH-generating system (composed of 10 mM glucose 6-phosphate, 1.0 IU/mL yeast glucose-6-phosphate dehydrogenase, and 0.5 mM NADP+) (45) in a final volume of 0.5 mL. At varying times, an aliquot of the incubation mixture (80 µL) was transferred to a 0.50 mL mixture (at 37 °C) of 10 mM glucose 6-phosphate, 1.0 IU/mL yeast glucose-6-phosphate dehydrogenase, and 0.5 mM NADP+. After incubation for 15 min at 37 °C, the reaction was stopped by the addition of 4 mL of ice-cold CH3OH. The mixture was mixed using a vortex device, and the protein was precipitated by brief centrifugation (3 × 103 × g, 10 min). The supernatant was analyzed directly for fluorescence emission at 585 nm with excitation at 530 nm using a spectrofluorometer (Varian model SF-330, Varian). Control experiments with purified P450 2B1 were performed in the absence of TCE following exactly the procedure described above. At varying times, aliquots of reaction mixtures of purified P450 2B1 (80 pmol in 100 µL of H2O) treated with 2.5 mM TCE oxide were swept with N2 for 5 min (to remove residual TCE) and reconstituted with b5 (0.056 µM), NADPH-P450 reductase (0.084 µM), and L-R-dilauroyl-sn-glycero-3-phosphocholine (0.84 µM) in 50 mM potassium phosphate buffer (pH 7.4) (total volume of 4.5 mL) for 45 min at room temperature. An assay of the P450 2B1 activity was performed using the described oxidation of 7-pentoxyresorufin O-depentylation to resorufin. At varying times, catalytic activities of unreacted P450 2E1 were also measured in control experiments. (4) Assays of P450 2E1 Activity after TCE Oxidation (47). Incubations (at 37 °C) included human liver microsomes (0.07 or 0.15 µM P450), P450 2E1/NADPH-P450 reductase membranes (1.0 µM P450), or purified P450 2E1 (0.21 µM P450) with 0.10 M potassium phosphate buffer (pH 7.4), an NADPHgenerating system (45), catalase (300 units/mL) (50), superoxide dismutase (80 units/mL) (51), and 0.15 mM TCE (final volume of 0.5 mL). In the case of the purified P450 2E1 system, P450 2E1 (0.21 µM P450) was reconstituted with b5 (0.5 µM), NADPH-P450 reductase (0.7 µM), and L-R-dilauroyl-sn-glycero3-phosphocholine (7 µM) in 100 mM potassium phosphate buffer (pH 7.4) (48). In all cases, the enzyme reactions were initiated with the NADPH-generating system. At varying times, an aliquot of the incubation mixture (80 µL) was transferred to a 0.50 mL mixture (at 37 °C) of 10 mM glucose 6-phosphate, 1.0 IU/mL yeast glucose-6-phosphate dehydrogenase, 0.5 mM NADP+, and 0.50 mM sodium chlorzoxazone (in 100 mM potassium phosphate buffer at pH 7.4). Assays of P450 2E1 activity were carried out by assessing chlorzoxazone 6-hydroxylation (vide supra). Control experiments with both purified P450

Chem. Res. Toxicol., Vol. 14, No. 4, 2001 453 2E1 and P450 2E1/NADPH-P450 reductase membranes were performed in the absence of TCE following the procedure described above. (5) Assays of P450 2B1 Activity after TCE Oxidation (47). Purified P450 2B1 (1.0 µM P450) was reconstituted with b5 (2.8 µM), NADPH-P450 reductase (3.3 µM), and l-R-dilauroyl-sn-glycero-3-phosphocholine (33 µM) in 100 mM potassium phosphate buffer (pH 7.4) (48) at room temperature for 45 min followed by addition of catalase (300 units/mL) (50), superoxide dismutase (80 units/mL) (51), and 0.15 mM TCE. The enzyme reaction was initiated at 37 °C with an NADPH-generating system (45), in a final volume of 0.5 mL. At varying times, an aliquot of the incubation mixture (80 µL) was transferred to a 0.50 mL mixture (at 37 °C) of 10 mM glucose 6-phosphate, 1.0 IU/mL yeast glucose 6-phosphate dehydrogenase, 0.5 mM NADP+, and 50 µM pentoxyresorufin (added in 5 µL of Me2SO). Assays of P450 2B1 activity were carried out by assessing fluorescence emission at 585 nm (vide supra). Control experiments with purified P450 2B1 were performed in the absence of TCE following the procedure described above. MALDI-TOF Analysis of Tryptic Peptides. TCE oxide (in dry CH3CN, added to a final concentration of 1.0 mM) was reacted with a solution of purified human P450 2E1 (1.4 µM) in 100 µL of 10 mM NH4CH3CO2 (the P450 2E1 had been dialyzed against this buffer). After 15 min, the mixture was evaporated under a stream of N2 (to remove TCE and CH3CN). A buffer of 100 mM NH4HCO3 (100 µL) was added followed by addition of 15 µL of trypsin solution (final concentration of 0.05 µg/µL). The mixture was incubated at 37 °C overnight. A small amount of the final solution (10 µL) was extracted using a C18 ZipTip (Millipore Corp., Bedford, MA) and analyzed by MALDITOF/MS (vide supra). Purified P450 2E1 (0.21 µM P450) was reconstituted at 37 °C for 45 min with b5 (0.5 µM), NADPH-P450 reductase (0.7 µM), and L-R-dilauroyl-sn-glycero-3-phosphocholine (7.0 µM) in 100 mM potassium phosphate buffer (pH 7.4) (48). Incubation (at 37 °C) included the reconstituted P450 2E1 mixture, E. coli superoxide dismutase (80 units/mL) (51), bovine catalase (300 units/mL) (50), and 0.15 mM TCE (final volume of 0.5 mL). The enzyme reaction was initiated with an NADPH-generating system (composed of 10 mM glucose 6-phosphate, 1.0 IU/mL yeast glucose-6-phosphate dehydrogenase, and 0.5 mM NADP+). After incubation for 45 min, P450 2E1 was separated from the mixture using HPLC. The conditions (Vydac protein C4 column, 2.1 mm × 150 mm, The Separation Group, Hesperia, CA) were as follows: flow rate, 0.50 mL/min; solvent A, 20 mM NH4CH3CO2 in H2O (pH 6.5); solvent B, 20 mM NH4CH3CO2 (pH 6.5) and CH3CN (pH 7.3) (5:95, v:v); 60% A and 40% B at time zero, 50% A and 50% B at 15 min, 20% A and 80% B at 30 min, 20% A and 80% B at 35 min, 60% A and 40% B at 45 min, and 60% A and 40% B at 50 min. Absorbance was monitored at 220 nm. The tR of P450 2E1 was ∼31 min. The P450 2E1 peak was collected, dried in vacuo, and subsequently dissolved in 100 mM NH4HCO3 (100 µL) followed by an addition of 10 µL of trypsin solution (final concentration of 0.05 µg/µL). The mixture was digested at 37 °C overnight (under Ar) and analyzed by MALDITOF/MS (vide supra). HPLC/ESI-MS Analysis of Tryptic Peptides. Tryptic peptides from P450 2E1 and TCE oxide-treated P450 2E1 were analyzed by HPLC/ESI-MS. The HPLC conditions (Vydac C18 column, 2.1 mm × 250 mm) were as follows: flow rate, 0.20 mL min-1; solvent A, 0.05% HCO2H and 0.005% CF3CO2H in a 90:10 (v:v) H2O/CH3OH mixture; solvent B, 0.05% HCO2H and 0.005% CF3CO2H in a 10:90 (v:v) H2O/CH3OH mixture; 100% A and 0% B at time zero, 0% A and 100% B at 45 min, 0% A and 100% B at 60 min, and 100% A and 0% B at 65 min. Absorbance was monitored at 214 nm. Analysis of N6-AcylLys Residues of P450 2E1 or NADPH-P450 Reductase Modified with TCE Oxide (24). The indicated amounts of TCE oxide (in dry CH3CN) were added to a solution of purified human P450 2E1 (1.4 µM) in 100 µL of 10 mM NH4CH3CO2 (dialyzed against this buffer) or a solution of

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purified NADPH-P450 reductase (2.0 µM). After 15 min, the mixture was evaporated under a stream of N2 (to remove TCE and CH3CN). A buffer of 250 mM potassium MOPS (pH 7.5, 200 µL) was added. The final mixture {with addition of internal standards [25 µL of N5-(dichloroacetyl)-L-ornithine (4 mM) and 25 µL of N5-formyl-L-ornithine (4 mM)]} was bubbled with Ar for 5 min (to remove O2 to minimize microbial contamination) and digested with 100 µL of a proteinase K solution in H2O (10 mg/mL) for 14 h at 37 °C, with the addition of a further 50 µL of a proteinase K stock after digestion of 7 h. After digestion, the mixture was taken to dryness in vacuo. A coupling buffer [10:5:2:3 (v:v:v:v) CH3CN/pyridine/(C2H5)3N/H2O mixture, 0.25 mL] was used to dissolve the residue, and then phenyl isothiocyanate (75 µL, 0.63 mmol) was added. After 15 min, the reaction mixture was taken to dryness in vacuo. The residue was dissolved in 0.25 mL of a solution of 0.10 M NH4CH3CO2 (pH 7.4) and CH3CN (3:7, v:v) for HPLC/MS analysis (23, 52). HPLC conditions (Zorbax Rx-C8 column, 2.1 mm × 150 mm, Mac-Mod, Chadds Ford, PA) were as follows: flow rate, 0.2 mL min-1; solvent A, 10 mM NH4CH3CO2 in H2O (pH 4.4); solvent B, 10 mM NH4CH3CO2/CH3CN (pH 7.4, 3:7, v:v); 95% A and 5% B at time zero, 85% A and 15% B at 10 min, 50% A and 50% B at 25 min, 50% A and 50% B at 32 min, 0% A and 100% B at 35 min, 0% A and 100% B at 41 min, 95% A and 5% B at 42.5 min, and 95% A and 5% B at 50 min. In control experiments, TCE oxide was added to 0.20 mL of potassium MOPS buffer (pH 7.5, 125 mM) and allowed to hydrolyze. After 15 min, P450 2E1 or NADPH-P450 reductase was added to the reaction mixture. The rest of the analytical procedure was carried out as described for the direct reaction with TCE oxide.

Results Reaction of TCE Oxide with Enzymes. Direct reaction of TCE oxide with purified human P450 2E1 led to inactivation of the enzyme in a concentration-dependent manner (Figure 1). Activity was lost toward both chlorzoxazone, a two-ring substrate, and the smaller substrate TCE. More than 90% of the catalytic activity could be inhibited at high TCE oxide concentrations. A concentration of TCE oxide (3 mM) was selected that would only partially inhibit P450 2E1 (based on panels A and C of Figure 1), and the reversibility of the inhibition was examined, because previous work indicated that inhibiton of some enzymes with TCE oxide is reversible (25). No recovery of P450 2E1 activity was observed, in contrast to what has been found with some other enzymes (25). The catalytic activity of P450 2B1 also decreased with increasing concentrations of TCE oxide (Figure 2A), and there was no recovery of catalytic activity (Figure 2B). The same phenomenon occurred with NADPH-P450 reductase (Figure 3). Activity was lost with both large (cytochrome c) and small (DCPIP) substrates. Analysis of Lys adducts was also carried out (Table 1). In the case of P450 2E1, the amount of N6-formylLys formed was 5-7 times that of N6-dichloroacetylLys at varying concentrations of TCE oxide (from 0.03 to 3.0 mM). The amount of Lys adducts increased with increasing TCE oxide concentration. When 3.0 mM TCE oxide was reacted with NADPH-P450 reductase, the amounts of N6-formylLys and N6-dichloroacetylLys were 6.5 and 0.6 nmol/nmol of protein, respectively. Assays of Mechanism-Based Inactivation of P450s. A significant amount of inactivation was observed with purified P450 2B1 during TCE oxidation compared to control assays (Figure 4). P450 2E1 was not inactivated during TCE oxidation with either human liver microsomes (Figure 5A), P450 2E1/NADPH-P450 reduc-

Cai and Guengerich

Figure 1. Inhibition of P450 2E1 by treatment with TCE oxide (0). (A) TCE oxide concentration-dependent inactivation of chlorzoxazone 6-hydroxylation activity. (B) Time dependence of chlorzoxazone 6-hydroxylation activity of P450 2E1 treated with 3 mM TCE oxide (0). The control experiment was carried out with TCE oxide (O). (C) TCE oxide concentration-dependent inactivation of conversion of P450 2E1-catalyzed TCE to chloral. (D) Time dependence of TCE oxidation to chloral by P450 2E1 treated with 3 mM TCE oxide (0). The control experiment was carried out without TCE oxide (O). The uninhibited rates of chlorzoxazone 6-hydroxylation and chloral formation were 4.6 and 1.5 nmol min-1 (nmol of P450)-1, respectively.

Figure 2. Inhibition of P450 2B1 by treatment with TCE oxide (0). (A) TCE oxide concentration-dependent inactivation of P450 2B1 activity [pentoxyresorufin O-dealkylation, uninhibited rate of 3.5 nmol min-1 (nmol of P450)-1]. (B) Time dependence of pentoxyresorufin O-dealkylation by P450 2B1 treated with 2.5 mM TCE oxide (0). The control experiment was carried out without TCE oxide (O).

tase membranes (Figure 5B), or purified P450 2E1 (Figure 5C), compared to control experiments. HPLC/ESI-MS Analysis of P450 2E1 Tryptic Peptides. ESI-MS analysis of intact P450 2E1 was not successful.2 When P450 2E1 was digested with trypsin and analyzed, 42 of the predicted 68 peptides were found. P450 2E1 was directly modified with TCE oxide and digested with trypsin. Modification of a Lys will block trypsin digestion and yield a peptide that would have 2 In discussions with P. F. Hollenberg (University of Michigan, Ann Arbor, MI), he also indicated difficulty in obtaining mass spectra of intact P450 2E1.

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Figure 3. Inhibition of NADPH-P450 reductase by treatment with TCE oxide (0). (A) TCE oxide concentration-dependent inactivation of NADPH-cytochrome c reduction activity of NADPH-P450 reductase. (B) Time dependence of NADPHcytochrome c reduction activity of NADPH-P450 reductase treated with 0.2 mM TCE oxide (0). The control experiment was carried out without TCE oxide (O). (C) TCE oxide concentration dependence of NADPH-DCPIP reduction activity of NADPHP450 reductase. (D) Time dependence of NADPH-DCPIP reduction activity of NADPH-P450 reductase treated with 0.2 mM TCE oxide (0). The control experiment was carried out without the addition of TCE oxide (O). The uninhibited rates for cytochrome c and DCPIP reduction were 3560 and 4010 nmol min-1 (nmol of reductase)-1, respectively.

Figure 4. Time-dependent loss of 7-pentoxyresorufin Odealkylation activity of P450 2B1 during enzymatic TCE oxidation by P450 2B1 (0). Control experiments (O) were carried out in the absence of TCE. The error bars denote the SD (n ) 3; when not visible, the SD was within the size of the symbol). The uninhibited pentoxyresorufin O-dealkylation activity was 3.5 nmol min-1 (nmol of P450)-1. Table 1. Lys Adducts of Purified P450 2E1 and NADPH-P450 Reductase Treated with TCE Oxide [TCE oxide] (mM)

N6-formylLys (nmol/nmol of protein)

N6-dichloroacetylLys (nmol/nmol of protein)

0.03 0.3 3.0

Purified P450 2E1 0.5 2.2 11.9

0.08 0.3 2.3

3.0

NADPH-P450 Reductase 6.5

0.6

been at least partially digested (due to Lys modification) and add either 28 (formyl) or 110 (dichloroacetyl) amu to what should be an internal Lys residue (in the

Figure 5. Time-dependent loss of chlorzoxazone 6-hydroxylation activity of P450 2E1 during enzymatic TCE oxidation by P450 2E1 (0). Control experiments (O) were carried out in the absence of TCE: (A) human liver microsomes, (B) human P450 2E1/NADPH-P450 reductase membranes, and (C) purified human P450 2E1. The error bars denote the SD (n ) 2 in panel A, n ) 3 in panels B and C). The uninhibited chlorzoxazone 6-hydroxylation activities were 1.5, 5.2, and 4.6 nmol min-1 (nmol of P450)-1 in panels A-C, respectively.

peptide). No modification was detected with the tryptic peptides from TCE oxide-treated purified human P450 2E1, compared to untreated P450 2E1 (results not shown). (See the Supporting Information for assignments of peptides observed in the HPLC/ESI-MS experiments.) MALDI-TOF MS Analysis of P450 2E1 Tryptic Peptides. The same approach to analysis of tryptic peptides (vide supra) was used with direct MALDI-TOF analysis of the mixture instead of HPLC/ESI-MS. Fewer peaks were identified, but three modified peptides were consistently observed in the TCE oxide-treated samples. Direct reaction of TCE oxide with purified human P450 2E1 resulted in modification of what appears to be Lys87 (AVKEALLDYK) with either a formyl or dichloroacetyl group, Lys251 (VKEHHQSLDPNCPR) with a formyl group, and Lys487 (YKLCVIPR) with a dichloroacetyl group (Figure 6). Lys87 (dichloroacetyl group) and Lys487 (formyl group) of human P450 2E1 were also adducted during the oxidation of TCE, which resulted in less extensive modification (Figure 7). (See the Supporting Information for the list of assignments of m/z peaks.)

Discussion Inactivation of P450 2E1, P450 2B1, and NADPHP450 Reductase by TCE Oxide. Cytochrome P450 2E1 has been shown to be a major P450 involved in the metabolism of TCE (13, 29, 30, 53, 54). Inactivation of P450 2E1 by direct reaction with TCE oxide was irreversible (Figure 1). TCE oxide inactivation of P450 2B1 (Figure 2) and NADPH-P450 reductase (Figure 3) was also irreversible. Similar inhibition profiles were seen with large and small substrates for both enzymes. For these inhibitions, millimolar concentrations of TCE oxide were required. These results, coupled with our earlier work on the instability of O- and S-acyl adducts (25),

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Figure 6. Mass spectra (MALDI-TOF) of TCE oxide-treated human P450 2E1 (after trypsin digestion at 37 °C): (A) control P450 and (B) P450 2E1 following reaction with 1 mM TCE oxide. See the Supporting Information for peak assignments. Asterisks denote peaks that could not be assigned to any predicted peptides.

Figure 7. Mass spectrum (MALDI-TOF) of human P450 2E1 after NADPH-dependent oxidation of TCE. P450 2E1 was separated from the incubation mixture with TCE by HPLC as described. The peaks assigned to adducted peptides are indicated with the molecular ions (MH+) and correspond to m/z 1103 and 1180 in Figure 6. See the Supporting Information for assignments. Asterisks denote peaks that could not be assigned to any predicted peptides.

suggest that N-acylLys adducts are formed and contribute to the inactivation. Previously, we detected formation of TCE-derived protein Lys adducts in purified P450 2E1 systems (24), suggesting that P450 2E1 is a target for reactive intermediates formed during TCE oxidation. However, we did not distinguish between P450 2E1 and NADPH-P450 reductase adducts previously (24). Pumford et al. (31) reported formation of TCE adducts with liver P450 2E1 in male B6C3F1 mice using a polyclonal antibody that could recognize N6-dichloroacetylLys-protein adducts in tissues. When purified P450 2E1 was treated with TCE oxide, protein Lys adducts were detected, with N6formylLys being the major product compared to N6dichloroacetylLys (Table 1). This finding, combined with the previous results in the literature, indicates that P450 2E1 is accessible to attack by intermediates formed in TCE oxidation, presumably acyl chloride intermediates formed from TCE oxide hydrolysis (23). The concentration of TCE oxide needed to inhibit 50% of P450 2E1 activity (3 mM) was similar with P450 2B1 but ∼10 times higher than for NADPH-P450 reductase (0.2 mM). However,

Cai and Guengerich

fewer Lys adducts were formed with the reductase than with P450 2E1 at a given concentration of TCE oxide (Table 1). These results can be interpreted as an indication that the reductase has Lys residues (susceptible to acylation) more critical to catalytic function than does human P450 2E1. However, the need for relatively high concentrations of TCE oxide should be noted. Inactivation of P450s during TCE Oxidation. A significant amount of P450 2B1 inhibition was detected during TCE oxidation (Figure 4), in line with our previously observed mechanism-based destruction of P450 2B1 heme (21, 55). Although the assays show inherently more variability and the “control” reactions have a loss of activity, no P450 2E1 inhibition was observed during TCE oxidation (Figure 5). In P450 systems, the amount of TCE oxide (formed from TCE oxidation) is usually in the low micromolar range (21, 24). Therefore, it is very unlikely that TCE oxide generation would be responsible for decreased catalytic activity in the inactivation of P450 2B1. We attribute the loss of P450 2B1 catalytic activity to the modification of heme, which had been reported previously (55), but the mechanism of this has not been fully characterized. This process is favored in rat P450 2B1 but apparently not with human P450 2E1 (Figure 5). Halmes et al. (32) reported a time-dependent loss of p-nitrophenol hydroxylation activity in liver microsomes of mice (treated with acetone to induce P450 2E1). The time-dependent loss was seen at a substrate concentration of 2.0 mM but not at 0.5 mM, and the significance of these results is not clear in that the Km for TCE oxidation to chloral in mice has been reported to be 2-35 µM (15, 56) and 28 µM in human liver microsomes (18). In summary, mechanism-based inactivation does not appear to be an issue with human P450 2E1. Sites of Modification of P450 2E1. Locating the positions of any O-acyl adducts is probably unrealistic in light of the known instability of derivatives (25). Our search was directed to identifying the positions of N6acylLys derivatives, because the available evidence indicated that (i) these adducts were formed (Table 1), (ii) such adducts were likely to be responsible for the inactivation of P450 2E1, as judged by the irreversibility, and (iii) these adducts might be relevant to the mouse TCE adducts reported by Pumford and his associates (31-34). Trypsin (or endolys C) digests provide a sensitive means of identifying Lys-adducted peptides by mass spectrometry in that acylation at a Lys blocks cleavage and adds, in this case, either 28 or 110/112 to the m/z of each peptide. HPLC/ESI-MS of the tryptic peptides of P450 2E1, without derivatization, yielded 42 identified peaks, of a predicted total of 68 (see the Supporting Information; note that 14 of the 42 identified peptides contained an internal Lys or Arg, indicative of a lack of complete digestion). When a sample of P450 2E1 was treated with 1.0 mM TCE oxide, which should be sufficient to form approximately four or five Lys adducts, no additional peaks could be identified. We used an alternate method of analysis, MALDI-TOF MS without preseparation of the peptides. Fewer peptides were identified using this approach (see the Supporting Information), but three additional peptides corresponding to those that might be modified were identified (Figure 6). In all three cases, a peak corresponding to the unmodified dipeptide could also be detected in the digest of unmodified P450 2E1. These results could be repeated in separate experiments

Trichloroethylene-P450 Interaction

(incubation with TCE oxide, digestion, and MS). Analysis of P450 2E1 modified during the course of TCE oxidation was more difficult because of the lower level of adducts, but we were able to consistently locate the Lys87-formyl and Lys487-dichloroacetyl adducts (Figure 7). From the analysis of the proteinase K digests (Table 1), we would expect approximately four N6-formyl- and one N6-dichloroacetylLys adduct to be formed after the reaction of P450 2E1 with 1.0 mM TCE oxide. The recovery of one formyl adduct, one dichloroacetyl adduct, and one residue modified with some of each group is not unreasonable but cannot be considered to necessarily represent all Lys adducts. The available results do not allow the determination of fractional modification of each Lys. Further, the results cannot be used to draw conclusions about which of these Lys groups is involved in P450 2E1 function or stability or which can be recognized by antibodies. The locations of Lys residues 87, 251, and 487 in the three-dimensional structure of human P450 2E1 are a matter of speculation. Two models of the structure have been presented (57, 58), both based upon homologue modeling with bacterial P450 102. In neither model is residue 87, 251, or 487 in what is construed to be the substrate binding site. The alignment of Tan et al. (57) would place Lys87 in the B helix, Lys251 immediately after the H helix, and Lys487 following the β12 β-sheet. Lys487 is only 10 residues away from the C-terminus and may be exposed, which might render this site accessible to antibodies. Conclusions and Relevance to Issues of TCE Toxicity. Rat P450 2B1 exhibits mechanism-based inactivation during oxidation of TCE, a confirmation of previous results in which only heme loss was examined (21, 55) but human P450 2E1 does not. P450 2E1 also produces considerably more TCE oxide than human P450 2E1 (24), although the epoxide does not appear to be responsible for P450 2B1 inactivation. A general conclusion is that high concentrations of TCE oxide are required for protein modification and inactivation of enzymes (vide supra and ref 25). Immunochemical studies with rats and mice indicate that N6-dichloroacetylLys adducts can be formed during TCE metabolism (31-33). However, in human hepatocytes incubated with TCE, the major protein recognized by anti-N6-dichloroacetylLys had an apparent mobility 3 kDa faster than P450 2E1. Our work shows that both N6-dichloroacetyl- and N6-formylLys adducts can be formed in human P450 and can be located. Such modified P450 2E1 may find use as a marker of P450 exposure. However, analysis of N6-dichloroacetylLys underestimates the level of total binding to P450 because of the contribution of N6-formylLys and any unstable adducts.

Acknowledgment. We thank Prof. D. Hachey for helpful suggestions regarding the HPLC/MS analyses. This work was supported in part by U.S. Public Health Service Grants R35 CA44353 and P30 ES00267. H.C. was supported in part by U.S. Public Health Service Grant F32 ES05919. Supporting Information Available: Table of peptide peaks observed in the HPLC/ESI-MS experiments and tables of assignments of peaks observed in Figures 6 and 7. This material is available free of charge via the Internet at http:// pubs.acs.org.

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References (1) Seltzer, R. J. (1975) Reactions grow to trichloroethylene alert: industry and federal agencies respond to NCI preliminary report showing widely used solvent to be carcinogenic in mice. Chem. Eng. News, 41-43. (2) Oak Ridge National Laboratory (1998) Toxicological Profile for Trichloroethylene, Oak Ridge, TN. (3) Defalque, R. J. (1961) Pharmacology and toxicology of trichloroethylene: a critical review of the world literature. Clin. Pharmacol. Ther. 2, 665-688. (4) Huff, J. E. (1971) New evidence on the old problems of trichloroethylene. Ind. Med. 40, 25-33. (5) Smith, G. F. (1966) Trichloroethylene: a review. Br. J. Ind. Med. 23, 249-262. (6) Stewart, R. D., Dodd, H. C., Gay, H. H., and Erley, D. S. (1970) Experimental human exposure to trichloroethylene. Arch. Environ. Health 20, 64-71. (7) Van Dyke, R. A., and Chenoweth, M. B. (1965) Metabolism of volatile anesthetics. Anesthesiology 26, 348-357. (8) Marshall, E. (1986) Woburn case may spark explosion of lawsuits. Science 234, 418-420. (9) Abelson, P. H. (1990) Volatile contaminants of drinking water. Science 247, 141. (10) Davidson, I. W. F., and Beliles, R. P. (1991) Consideration of the target organ toxicity of trichloroethylene in terms of metabolite toxicity and pharmacokinetics. Drug Metab. Rev. 23, 493-599. (11) Forkert, P. G., and Birch, D. W. (1989) Pulmonary toxicity of trichloroethylene in mice: covalent binding and morphological manifestations. Drug Metab. Dispos. 17, 106-113. (12) Goeptar, A. R., Commandeur, J. N. M., van Ommen, B., van Bladeren, P. J., and Vermeulen, N. P. E. (1995) Metabolism and kinetics of trichloroethylene in relation to toxicity and carcinogenicity. Relevance to the mercapturic acid pathway. Chem. Res. Toxicol. 8, 3-21. (13) Bru¨ning, T., and Bolt, H. M. (2000) Renal toxicity and carcinogenicity of trichloroethylene: key results, mechanisms, and controversies. Crit. Rev. Toxicol. 30, 253-285. (14) Leibman, K. C., and Ortiz, E. (1977) Metabolism of halogenated ethylenes. Environ. Health Perspect. 21, 91-97. (15) Lash, L. H., Fisher, J. W., Lipscomb, J. C., and Parker, J. C. (2000) Metabolism of trichloroethylene. Environ. Health Perspect. 108, 177-200. (16) Steinberg, A. D., and DeSesso, J. M. (1993) Have animal data been used inappropriately to estimate risks to humans from environmental trichloroethylene? Regul. Toxicol. Pharmacol. 18, 137-153. (17) Gonzalez-Leon, A., Merdink, J. L., Bull, R. J., and Schultz, I. R. (1999) Effect of pre-treatment with dichloroacetic or trichloroacetic acid in drinking water on the pharmacokinetics of a subsequent challenge dose in B6C3F1 mice. Chem.-Biol. Interact. 123, 239-253. (18) Lipscomb, J. C., Garrett, C. M., and Snawder, J. E. (1997) Cytochrome P450-dependent metabolism of trichloroethylene: interindividual differences in humans. Toxicol. Appl. Pharmacol. 142, 311-318. (19) Henschler, D., and Bonse, G. (1977) Metabolic activation of chlorinated ethylenes: dependence of mutagenic effect on electrophilic reactivity of the metabolically formed epoxides. Arch. Toxicol. 39, 7-12. (20) Uehleke, H., Poplawski, S., Bonse, G., and Henschler, D. (1977) Spectral evidence for 2,2,3-trichloro-oxirane formation during microsomal trichloroethylene oxidation. Xenobiotica 7, 94-95. (21) Miller, R. E., and Guengerich, F. P. (1982) Oxidation of trichloroethylene by liver microsomal cytochrome P-450: evidence for chlorine migration in a transition state not involving trichloroethylene oxide. Biochemistry 21, 1090-1097. (22) Miller, R. E., and Guengerich, F. P. (1983) Metabolism of trichloroethylene in isolated hepatocytes, microsomes, and reconstituted enzyme systems containing cytochrome P-450. Cancer Res. 43, 1145-1152. (23) Cai, H., and Guengerich, F. P. (1999) Mechanism of aqueous decomposition of trichloroethylene oxide. J. Am. Chem. Soc. 121, 11656-11663. (24) Cai, H., and Guengerich, F. P. (2000) Acylation of protein lysines by trichloroethylene oxide. Chem. Res. Toxicol. 13, 327-335. (25) Cai, H., and Guengerich, F. P. (2001) Reaction of trichloroethylene oxide with proteins and DNA: instability of adducts and modulation of functions. Chem. Res. Toxicol. 14, 54-61. (26) Stevens, D. K., Eyre, R. J., and Bull, R. J. (1992) Adduction of hemoglobin and albumin in vivo by metabolites of trichloroethylene, trichloroacetate, and dichloroacetate in rats and mice. Toxicol. Appl. Pharmacol. 19, 336-342.

458

Chem. Res. Toxicol., Vol. 14, No. 4, 2001

(27) Kautianinen, A., Vogel, J. S., and Turteltaub, K. W. (1997) Dosedependent binding of trichloroethylene to hepatic DNA and protein at low doses in mice. Chem.-Biol. Interact. 106, 109-121. (28) Nakajima, T., Okino, T., Okuyama, S., Kaneko, T., Yonekura, I., and Sato, A. (1988) Ethanol-induced enhancement of trichloroethylene metabolism and hepatotoxicity: difference from the effect of phenobarbital. Toxicol. Appl. Pharmacol. 94, 227-237. (29) Nakajima, T., Wang, R. S., Murayama, N., and Sato, A. (1990) Three forms of trichloroethylene-metabolizing enzymes in rat liver induced by ethanol, phenobarbital, and 3-methylcholanthrene. Toxicol. Appl. Pharmacol. 102, 546-552. (30) Guengerich, F. P., Kim, D.-H., and Iwasaki, M. (1991) Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol. 4, 168179. (31) Halmes, N. C., McMillan, D. C., Oatis, J. E., Jr., and Pumford, N. R. (1996) Immunochemical detection of protein adducts in mice treated with trichloroethylene. Chem. Res. Toxicol. 9, 451-456. (32) Halmes, N. C., Samokyszyn, V. M., and Pumford, N. R. (1997) Covalent binding and inhibition of cytochrome P4502E1 by trichloroethylene. Xenobiotica 27, 101-110. (33) Halmes, N. C., Perkins, E. J., McMillan, D. C., and Pumford, N. R. (1997) Detection of trichloroethylene-protein adducts in rat liver and plasma. Toxicol. Lett. 92, 187-194. (34) Griffin, J. M., Lipscomb, J. C., and Pumford, N. R. (1998) Covalent binding of trichloroethylene to proteins in human and rat hepatocytes. Toxicol. Lett. 95, 173-181. (35) Epstein, J., Rosenthal, R. W., and Ess, R. J. (1955) Use of p-(4nitrobenzyl)pyridine as analytical reagent for ethylenimines and alkylating agents. Anal. Chem. 27, 1435-1439. (36) Gillam, E. M. J., Guo, Z., and Guengerich, F. P. (1994) Expression of modified human cytochrome P450 2E1 in Escherichia coli, purification, and spectral and catalytic properties. Arch. Biochem. Biophys. 312, 59-66. (37) Bell-Parikh, L. C., Hosea, N. A., Martin, M. V., and Guengerich, F. P. (1999) Purification of cytochrome P450 enzymes. In Current Protocols in Toxicology, Chapter 4.2.1, John Wiley & Sons, New York. (38) Parikh, A., Gillam, E. M. J., and Guengerich, F. P. (1997) Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat. Biotechnol. 15, 784-788. (39) Hanna, I. H., Teiber, J. F., Kokones, K. L., and Hollenberg, P. F. (1998) Role of the alanine at position 363 of cytochrome P450 2B2 in influencing the NADPH- and hydroperoxide-supported activities. Arch. Biochem. Biophys. 350, 324-332. (40) Yasukochi, Y., and Masters, B. S. S. (1976) Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography. J. Biol. Chem. 251, 5337-5344. (41) Shimada, T., Misono, K. S., and Guengerich, F. P. (1986) Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction. J. Biol. Chem. 261, 909-921. (42) Imaoka, S., and Funae, Y. (1986) Ion-exchange high-performance liquid chromatography of membrane-bound protein cytochrome P-450. J. Chromatogr. 375, 83-90. (43) Phillips, A. H., and Langdon, R. G. (1962) Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. J. Biol. Chem. 237, 2652-2660.

Cai and Guengerich (44) Massey, V. (1959) The microestimation of succinate and the extinction coefficient of cytochrome c. Biochim. Biophys. Acta 34, 255-256. (45) Guengerich, F. P. (1994) Analysis and characterization of enzymes. In Principles and Methods of Toxicology (Hayes, A. W., Ed.) 3rd ed., pp 1259-1313, Raven Press, New York. (46) Dignam, J. D., and Strobel, H. W. (1975) Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat liver. Biochem. Biophys. Res. Commun. 63, 845-852. (47) Peter, R., Bo¨cker, R. G., Beaune, P. H., Iwasaki, M., Guengerich, F. P., and Yang, C.-S. (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450 IIE1. Chem. Res. Toxicol. 3, 566-573. (48) Bell, L. C., and Guengerich, F. P. (1997) Oxidation kinetics of ethanol by human cytochrome P450 2E1. Rate-limiting product release accounts for effects of isotopic hydrogen substitution and cytochrome b5 on steady-state kinetics. J. Biol. Chem. 272, 29643-29651. (49) Guengerich, F. P., Dannan, G. A., Wright, S. T., Martin, M. V., and Kaminsky, L. S. (1982) Purification and characterization of liver microsomal cytochromes P-450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or β-naphthoflavone. Biochemistry 21, 6019-6030. (50) Roberts, E. S., Alworth, W. L., and Hollenberg, P. F. (1998) Mechanism-based inactivation of cytochromes P450 2E1 and 2B1 by 5-phenyl-1-pentyne. Arch. Biochem. Biophys. 354, 295-302. (51) Guengerich, F. P., and Strickland, T. W. (1977) Metabolism of vinyl chloride: destruction of the heme of highly purified liver microsomal cytochrome P-450 by a metabolite. Mol. Pharmacol. 13, 993-1004. (52) Ng, L. T., Pascaud, A., and Pascaud, M. (1987) Hydrochloric acid hydrolysis of proteins and determination of tryptophan by reversedphase high-performance liquid chromatography. Anal. Biochem. 167, 47-52. (53) Nakajima, T., Wang, R. S., Elovaara, E., Park, S. S., Gelboin, H. V., and Vainio, H. (1992) A comparative study on the contribution of cytochrome P450 isozymes to metabolism of benzene, toluene and trichloroethylene in rat liver. Biochem. Pharmacol. 43, 251257. (54) Griffin, J. M., Gilbert, K. M., and Pumford, N. R. (2000) Inhibiton of CYP2E1 reverses CD4+ T-cell alterations in trichloroethylenetreated MRl+/+ mice. Toxicol. Sci. 54, 384-389. (55) Guengerich, F. P. (1986) Covalent binding to apoprotein is a major fate of heme in a variety of reactions in which cytochrome P-450 is destroyed. Biochem. Biophys. Res. Commun. 138, 193-198. (56) Clewell, H. J., III, Gentry, P. R., Covington, T. R., and Gearhart, J. M. (2000) Development of a physiologically based pharmacokinetic model of trichloroethylene and its metabolites for use in risk assessment. Environ. Health Perspect. 108, 283-305. (57) Tan, Y., White, S. P., Paranawithana, S. R., and Yang, C. S. (1997) A hypothetical model for the active site of human cytochrome P4502E1. Xenobiotica 27, 287-299. (58) Lewis, D. F. V., Bird, M. G., Dickins, M., Lake, B. G., Eddershaw, P. J., Tarbit, M. H., and Goldfarb, P. S. (2000) Molecular modelling of human CYP2E1 by homology with the CYP102 haemoprotein domain: investigation of the interactions of substrates and inhibitors within the putative active site of the human CYP2E1 isoform. Xenobiotica 30, 1-25.

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