Introduction

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We have reported previously that pretreatment of mice with the garlic derivative diallyl sulfone (DASO₂) protected them from pulmonary damage induced by 1,1,-dichloroethylene (DCE), a compound that is used in the manufacture of plastics and that is a prevalent water contaminant (1). The chloroethylene elicits preferential Clara cell injury in the lungs of mice treated with DCE (2). The protective effects provided by DASO₂ against DCE-induced Clara cell cytotoxicity appear to be linked to the mechanism by which the cellular injury is mediated. The toxic effects have been ascribed to oxidative metabolism by the cytochrome P450 enzyme CYP2E1, leading to production of an epoxide from DCE as assessed by conjugated products of the epoxide with glutathione (GSH) (3, 4). These products have been identified as 2-(S-glutathionyl)-acetyl glutathione ([B]) and 2-S-glutathionyl acetate ([C]). Substantial evidence has accrued to link the formation of the epoxide to the DCE-induced Clara cell lesion (4). The chemoprotective effects evoked by DASO₂ against the Clara cell damage are associated with a marked reduction in generation of conjugates [B] and [C] in lung microsomes isolated from mice treated with DASO₂ (1). The decreased formation of the GSH conjugates correlated with significantly diminished amounts of immunodetectable CYP2E1 protein and p-nitrophenol (PNP) hydroxylation, a catalytic activity associated with the CYP2E1 enzyme, in lung microsomes of mice treated with DASO₂. More recently, we have shown that pretreatment of mice with DASO₂ produced an 85% inhibition in the formation of conjugates [B] and [C] in lung cytosolic fractions isolated from mice treated with DCE (6).

The mechanisms involved in the protective action of DASO₂ against chemically induced carcinogenesis have been investigated in conjunction with the organosulfur compounds diallyl sulfide (DAS) and diallyl sulfoxide (DASO) (7). These studies have shown that DASO and DASO₂ are detectable in extracts of liver, blood, and urine from rats treated with DAS, suggesting that DASO and DASO₂ are metabolites derived from the metabolism of DAS. Moreover, findings from preliminary experiments using microsomal incubations revealed nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation of DASO from DAS and DASO₂ from DASO. These data are consistent with a metabolic pathway involving the sequential metabolism of DAS to DASO and subsequently to DASO₂. Results from recent studies, which identified oxidative products in the bile of rats treated with DAS, DASO, or DASO₂, indicated that all three compounds undergo metabolism and supported the contention that DAS and DASO are metabolized to DASO₂ (8).

The role of CYP2E1 in the metabolism of DAS, DASO, and DASO₂ has been investigated. Treatment of rats with DAS produced time- and dose-dependent decreases in hepatic CYP2E1-mediated catalytic activity and in content of immunodetectable CYP2E1 protein (7, 9). Time-dependent loss of CYP2E1 activity was also observed in rat liver after treatment with DASO and DASO₂ (7). However, the inhibition of CYP2E1 by DASO₂ was more pronounced and was manifested more rapidly than that produced by either DAS or DASO. Moreover, CYP2E1 activity was decreased in liver microsomes from acetone-treated rats that were incubated with DASO₂ and an NADPH-generating system, but this effect was not observed in incubations with either DAS or DASO. The efficacy of DASO₂ as a CYP2E1 inhibitor has been proposed to be due to mechanism-based inactivation of this P450, and a reactive intermediate generated from oxidation of DASO₂ has been implicated in CYP2E1 inactivation (7, 8). This action of DASO₂ on the CYP2E1 enzyme has been postulated to be responsible for the chemoprotective effects of DAS found in vivo.

The available findings led us to postulate that the mechanism mediating the protective effects of DASO₂ against DCE-induced Clara cell cytotoxicity is associated with the inhibition of CYP2E1-dependent metabolism of DCE through formation of a reactive intermediate from DASO₂. Here we have hypothesized that the metabolite generated from P450-mediated oxidation of DASO₂ is an epoxide (1,2-epoxypropyl-3,3'-sulfonyl-1'-propene [DASO₃]). Because of the reactivity of the epoxide, we have trapped the electrophilic epoxide with GSH and identified the products as GSH conjugates. The formation of DASO₃-GSH conjugates from DASO₂ was determined in incubations of lung microsomes from mice, and the results were compared with those obtained in incubations of human lung microsomes. Formation of DASO₃ was confirmed by detecting it as a 4-(p-nitrobenzyl)pyridine (NBP) derivative. The role of CYP2E1 in the generation of DASO₃ was investigated by identifying the DASO₃-GSH conjugates in incubations with CYP2E1-expressed lymphoblastoid microsomes, by determining the effects of DASO₂ on the CYP2E1 enzyme, and by performing immunoinhibition studies with a CYP2E1 inhibitory antibody. It was anticipated that the results of these studies would aid in elucidation of the mechanism by which the protective effects of DASO₂ against toxicities induced by chemicals including DCE are produced in the lung.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

Chemicals and reagents were obtained from the suppliers as indicated: acetonitrile and methanol, high performance liquid chromatography (HPLC) grade (EM Science Inc., Gibbstown, NJ); diethyl ether (BDH Inc., Toronto, ON, Canada); NADP⁺, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase (Sigma Chemical Co., St. Louis, MO); NBP, GSH, phosphoric acid (85%), acetone, and ethyl acetate (Aldrich Chemical Co., Montreal, PQ, Canada); sodium acetate and 4Å molecular sieve (Mallinckrodt Inc., Paris, KY); [³H]GSH, specific activity 43.8 Ci/mmol (DuPont Canada, NEN Ltd., Mississauga, ON, Canada); and rat CYP2E1-expressed human B-lymphoblastoid microsomes (GENTEST Corp., Woburn, MA). The activity of CYP2E1 is expressed in the human lymphoblast cell line AHH-1 from a transfected rat CYP2E1 complementary DNA (cDNA) (10). The CYP2E1 enzyme was induced by dimethylsulfoxide (0.2%) and the P450 reductase (cDNA derived) was induced by dexamethasone (1 µM) before preparation of the microsomes. DASO₂ was synthesized as described (11). DASO₃ was synthesized from DASO₂ by oxidation with freshly distilled dimethyldioxirane and was prepared as described previously (12). The inhibitory CYP2E1 monoclonal antibody (mAb 1-91-3) (13) was donated by Dr. S. S. Park (Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick, MD). All other chemicals used in this study were purchased from standard commercial suppliers.

Treatment of Animals

Female CD-1 mice weighing 24 to 28 g were obtained from Charles River (St. Constant, PQ, Canada). The mice were maintained on a 12-h light/dark cycle, provided freely with water and food (Mouse Diet 5015; PMI Nutrition International, Inc., Brentwood, MO), and equilibrated to laboratory conditions before being assigned to an experiment. The mice were killed by cervical dislocation for preparation of lung microsomes. To determine the effects of DASO₂ on PNP hydroxylase activity in vivo, mice were treated with DASO₂ (25 to 200 mg/kg, orally) and killed 2 h later for preparation of lung microsomes.

Preparation of Lung Microsomes

Human lung tissue was obtained from Kingston General Hospital (Kingston, ON, Canada) from consenting patients undergoing surgical lobectomies. The human lung experiments were performed according to a protocol approved by the Human Ethics Committee of Queen's University. Tissues distant from the primary lesions were excised, placed on ice, and transferred immediately to a biohazard facility.

For preparation of murine lung microsomes, lungs from 30 mice were pooled and homogenized in four volumes of cold phosphate-buffered KCl (1.15% KCl, 100 mM K₂PO₄, 1.5 mM ethylenediaminetetraacetic acid [EDTA], pH 7.4). Murine and human lung microsomes were prepared using procedures described previously (14, 15). Microsomal pellets were resuspended in 100 mM phosphate buffer (100 mM K₂PO₄, 1.5 mM EDTA, pH 6.8) in a volume of 0.2 ml/g tissue weight (mouse) or 0.1 ml/g tissue weight (human), and stored at 70°C. Protein concentrations were determined by the method of Lowry and coworkers (16) using bovine serum albumin as the standard.

Microsomal Incubations

Lung microsomes of mice were suspended in 100 mM K₂PO₄, pH 7.4, at a protein concentration of 3.0 mg/ml, whereas human lung microsomes were suspended at a protein concentration of 5.0 mg/ml. Reaction mixtures in a final volume of 1 ml consisted of 100 mM K₂PO₄ buffer containing 1.5 mM EDTA, 3 mg of microsomal protein, 5.0 mM MgCl₂, an NADPH-generating system (7.5 mM glucose-6-phosphate, 4 U glucose-6-phosphate dehydrogenase, 0.4 mM NADP⁺, and [³H]GSH (0.1 µCi, 1.0 mM). After preincubation of reaction mixtures for 3 min at 37°C in a shaking water bath, DASO₂ (0.25 to 1 mM) was added and the reaction was allowed to proceed for 30 min. The incubation time was extended to 2 h in experiments performed for detection of the DASO₃-GSH conjugates. After the incubations, proteins in the samples were precipitated with perchloric acid (70%) and centrifugation.

The role of CYP2E1 in mediating the formation of DASO₃ was determined in incubations performed with rat CYP2E1- expressed human B-lymphoblastoid microsomes co-expressed with NADPH-cytochrome P450 reductase. The reaction mixtures in a final volume of 100 µl contained 100 mM K₂PO₄ buffer, pH 7.4, microsomes containing 1.5 pmol of cytochrome P450, DASO₂ (0.5 mM), [³H]GSH (5.0 mM, 0.5 µCi), and an NADPH-generating system (3.3 mM MgCl₂, 3.3 mM glucose-6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, and 1.3 mM NADP⁺). After preincubation of reaction mixtures for 3 min at 37°C, DASO₂ was added and the incubations were carried out for 1.5 h. Proteins were then precipitated with trichloroacetic acid (20 µl/ ml) and centrifugation.

In the immunoinhibition experiments, lung microsomes were preincubated with an inhibitory CYP2E1 mAb according to procedures described previously (17). A mAb protein:microsomal protein ratio of 0.5 was used. This ratio has been shown previously to inhibit 70% of CYP2E1-mediated catalytic activity and 50% of the metabolism of CYP2E1-selective substrates (4, 18- 20). In control experiments, microsomes were preincubated with mAb HyHel 9, an antibody specific for egg white lysozyme (21). After reaction with the mAb, microsomes were incubated with DASO₂ and levels of DASO₃-GSH conjugates were determined.

In the microsomal samples used for HPLC analyses, the supernatants were lyophilized in vacuo and stored at 70°C. The samples were resuspended in degassed H₂O just before HPLC analysis. For protein immunoblotting and for measurement of PNP hydroxylase activity, microsomal pellets were washed three times with 5.0 ml of cold 100 mM K₂PO₄ buffer, pH 6.8, to remove residual DASO₂ and suspended in 2.0 ml of buffer.

Synthesis of DASO₃-GSH Conjugate Standards

[³H]GSH (1.2 mM, 0.2 µCi/ml) was dissolved in H₂O (10 ml) and the pH of the solution was adjusted to 7.8 with 0.25 N NaOH. The synthesized DASO₃ was dissolved in dry methanol (10 ml) and added immediately to the GSH solution in equimolar amounts. The mixture was stirred at 10°C in the dark for 18 h, lyophilized in vacuo, redissolved in 2.0 ml H₂O, and stored at 70°C. The products of this synthesis (100 µl) were analyzed with a reversed-phase C₁₈ column (5 µm, 4.6 × 250 mm; Phenomenex, Torrance, CA). The isocratic mobile phase was 5% aqueous methanol containing 0.06% trifluoroacetic acid (TFA) at a flow rate of 1.0 ml/ min as described (8). The column effluent was monitored at 200 nm. For detection of GSH-containing peaks, 0.25-ml aliquots of the effluent were collected over 60 min and levels of radioactivity were determined. The retention time of the DASO₃-GSH peak was determined to be at 20.5 min. A subsequent synthesis was performed using GSH and the synthesized DASO₃. The mixture (0.5 ml) was subjected to a semipreparative HPLC analysis using an Ultrasphere ODS column (5 µm, 10 × 250 mm; Beckman, Palo Alto, CA) and a flow rate of 5.0 ml/min. The peak eluting at 20.5 min was collected, lyophilized in vacuo, stored at 70°C, and subjected to nuclear magnetic resonance (¹H-NMR) analysis for confirmation of the identities of the GSH conjugates formed. The synthesized DASO₃-GSH conjugates were used subsequently as analytical standards.

HPLC Analysis of DASO₃-GSH Conjugates

The products obtained from the lung microsomal incubations (60 µl) were analyzed by HPLC as described. The mobile phase consisted of solvent A (0.06% aqueous TFA) and solvent B (acetonitrile containing 0.06% TFA) at a constant flow rate of 1.0 ml/min. The gradient started at 100% solvent A was followed by a linear increase to solvent B in 30 min and then 5% B min¹ to 90% B. The column effluent was monitored at 200 nm. For detection of GSH-containing peaks, 0.25-ml aliquots of the column effluent were collected over 60 min. The relative size of the peaks was estimated by summation and transformation of radioactive counts. The incubation mixtures containing DASO₂ and GSH were subjected to semipreparative HPLC analysis as described. The peak corresponding to the retention time of the standard was collected, and the solution was adjusted to pH 7.0 with ammonium hydroxide, lyophilized in vacuo, and stored at 70°C.

Formation of DASO₃ in Microsomal Incubations

Formation of DASO₃ in incubations of human and murine lung microsomes was estimated by measuring in DASO₃ derivatized with NBP (22, 23). The derivatized products has been used in previous studies for detection of alkylating products, including epoxides generated from trichloroethylene (24, 25) and DCE (3). The reaction mixtures contained 1.5 ml of 100 mM K₂PO₄ buffer, pH 7.4 (pH-adjusted with 70% phosphoric acid), 1.5 mg of microsomal protein/ml, DASO₂, and an NADPH-generating system as described previously. In the time-course studies, the reaction mixtures were preincubated for 3 min at 37°C after which DASO₂ (1 mM) was added and the reaction carried out from 5 to 50 min. In the concentration-response studies, DASO₂ (0.25 to 2.0 mM) was added and the incubations were conducted for 30 min. In the control reactions, NADP⁺ or DASO₂ was omitted from the reaction mixtures.

A mixture containing 0.5 ml 100 mM acetate buffer, pH 4.6, and 0.2 ml NBP (5% wt/vol in acetone) was preheated to 60°C and reacted for 10 min at 80°C with 1 ml of the microsomal incubation mixtures. Trichloroacetic acid (20 µl/ml) was then added, the pH was adjusted to < 5.0, and the incubation was performed for an additional 30 min. The mixtures were cooled by immersion in an ice bath (10 min), centrifuged at 5,000 × g for 4 min, and returned to the ice bath for 5 min. The supernatants (1.0 ml) were dispensed into prechilled (20°C) mixtures of acetone (1.0 ml) and ethyl acetate (2.5 ml), and kept at 20°C to prevent product degradation. A total of 10 N NaOH (1.5 ml) was added to the mixture while still maintained at 20°C, and formation of the alkylated NBP product was determined spectrally at 540 nm. Individual samples were warmed to 37°C with gentle mixing to achieve color development, which peaked within 3 to 5 min but degraded rapidly thereafter. Results were expressed with reference to a standard calibration curve that related absorbance at 540 nm against known quantities of the synthesized and purified DASO₃. Assays were performed under optimal conditions of linearity for time and substrate concentrations.

PNP Hydroxylase Activity

PNP hydroxylase activity was used as an index of CYP2E1 catalytic activity and was measured as described in our previous studies (1). Hydroxylase activity was determined in lung microsomes from untreated or DASO₂-treated mice or from human and murine lung microsomes incubated with DASO₂. Hydroxylase activity was estimated by the conversion of PNP to 4-nitrocatechol determined spectrally at 546 nm.

Instrumentation

HPLC experiments were performed on a Beckman System Gold Programmable Solvent Module 126 HPLC with a Beckman System Gold Module 168 ultraviolet detector. Spectral analyses for enzyme assays were performed on a Beckman Model DU 640B spectrophotometer. ¹H-NMR spectra were obtained with a Bruker Avance spectrometer (Rheinstetten, Germany) at 500 MHz.

Statistical Analysis

Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed by two-way analysis of variance followed by the Student-Newman-Keuls test to identify significant differences between experimental groups. The level of significance was set at P < 0.05.

	Results

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Synthesis of DASO₃-GSH Conjugate Standards

HPLC analysis of the reaction products of DASO₃ with GSH detected a major peak eluting at 20.5 min (Figure 1A). This peak was not present in reactions in which either DASO₃ or GSH was omitted. ¹H-NMR analysis revealed the identities of the products contributing to this peak as the epoxide-derived GSH conjugates S-(1R,S-[[1-hydroxymethyl-2,3'-sulfonyl]-1'-propenyl]ethyl)glutathione (conjugates [D] and [E], diastereomers) and S-(1-[[2R,S-hydroxypropyl]-3,3'-sulfonyl]-1'-propenyl)glutathione (conjugates [F] and [G], diastereomers). The spectral data of conjugates [D], [E], and [F] were consistent with those reported previously (8). ¹H-NMR (D₂O) for conjugate [F]: : 1.97 (2H, m, Glu-, ¹), 2.55 (2H, m, Glu-, ¹), 2.84 (2H, m, CH₂SG), 2.95 (1H, m, Cys-), 3.14 (1H, m, Cys-¹), 3.48 (2H, m, CH(OH) CH₂SO₂), 3.80 (1H, m, Glu-CH), 3.92 (2H, s, Gly-CH₂), 4.08 (2H, m, CH₂-CH = CH₂), 4.39 (1H, m, CH(OH)-CH₂SG), 4.60 (1H, m, Cys-), 5.60 (2H, m, CH = CH₂), 5.95 (1H, m, CH = CH₂). The presence of conjugates [D]/[E] was confirmed by a multiplet at : 3.42 (1H, m, S-CH-CH₂OH), which was shown by correlated spectroscopy (COSY) to be coupled to multiplets at 3.75 (1H, m, CH-CH₂-OH) and 3.80 (1H, m, CH-CH₂-OH), and also to a multiplet at 3.62 (2H, m, S-CH-CH₂SO₂). The cysteinyl methine proton of conjugates [D]/[E] at  4.65 (1H, m, Cys-) is also partially resolved from that of conjugates [F]/[G] and shown by COSY to be coupled to multiplets at 3.05 (1H, m, Cys-) and 3.19 (1H, m, Cys-¹). All other resonances were unresolved from those of conjugates [F]/[G].

View larger version (20K): [in this window] [in a new window]   Figure 1.   Radiochromatograms of DASO3-GSH conjugates subjected to reversed-phase HPLC analysis. Aliquots of the column effluent were collected, and levels of radioactivity were determined. The standard (A) was synthesized by reacting DASO3 (1.2 mM) with [3H]GSH (1.2 mM, 0.2 µCi/ml). The GSH conjugates were identified in incubations containing CYP2E1-expressed human B-lymphoblastoid microsomes (1.5 pmol cytochrome P450), [3H]GSH (0.5 mM, 0.5 µCi/ml), DASO2 (0.5 mM), and an NADPH-generating system in a final volume of 100 µl (B). The DASO3-GSH conjugates were also identified in murine (C) and human (D) lung microsomal incubations containing DASO2 (0.5 mM), [3H]GSH (1.0 mM, 0.1 µCi), and an NADPH-generating system in a final volume of 1 ml. Methods for the chemical synthesis and the microsomal incubations are detailed in MATERIALS AND METHODS.

Microsomal Incubations

Radiochromatograms obtained from HPLC analysis of supernates from incubations of murine lung microsomes conducted in the presence of DASO₂ and an NADPH-generating system revealed a major peak with a retention time (20.5 min) corresponding to that of the synthesized DASO₃-GSH standards (Figures 1A and 1C). The relative size of the 20.5-min peak was used to estimate for magnitudes of formation of the DASO₃-GSH conjugates. The DASO₃-GSH peak was also detected in incubations of human lung microsomes incubated with DASO₂ and an NADPH-generating system (Figure 1D). However, the amounts of GSH conjugates formed in human lung microsomal incubations were lower than those generated in incubations of lung microsomes from mice (Figures 1C and 1D). The GSH conjugates were not detected in murine and human lung microsomal incubations performed under conditions in which DASO₂ or an NADPH-generating system was omitted (data not shown).

Time-course experiments, using a concentration of 0.5 mM of DASO₂, revealed that formation of the DASO₃-GSH conjugates in murine lung microsomal incubations was time-dependent and was incremental from 0.5 to 2.0 h (Figure 2A). Extending the duration of the incubations did not produce any further increase in formation of the DASO₃-GSH conjugates. Similar results were obtained in time-course studies in which incubations of human lung microsomes with DASO₂ (0.5 mM) were carried out between 0.5 and 3.0 h (Figure 1C). As found in the incubations of lung microsomes from mice (Figure 1A), saturation was achieved at an incubation time of 2.0 h (Figure 2C). The production of the GSH conjugates was also concentration-dependent at DASO₂ amounts ranging from 0.25 to 0.75 mM. Peak levels were detected at a DASO₂ concentration of 0.75 mM, and no additional increase in GSH conjugate formation occurred when the concentration was raised to 1.0 mM (Figure 2B). A concentration-dependent response was also evoked in experiments using human lung microsomal incubations, with peak levels of GSH conjugates being produced at a DASO₂ concentration of 0.75 mM (Figure 2D). However, in both the time- and concentration-response studies, the levels of DASO₃-GSH conjugates formed by murine lung microsomes were considerably higher than those formed by human lung microsomes.

View larger version (24K): [in this window] [in a new window]   Figure 2.   Time- and concentration-dependent formation of DASO3-GSH conjugates in murine (A and B) and human (C and D) lung microsomal incubations containing DASO2. In the incubations, the reaction mixtures contained 3.0 mg microsomal protein, [3H]GSH (1.0 mM, 0.1 µCi), DASO2, and an NADPH-generating system in a final incubation volume of 1 ml. In the time-course experiments (A and C), the reactions were performed with 0.5 mM of DASO2. In the concentration-response studies (B and D), DASO2 was used in concentrations ranging from 0.25 to 1.0 mM (mouse) and 0.25 to 2.0 mM (human); an incubation time of 1.5 h was used. After precipitation of proteins, the supernates (100 µl) were subjected to reversed-phase HPLC analysis. Aliquots of the column effluent were collected, and levels of radioactivity were determined. Values present the relative size of the major peak at 20.5 min measured by the summation of the radioactive counts and conversion using the specific activity of GSH. Data are expressed as the mean ± SD of triplicate determinations performed on three different microsomal preparations.

The involvement of CYP2E1 in mediating the formation of the GSH conjugates was determined by performing incubations of CYP2E1-expressed human lymphoblastoid microsomes with DASO₂ in the presence of GSH and an NADPH-generating system. As found in the incubations with lung microsomes from mice and humans, HPLC analysis of the supernates detected the 20.5-min peak (Figure 1B). This peak was absent when DASO₂ or the NADPH-generating system was omitted from the incubation mixtures (data not shown). The role of CYP2E1 was also evaluated in immunoinhibition experiments using a CYP2E1 mAb. This mAb has been shown in our previous studies to be effective in inhibiting the CYP2E1 enzyme in the murine lung microsomal incubations (18). Preincubation of lung microsomes with the CYP2E1 mAb followed by incubation with DASO₂ resulted in about a 50% inhibition in generation of the DASO₃-GSH conjugates (Table 1). This inhibitory effect was not manifested in lung microsomes preincubated with a nonspecific mAb (HyHel 9).

Formation of DASO₃ in Lung Microsomal Incubations

Formation of DASO₃ was determined in murine lung microsomal incubations by measuring the formation of an NBP derivative. A standard curve was generated from the derivatized DASO₃ synthesized standard and was used to estimate the amounts of DASO₃ generated in the microsomal incubations. Lung microsomes were incubated with DASO₂ together with an NADPH-generating system. Time-course studies showed that DASO₃ was formed rapidly and was detectable at 5 min after reaction of murine lung microsomes with 0.5 mM of DASO₂ (Figure 3A). Formation of DASO₃ continued to increase with longer incubation times; peak levels were manifested at 35 min and declined when the incubation time was continued up to 50 min. Reaction of murine lung microsomes with concentrations of DASO₂ ranging from 0.25 to 1.0 mM yielded a concentration-dependent response, and the production of DASO₃ was proportional to the amounts of DASO₂ used in the incubations (Figure 3B). Saturation was attained at a concentration of 1.0 mM of DASO₂, and increase of the DASO₂ concentration to 2 mM produced no further increase in the levels of DASO₃ formed in the incubations. Formation of the derivatized DASO₃ was not detectable in incubations of human lung microsomes with even high concentrations of DASO₂. Hence, DASO₃ was readily produced from DASO₂ in incubations of murine lung microsomes in a time- and concentration-dependent manner, and was not detectable in reactions in which NADPH or DASO₂ was absent. In contrast, the formation of the derivatized epoxide was not detectable in incubations of human lung microsomes.

View larger version (17K): [in this window] [in a new window]   Figure 3.   Time- and concentration-dependent formation of the derivatized DASO3 from DASO2 in murine lung microsomal incubations. The formation of DASO3 was determined from production of the NBP derivative and estimated by relating absorbance at 540 nm to a reference standard curve constructed from the synthesized DASO3. The reaction mixtures in the incubations contained 2.25 mg of microsomal protein, DASO2, and an NADPH-generating system. In the time-course studies (A), DASO3 formation from DASO2 (1 mM) was determined at incubation times ranging from 5 to 50 min. In the concentration-response experiments (B), the amounts of DASO2 used in the incubations ranged from 0.25 to 2.0 mM of DASO2; an incubation time of 35 min was used. The alkylated NBP derivative was determined as described in MATERIALS AND METHODS. Data are expressed as the mean ± SD of triplicate determinations performed on three different microsomal preparations.

PNP Hydroxylase Activity

Control levels of PNP hydroxylase activity in the lungs of mice were determined in microsomes that were not incubated with DASO₂ or an NADPH-generating system. Incubation of DASO₂ with lung microsomes from mice produced decreases in levels of PNP hydroxylase activity. The decreases were concentration-dependent and were elicited in incubations of lung microsomes with amounts of DASO₂ ranging from 0.25 to 1.0 mM (Figure 4A). The reduction in hydroxylase activity was most pronounced at concentrations of 0.25 to 0.5 mM of DASO₂ and was less marked at substrate concentrations of 0.75 to 1.0 mM. No inhibitory effects were observed in microsomes incubated with DASO₂ in the absence of the NADPH-generating system. In in vivo studies, PNP hydroxylase activity was determined in lung microsomes from control and DASO₂-treated mice. Mice were treated with doses of DASO₂ ranging from 25 to 200 mg/kg and hydroxylase activity was measured 2 h after DASO₂ exposure. Our previous studies have shown that lung hydroxylase activity was significantly inhibited 2 h after treatment with 100 mg/kg of DASO₂ (1). Treatment of mice with doses of DASO₂ ranging from 25 to 200 mg/kg produced marked inhibition of enzyme activity that was maximal 2 h after treatment with a dose of 25 mg/kg (Figure 4B). The decreases seen at doses ranging from 50 to 200 mg/kg of DASO₂ were less pronounced. Levels of PNP hydroxylase activity in human lung microsomes were markedly lower than those detected in murine lung microsomes (Figure 4C) and were within the range of values reported in our previous studies (15). Incubation of human lung microsomes with DASO₂ produced an inhibition of hydroxylase activity that was most pronounced at a concentration of 0.25 mM. The inhibitory effect was less marked at a substrate concentration of 0.50 mM; however, no further decreases were detected at DASO₂ concentrations of 0.50 to 1.00 mM (Figure 4C). Thus, exposure to DASO₂ produced marked decreases in CYP2E1-associated PNP hydroxylase activity in the lungs of mice under both in vitro and in vivo conditions, and in the lungs of humans under in vitro conditions.

View larger version (20K): [in this window] [in a new window]   Figure 4.   Concentration- and dose-dependent effects of DASO2 on PNP hydroxylase activity. In the in vitro experiments, murine (A) and human (C) lung microsomal incubations were performed with concentrations of DASO2 ranging from 0.25 to 1.0 mM. Reaction mixtures contained 3.0 mg of microsomal protein, an NADPH-generating system, and DASO2. In the in vivo studies, mice were treated with DASO2 (25 to 200 mg/kg, orally), and PNP hydroxylase activity of lung microsomes was determined 2 h after treatment. PNP hydroxylase activity was estimated by measuring the formation of 4-nitrocatechol from PNP as described in MATERIALS AND METHODS. Data are expressed as mean ± SD of triplicate determinations for each DASO2 concentration or dose, and was performed on three separate microsomal preparations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have used a murine model to investigate the potential of DASO₂ to protect against chemically induced pulmonary toxicity. In this model, mice are treated with DCE to elicit lung damage that selectively targets the bronchiolar Clara cells (2). Substantial evidence has accrued to support an important role for CYP2E1 in the generation of the DCE epoxide, which has been proposed to be the ultimate toxic species (3, 4, 25). Importantly, damage of the Clara cells coincided with localization of CYP2E1 and the DCE epoxide within this cell type, and strongly supported the contention that the epoxide is generated in the target Clara cells (6, 14). Pretreatment of mice with DASO₂ decreased levels of the CYP2E1 enzyme, as assessed by protein content and catalytic activity, and decreased the amounts of the epoxide found within the Clara cells compared with the levels detected in the lungs of control mice (1, 6). These data indicated that the protective action of DASO₂ against DCE-induced Clara cell cytotoxicity is associated with inhibition of the CYP2E1 enzyme and decreased in situ formation of DCE metabolites, including the epoxide.

We were interested in identifying the mechanism responsible for the protective effects of DASO₂ against DCE-induced cytotoxicity in the lungs of mice. Previous studies in rat liver reported that the organosulfur compounds DAS, DASO, and DASO₂ are all competitive inhibitors of CYP2E1 (7, 9). However, inhibition of CYP2E1 by DASO₂ occurs more rapidly and is more pronounced than those elicited by DAS or DASO. The effectiveness of DASO₂ as a CYP2E1 inhibitor has been proposed to be due to mechanism-based inactivation of the P450, and a reactive intermediate generated from oxidation of DASO₂ is implicated in the inactivation of CYP2E1 (7, 8). Here we have extended the findings of these previous studies and have undertaken to investigate and to identify the reactive metabolite formed from DASO₂ metabolism in the lung. Our results showed that DASO₃ was formed from DASO₂ and conjugated with GSH in incubations of murine lung microsomes performed in the presence of NADPH and GSH (Figure 1C). Conjugation with GSH appeared to be achieved nonenzymatically inasmuch as inclusion of GSH-S-transferases in the incubation mixtures did not alter conjugation levels (data not shown). The DASO₃-GSH conjugates were generated under time- and concentration-dependent conditions (Figures 2A and 2B). These GSH conjugates were also formed in incubations of human lung microsomes in a time- and concentration-dependent fashion, although the levels were lower than those formed in lung microsome from mice (Figures 2C and 2D). The formation of DASO₃ in incubations of murine lung microsomes was confirmed in experiments that measured levels of the NBP derivative using the synthesized DASO₃ derivatized with NBP as a standard (Figure 3). The derivatized DASO₃ was produced under time- and concentration-dependent conditions (Figures 3A and 3B) but was not detectable in incubations of human lung microsomes, presumably because of the low DASO₃ levels generated. These findings demonstrated that metabolism of DASO₂ results in the formation of DASO₃ in the lungs of mice and humans, and this is likely the reactive species responsible for the protective effects against DCE-induced toxicity manifested in the lungs of mice after DASO₂ administration.

Relevant also in the context of regulatory mechanisms are issues regarding whether induction of phase II enzymes and/or induction of P450 enzymes by DASO₂ are implicated in its protective action against DCE-induced lung toxicity. Previous studies have shown that DAS, a precursor of DASO₂, induces GSH-S-transferases in murine lung (26). Also, studies in liver have found that treatment of mice with fresh garlic extracts does not produce any alterations in GSH levels (27). In addition, both DAS and DASO₂ produce induction of CYP2B1/2 in rat liver, with significant increases in pentoxyresorufin dealkylase activity and immunodetectable protein (7, 9, 28). However, previous studies have found that addition of GSH-S-transferase enzymes to reaction mixtures of microsomal incubations with DCE does not lead to increased formation of epoxide-derived GSH conjugates, suggesting that conjugation of DCE metabolites is mediated via a nonenzymatic reaction (29). In regard to CYP2B1, our previous studies have indicated that this P450 enzyme does not play a pertinent role in DCE metabolism (20). In view of these data, we believe that the inductive effects of DASO₂ are not likely to lead to modification of the metabolism of DCE.

The role of CYP2E1 in the formation of DASO₃ from DASO₂ oxidation was confirmed by data derived from three sets of experiments. First, DASO₃-GSH conjugates were detected in incubations of CYP2E1-expressed human lymphoblastoid microsomes performed in the presence of GSH and NADPH (Figure 1B). Second, PNP hydroxylase activity was decreased in a concentration-dependent pattern in both human and murine lung microsomes incubated with DASO₂ (Figures 4A and 4C). Moreover, dose-dependent decreases in hydroxylase activity were manifested in lung microsomes from mice treated orally with DASO₂ (Figure 4B). Third, formation of DASO₃-GSH conjugates was significantly inhibited in murine lung microsomes preincubated with a CYP2E1 inhibitor mAb before incubation with DASO₂ (Table 1). Taken together, these data supported a mechanism in which DASO₂ undergoes a CYP2E1-mediated oxidative metabolism to DASO₃, an event that in turn leads to inactivation of this P450. These findings suggested that the epoxide is involved in CYP2E1 alkylation at the site of formation, and this assumption is based, in part, on the high reactivity of epoxides.

It is of note that there was approximately a 50% inhibition of formation of the DASO₃-GSH conjugates when preincubation with the CYP2E1 inhibitory mAb was performed at a mAb protein:microsomal protein ratio of 0.5, suggesting that CYP2E1 mediated the metabolism of DASO₂ by at least 50% in murine lung microsomes. These findings suggested participation of other P450 isozymes in DASO₂ metabolism and/or incomplete inhibition of the CYP2E1 enzyme due possibly to inaccessibility of the epitope recognized by the mAb. Relevant in this context is the level of inhibition produced by the mAb in mediating metabolism of the CYP2E1 substrate DCE (4). Preincubation of lung microsomes with the mAb at a ratio of 0.5 produced 50% inhibition in DCE metabolism and using a ratio of 1.0 did not exacerbate the inhibitory effect of the mAb on DCE metabolism. Similar magnitudes of metabolic inhibition were also manifested for other CYP2E1 substrates, including ethyl carbamate (18) and vinyl carbamate (19). This lack of complete inhibition by high concentrations of the mAb is consistent with findings reported in studies with liver microsomes (30, 31). Interestingly, the use of purified CYP2E1 in a reconstituted system produced complete inhibition of CYP2E1 catalytic activity (30). These findings suggested that it may not be possible to completely inhibit CYP2E1 catalytic activity in a microsomal incubation system, and this has been postulated to be due to interference by NADPH-cytochrome P450 reductase or inaccessibility of a portion of CYP2E1 to the antibody (30). Nevertheless, immunoinhibition experiments are highly specific and represent a valuable tool for clarifying the role of particular P450 isozymes in the metabolism of chemicals and drugs.

It is of interest to compare the metabolism of DASO₂ in lung tissues from mice and from humans. Our results showed that the DASO₃-GSH conjugates were formed in human lung microsomal incubations, but at levels that were markedly lower than those formed in murine lung microsomal incubations (Figure 2), indicating that DASO₃ is generated to a lesser extent in human than in murine lung. As was found in the murine lung microsomal incubations, the production of the DASO₃-GSH conjugates in the human lung microsomal incubations was time- and concentration-dependent (Figure 2). At a DASO₂ concentration of 0.5 mM, maximal formation of the DASO₃-GSH conjugates by both human and murine lung microsomes was detected at an incubation time of 2 h, after which a plateau was achieved. The concentration response for DASO₂ was also similar in both human and murine lung microsomal incubations: the amounts of DASO₃-GSH conjugates formed were incremental with increasing DASO₂ concentrations, and saturation was attained at 0.75 mM (Figure 2). These findings indicated that DASO₃ formation in human lung was markedly lower than that in murine lung, and this is affirmed by our inability to detect the derivatized DASO₃ in the human lung microsomal incubations. Nevertheless, the DASO₃ metabolite is an efficacious inhibitor of PNP hydroxylation, and enzyme activities in both human and murine lung microsomes were significantly decreased (Figure 4). These data indicated that DASO₂ is an effective inhibitor of the CYP2E1 enzyme in human lung and, furthermore, that the mouse is an appropriate model for investigation of mechanisms involved in the metabolism of CYP2E1-selective substrates in conjunction with DASO₂ exposure.

The coincidental formation of DASO₃ and CYP2E1 inactivation supported an assumption that DASO₃ is the reactive species responsible for CYP2E1 inhibition. However, studies with 2-isopropyl-4-pentenamide and its methyl ester analog methyl 2-isopropyl-4-pentenoate as well as the therapeutic drug novonal indicated that the reactive species responsible for P450 alkylation is not an epoxide or secondary metabolites derived from the epoxide, but rather is mediated by a cationic intermediate species (32- 34). Hence, while it is likely that CYP2E1 alkylation may occur via the action of DASO₃, the precise reactive species that is involved requires further investigation and remains to be established. Nevertheless, the inverse relationship between the formation of DASO₃ and the diminution of PNP hydroxylase activity supported the premise that the magnitude of DASO₃ formation is a good indicator of the extent of DASO₂ metabolism and subsequent CYP2E1 inactivation (Figures 2-4).

In summary, these studies have produced data to demonstrate that DASO₃ is the reactive intermediate produced from CYP2E1-mediated metabolism of DASO₂, suggesting that this metabolite is likely responsible for CYP2E1 alkylation. Furthermore, the chemoprotective effects evoked by DASO₂ against toxicities and carcinogenicities caused by CYP2E1-selective substrates such as DCE are related to the capacity to metabolize DASO₂ to DASO₃.