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Oxidation of Vinyl Carbamate and Formation of 1,N⁶-Ethenodeoxyadenosine in Murine Lung

Departments of Anatomy and Cell Biology (P.-G.F., G.B., K.C., A.S.), Biochemistry (M.K., G.J.), and Chemistry (R.B.), Queen's University, Kingston, Ontario, Canada; and Food Research Division, Bureau of Chemical Safety, Health Canada, Ottawa, Ontario, Canada (H.C.)

(Received November 9, 2006; accepted February 9, 2007)

	Abstract

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Vinyl carbamate (VC) is derived from ethyl carbamate, a carcinogen formed in fermentation of food and alcoholic products. We have undertaken studies to test the hypothesis that an epoxide generated from VC oxidation leads to formation of 1,N⁶-ethenodeoxyadenosine (dAS). We have developed approaches using liquid chromatography-mass spectrometry and liquid chromatographytandem mass spectrometry for identification and quantitation of dAS. Scanning and fragment ion analyses confirmed the identity of dAS based on the molecular ion [M + H]⁺ m/z 276 and the specific fragment ion m/z 160. Chemical oxidation of VC in reactions containing 2'-deoxyadenosine produced dAS with ¹H NMR, chromatographic, and mass spectral characteristics identical to those of the authentic dAS, suggesting DNA alkylation by the VC epoxide. Subsequent studies evaluated formation of dAS in incubations of murine lung microsomes or recombinant CYP2E1 with VC. The formation of dAS in incubations of lung microsomes or recombinant CYP2E1 with VC was dependent on protein concentrations, CYP2E1 enzyme levels, and incubation time. The rates of dAS formation were highly correlated with VC concentrations. Peak rates were produced by lung microsomes and recombinant CYP2E1 at 3.0 and 2.5 mM VC, respectively. In inhibitory studies, incubations of VC were performed using lung microsomes from mice treated with the CYP2E1 inhibitor diallyl sulfone (100 mg/kg, p.o.). Results from these studies showed significantly decreased dAS formation in microsomes incubated with VC, with an inhibition of 70% at 3.0 mM. These findings suggested that CYP2E1 is a major enzyme mediating VC oxidation, leading to the formation of a metabolite that alkylates DNA to form the dAS adduct.

The carcinogenicity of EC and the formation of lung adenomas in animals were first reported in 1943 (Nettleship et al., 1943). Subsequent studies in mice demonstrated that EC induces tumors in a variety of tissues including the skin, liver, lung, mammary gland and lymphoid tissue (Mirvish, 1968). A long latent period of about 1 year was generally required for tumor development. An exception was in the lung, where tumors were formed more rapidly and were manifested in about 2 to 6 months (Mirvish, 1968; Shimkin and Stoner, 1975). A similar spectrum of tumors is induced by both EC and VC. However, comparative studies revealed that VC is more carcinogenic than EC; lung tumors induced by VC are 20- to 50-fold greater in number than are induced by EC, depending on the route of exposure (Dahl et al., 1978, 1980). Moreover, VC but not EC is mutagenic to Salmonella typhimurium strains TA 1535 and TA100 (Dahl et al., 1978, 1980; Leithauser et al., 1990). However, more recent studies have identified the mutagenicity of EC, using the transgenic Muta Mouse and lacZ as the reporter gene (Williams et al., 1998). Nevertheless, these findings indicated that VC has greater carcinogenic potential than EC, and further that the lung is an important and possibly a primary target tissue.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Proposed scheme of EC metabolism to VC and the formation of 1,N6-ethenodeoxyadenosine from reaction of VC epoxide with 2'-deoxyadenosine.

In this investigation, we have extended the findings of previous studies and adopted an in vitro approach for experiments designed to further elucidate the metabolism of VC and the mechanisms that mediated DNA alkylation. We have undertaken studies to test the hypothesis that oxidation of VC by CYP2E1 leads to the formation of an epoxide that alkylates DNA, resulting in the formation of dAS in murine lung. Using an approach based on high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), we have performed studies to determine whether chemical oxidation of VC in reactions supplemented with 2'-deoxyadenosine (dAS) leads to dAS formation. The proposed scheme for this metabolic pathway is illustrated in Fig. 1. We have subsequently carried out studies to investigate dAS formation in incubations of murine lung microsomes with VC. The involvement of CYP2E1 in VC metabolism was also investigated using incubations of recombinant rat CYP2E1 (rCYP2E1) and lung microsomes from mice treated with the CYP2E1 inhibitor, diallyl sulfone (DASO₂). Our results demonstrated that VC oxidation has a critical role in dAS formation. The dAS adduct was also generated from VC in incubations of lung microsomes and rCYP2E1. These results demonstrated that VC oxidation, mediated mainly by CYP2E1, and subsequent DNA alkylation leads to production of dAS in murine lung.

	Materials and Methods

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Chemicals and Reagents. Chemicals were purchased from suppliers as follows: 2'-deoxyadenosine, 1,N⁶-ethenodeoxyadenosine, NADPH, K₂HPO₄, and BSA, Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada); KCl, EDTA, and acetonitrile, Fisher Scientific (Ottawa, ON, Canada). Synthesis of DASO₂ was carried out by Colour Your Enzyme (Bath, ON, Canada). Recombinant rat CYP2E1 expressed in human ß-lymphoblastoid microsomes was obtained from BD Biosciences Discovery Labware (Bedford, MA). All solvents used for sample preparation and HPLC and LC-MS analyses were of HPLC grade and were purchased from Caledon Laboratories (Georgetown, ON, Canada). Details and results of the synthesis of VC and 2'-deoxy-2-deuteroethenoadenosine (DDAS) are described in the Supplemental Data.

Chemical Oxidation of VC and Synthesis of dAS. Oxidation of VC was performed according to the following method. VC (0.5 mM) was added to m-chloroperoxybenzoic acid (mCPBA) (0.51 mM) dissolved in CH₂Cl₂ (3.0 ml). The reaction mixture was stirred for approximately 25 min. During this time a saturated solution of dAS (0.2 mM) was prepared in water (3.0 ml), added to the reaction mixture, and stirred vigorously for 12 h at room temperature. The aqueous layer was then separated and the organic layer was extracted with water (three times, 3.0 ml). The combined aqueous layers were washed with ether (four times, 10 ml) to remove unreacted mCPBA and the side product m-chlorobenzoic acid. The aqueous layer was lyophilized in vacuo and resuspended in water containing 5% acetonitrile. Controls, comprising samples in which mCPBA or VC and mCPBA were omitted, were processed in parallel with the experimental samples and were subjected to identical protocols. All the samples were then subjected to HPLC analysis for purification of the dAS adduct and subsequent analyses by LC-MS, LC-MS/MS, and ¹H NMR.

Animal Treatment. Female CD-1 mice, weighing 25 to 28 g, were purchased from Charles River Canada (St. Constant, QC, Canada). The mice were maintained on a 12-h light/dark cycle and were allowed free access to water and food (Mouse Diet 5015; PMI Nutrition International Inc., Brentwood, MO). The mice were acclimatized to laboratory conditions for 1 week before being used for experiments. For inhibition studies, mice (n = 40) were treated with 100 mg/kg DASO₂ (p.o.) in water, whereas control mice (n = 40) were treated with only water. After 2 h, the mice were sacrificed, lung tissues excised, frozen in liquid nitrogen, and stored at –80°C.

Preparation of Microsomes. Lungs from 40 mice were pooled for each microsomal sample. This pooling is essential for obtaining sufficient tissue for preparation of the lung microsomes. Microsomes were prepared by differential centrifugation as described previously (Forkert et al., 2006). Lung tissue was minced and homogenized in 4 volumes of ice-cold phosphate-buffered KCl (139 mM KCl, 100 mM K₂HPO₄, 1.5 mM EDTA, pH 7.4). The homogenate was subjected to centrifugation at 12,000g for 20 min at 2°C. The supernatant was then centrifuged at 105,000g for 60 min. The final pellet was homogenized using 400 µl of buffer, and aliquots were frozen in liquid nitrogen and stored at –80°C. Microsomal protein concentrations were determined by using the Bradford assay, using bovine serum albumin as the standard (Bradford, 1976).

Formation of dAS in Lung Microsomal Incubations. In the lung microsomal incubations, reaction mixtures in a total volume of 250 µl contained microsomal protein in 0.1 M phosphate buffer, pH 7.4, 1.0 mM NADPH, and 12.5 mM dAS. The incubations were carried out at 37°C. After the incubation, the samples were placed on ice and 250 µl of ice-cold acetonitrile was added. The proteins were precipitated and removed by centrifugation at 15,000g for 3 min. Experiments for determination of the protein concentration-curve were carried out with VC (1.0 mM), an incubation time of 20 min, and 0 to 5 mg of microsomal protein per ml. The time course experiments were performed with VC (1.0 mM) and 2.0 mg/ml microsomal protein, using incubation times of 0 to 125 min. The concentration studies were carried out with 0 to 3.0 mM VC, 2 mg/ml microsomal protein, and an incubation time of 40 min.

Formation of dAS by rCYP2E1. Rates of formation of dAS by CYP2E1 were determined in incubations containing rCYP2E1 coexpressed with NADPH-cytochrome P450 reductase. Reaction mixtures in a final volume of 250 µl of 0.1 M phospate-buffered saline, pH 7.4, contained 10 pmol of rCYP2E1, 1.0 mM VC in water, and were preincubated for 3 min at 37°C. The reaction was initiated by addition of 1.0 mM NADPH and the incubations were carried out for an additional 20 min. Reactions were terminated by immersion into liquid nitrogen. To determine dAS formation as a function of rCYP2E1 enzyme levels, incubations of VC (1.0 mM) were performed with amounts of enzyme ranging from 0.25 to 60 pmol and an incubation time of 20 min. Time course studies were carried out in the incubations of VC (1.0 mM) with rCYP2E1 (10 pmol), using incubation times of 0 to 80 min. Concentration-response studies were performed using 10 pmol of rCYP2E1, an incubation time of 20 min, and VC concentrations ranging from 0 to 4.0 mM.

Formation of dAS by Lung Microsomes from DASO₂-Treated Mice. Incubations with VC were performed with lung microsomes from mice that were treated 2 h previously with DASO₂ (100 mg/kg p.o.) in water. Incubations with lung microsomes from mice treated with equivalent volumes of the vehicle served as controls. Reaction mixtures in a final volume of 250 µl contained microsomal protein (2.0 mg/ml) in 0.1 M phosphate buffer, pH 7.4, 1.0 mM NADPH, 12.5 mM dAS, and a range of VC concentrations (0–3.0 mM). The incubations were carried out at 37°C for 40 min.

HPLC Purification of dAS. Samples generated from the chemical synthesis of VCO and the rCYP2E1 incubations were redissolved in 95:5% water/acetonitrile and subjected to HPLC purification against the standards, dAS and dAS. HPLC analysis was conducted on an Alliance 2695 separations module and 996 photodiode array detector (Waters, Milford, MA) using a water/acetonitrile-based gradient system on a C₁₈ Ultrasphere ODS column (5 µm, 4.6 mm x 25 cm; Beckman Coulter, Fullerton, CA). The gradient was started at 95:5% water/acetonitrile and brought to 86:14% over 16 min at a flow rate of 0.4 ml/min. The mobile phase was returned to its initial conditions of 95:5% water/acetonitrile over 3 min at a flow rate of 0.4 ml/min and subsequently over 6 min at a flow rate of 1 ml/min. The duration of each run was 25 min. A 5.0-min time delay between samples was included to ensure proper reequilibration of the HPLC column. Under these conditions, the authentic dAS and the dAS adduct eluted at 18.93 min, whereas dAS eluted at 16.87 min. Fractions that possessed spectral and/or chromatographic properties identical to those of the authentic dAS were collected, and samples of each type were pooled separately and evaporated to dryness.

Identification of DNA Adducts. The pooled fractions from the chemical synthesis, lung microsomal incubations, and rCYP2E1 incubations were redissolved in 95:5% water/methanol and subjected to LC-MS analysis using an HPLC system similar to that described above, connected in-line with a Quattro Ultima mass spectrometer (Waters) in positive ion mode. Separation was facilitated by a gradient solvent system starting at 95:4:1% water/methanol/1% glacial acetic acid and terminating at 59:40:1 over 25 min on a Zorbax SB-C₁₈ column (3.5 µm, 2.1 x 150 mm; Agilent Technologies, Palo Alto, CA), using a flow rate of 0.12 ml/min. Initially, the instrument was set to establish the molecular weight of the fractions in scanning or MS1 mode (set to scan from 50 to 600 m/z), against the authentic dAS, dAS, and DDAS (synthesis described in Supplemental Data). MS2 analysis was subsequently used to establish the fragmentation pattern of the selected MS1 parent, using collision-induced dissociation with argon gas at a pressure of 30 mbar and a collision energy of 20 eV, in which specific molecular fragments were detected over a range of 50 to 450 m/z. The identification of the DNA adduct in our experimental preparations was based upon a comparison of UV spectral properties, chromatographic retention time, and characteristic ions from MS1 and MS2, with the authentic dAS.

View larger version (24K): [in this window] [in a new window]   FIG. 2. Standards used for identification and quantitation of the dAS adduct. Samples were analyzed by LC-MS/MS in multiple reaction mode, where the abundance of the characteristic MS2 ions (D–F) from selected parent MS1 ions (A–C) were measured for dAS (A), the authentic dAS used as a standard (B), and DDAS used as the internal standard (C).

Statistical Analysis. Data are expressed as mean ± S.E.M. Data analysis including curve-fitting analysis of VC concentration data was performed using GraphPad Prism version 4 (GraphPad Software Inc., San Diego, CA).

	Results

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Identification of dAS by LC-MS/MS. In this study, we have developed a LC-MS/MS protocol to establish the identity of the DNA adduct formed in incubations of VC with lung microsomes and rCYP2E1 as well as under conditions of VC oxidation using mCPBA. The MS1 mass spectral analysis of dAS revealed the molecular ion m/z 252 [M + H]⁺ (Fig. 2A). Similar analysis for the authentic dAS, which was used as the standard, was identified by the molecular ion m/z 276 (Fig. 2B). The synthesized DDAS, applied as the internal standard, was characterized by the production of the molecular ion m/z 277, which accounts for the introduction of a single deuterium atom at C2 (Fig. 2C). Subsequent MS2 analysis of the fragments for dAS, the authentic dAS, and DDAS yielded the specific fragment ions m/z 136 (Fig. 2D), m/z 160 (Fig. 2E), and m/z 161 (Fig. 2F), respectively. It should be noted that no ions corresponding to the sugar backbone cleavage fragment were detected in MS2 mode. Based upon the MS1 and MS2 data, the following MRM transitions were established to selectively identify and quantitate the nucleotides under investigation: dAS, 252136; dAS, 276160; DDAS, 277161. Mass spectral analysis of the DNA adduct generated in incubations of VC with lung microsomes in the presence of dAS, or similar incubations with rCYP2E1, produced the major molecular ion m/z 276 [M + H]⁺ (Fig. 3, A and B), identical to that of the authentic dAS (Fig. 2B). MS2 analysis revealed a specific fragment m/z 160 for the dAS adduct generated in incubations of VC with lung microsomes (Fig. 3D) or rCYP2E1 (Fig. 3E). The difference in the m/z ratio between dAS and dAS produced in the incubations of VC with lung microsomes and rCYP2E1 (24 mass units) corresponded to the change in molecular weight predicted for the formation of the 1,N⁶-etheno adduct of dAS. The same difference in m/z ratio was observed among the MS2 fragments, which resulted from cleavage of the anomeric C–N bond.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Mass spectral analyses of chemical and biological samples for formation of dAS. The dAS adduct was identified on the basis of its characteristic MS1 spectra (A–C) and MS2 fragmentation patterns (D–F) in positive ion mode. The dAS adduct was generated in incubations of VC with lung microsomes (A and D), in incubations of VC with rCYP2E1 (B and E), or synthesized by VC oxidation, using mCPBA, in the presence of dAS (C and F).

Representative MRM chromatograms of the standards and experimental preparations are shown in Fig. 4. Typically, DDAS (18.60 min) eluted 0.2 to 0.3 min earlier than the authentic dAS (18.93 min). The slight discrepancy between the retention time of the authentic dAS and the adduct detected by the same MRM transition in the microsomal and rCYP2E1 incubations (0.32–0.33 min) is due to the presence of excess dAS in the sample, even after an initial HPLC purification step before LC-MS/MS. Some contaminating dAS (m/z 252) in the prepurified adduct sample derived from incubations with rCYP2E1 was also evident (Fig. 4B). Taken together, the chromatographic retention times, and characteristic MS1 and MS2 ions confirmed that dAS is produced in incubations of VC and dAS with both lung microsomes and rCYP2E1.

View larger version (22K): [in this window] [in a new window]   FIG. 4. A, retention times were derived from LC-MS/MS analysis of the standards dAS (m/z 252136), authentic dAS used as the standard (m/z 276160), and DDAS used as the internal standard (m/z 277161). B, retention times were also derived for the products generated in the experimental preparations: VC oxidation (VCO) in the presence of dAS (m/z 276160), incubations of lung microsomes with VC (m/z 276160), and incubations of rCYP2E1 with VC (m/z 276160). The arrows represent the proposed sites of cleavage at the anomeric centers of dAS, dAS, or DDAS (A).

Chemical Oxidation of VC and Synthesis of dAS. The DNA adduct produced from dAS in the presence of VC oxidized with mCPBA produced identical MS1 and MS2 spectra (Fig. 3, C and F), similar to those for the authentic dAS (Fig. 2, B and E). The spectra were also similar to those obtained in the incubations of VC with lung microsomes (Fig. 3, A and D) and with rCYP2E1 (Fig. 3, B and E). The chromatographic retention time for dAS formed under the oxidative conditions of VC was 18.60 min, and was similar to those identified for the adduct produced in the lung microsomal and rCYP2E1 incubations, which were 18.61 min and 18.76 min, respectively (Fig. 4B). No dAS adduct was produced in the controls in which reactions were carried out in the absence of mCPBA or of VC and mCPBA.

The identity of the dAS adduct formed via chemical oxidation of VC in the presence of dAS was confirmed by the spectroscopic data. ¹H NMR (600 MHz, D₂O): 9.08 (1H, s, H₂), 8.37 (1H, s, H₈), 7.94 (1H, s, H₁₀ or H₁₁), 7.53 (1H, s, H₁₀ or H₁₁), 6.54 (1H, t, J = 6.6Hz, H_1'), 4.10 (1H, m, H_4'), 3.76 (1H, dd, J = 12.6, 3.4 Hz, H_5a'), 3.70 (1H, dd, J = 12.6, 4.7 Hz, H_5b'), 2.87 (1H, pentet, J = 13.8, 6.8 Hz, H_2a'), 2.55 (1H, m, H_2b'). These spectroscopic data are in agreement with those obtained for the authentic dAS. ¹H NMR (400 MHz, D₂O): 9.10 (1H, s, H₂), 8.42 (1H, s, H₈), 7.97 (1H, s, H₁₀ or H₁₁), 7.58 (1H, s, H₁₀ or H₁₁), 6.59 (1H, t, J = 6.6 Hz, H_1'), 4.70 (1H, m, H_3'), 4.19 (1H, m, H_4'), 3.85 (1H, dd, J = 12.5, 3.5 Hz, H_5a'), 3.79 (1H, dd, J = 12.5, 4.9 Hz, H_5b'), 2.94 (1H, pentet, J = 14, 6.8 Hz, H_2a'), 2.63 (1H, ddd, J = 14, 6.3, 3.9 Hz, H_2b').

View larger version (14K): [in this window] [in a new window]   FIG. 5. Formation of dAS in incubations of murine lung microsomes with VC. Studies of protein concentrations were carried out using 0 to 5 mg of microsomal protein, 1.0 mM VC, and an incubation time of 20 min (A). Time-dependent studies were performed using 2.0 mg of microsomal protein, 1.0 mM VC, and incubation times of 0 to 125 min (B). Concentration-dependent studies were carried out using 2.0 mg/ml microsomal protein, an incubation time of 40 min, and VC concentrations ranging from 0 to 3.0 mM (C). All incubations contained 1.0 mM NADPH and were carried out at 37°C.

Formation of dAS in Lung Microsomal Incubations.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Formation of dAS in incubations of rCYP2E1 with VC. Studies of rCYP2E1 concentrations were carried out with 0 to 60 pmol of enzyme, 1.0 mM VC, and an incubation time of 20 min (A). Time course studies were performed with 10 pmol of rCYP2E1, 1.0 mM VC, and incubation times of 0 to 80 min (B). Concentration-dependent studies were carried out with 10 pmol of rCYP2E1, VC concentrations ranging between 0 and 4.0 mM, and an incubation time of 20 min (C). All incubations contained 1.0 mM NADPH and were carried out at 37°C.

View larger version (18K): [in this window] [in a new window]   FIG. 7. Formation of dAS in incubations of VC with lung microsomes from control and DASO2-treated mice. Mice were treated with DASO2 (100 mg/kg p.o.), whereas control mice were treated with water. All mice were sacrificed 2 h later for isolation of lung microsomes. Reaction mixtures in a final volume of 250 µl contained microsomal protein (0.5 mg) in 0.1 M phosphate buffer, pH 7.4, 1.0 mM NADPH, 12.5 mM dAs, and VC (0–3.0 mM). The microsomal incubations were carried out for 40 min at 37°C. The correlation of variation was R2 = 0.9510 and R2 = 0.9883 for control and DASO2-treated mice, respectively.

	Discussion

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This study has produced definitive physicochemical data that have validated our working hypothesis that oxidation of VC leads to the formation of VCO that can alkylate DNA to form dAS (Fig. 1). We have adopted an approach using LC-MS and LC-MS/MS to investigate formation of dAS from VC. The identification of dAS was confirmed based on the MS1 (m/z 276) and MS2 (m/z 160) ions (Figs. 2, B and E; and Fig. 3), as well as the chromatographic retention times of the samples (Fig. 4). A critical component of this study was the synthesis of the internal standard DDAS ((Fig. 2, C and F), which was integral to quantitation of the formation of dAS under various experimental conditions. In conjunction with NMR analysis, the results have provided validation for the application of the LC-MS and LC-MS/MS approach for qualitative and quantitative characterization of the dAS adduct.

An important objective of this study was to confirm that oxidation of VC leads to the production of a metabolite, presumably the reactive VCO, which reacts with DNA to form dAS (Fig. 1). We have carried out chemical experiments in which VC was oxidized with mCPBA and then reacted with dAS. The DNA adduct produced from this reaction demonstrated mass spectral (Fig. 3, C and F) and chromatographic properties (Fig. 4B) similar to those of the authentic dAS (Fig. 2, B and E), thus confirming that dAS was formed. In the biological experiments, chromatographic and mass spectral analyses of samples from the lung microsomal incubations supplemented with dAS confirmed that the dAS adduct was generated from VC in a manner that was dependent on protein concentrations (Fig. 5A) and incubation time (Fig. 5B). The production of dAS was highly correlated (R² = 0.9940) with VC concentrations (Fig. 5C). These results have established the ability of lung microsomes to catalyze P450-dependent formation of dAS in our experiments. Furthermore, our results have shown that dAS production in incubations of VC with rCYP2E1 was dependent on enzyme concentrations (Fig. 6A) and incubation time (Fig. 6B), and was highly correlated (R² = 0.9108) with VC concentrations (Fig. 6C). Taken together, these findings have provided evidence to support a role for CYP2E1 in VC oxidation and the subsequent production of dAS (Fig. 1).

Previous studies have identified the garlic constituent DASO₂ as an inhibitor of the CYP2E1 enzyme in the lungs of mice (Forkert et al., 1996). Treatment of mice with DASO₂ (100 mg/kg p.o.) elicited a 75% inhibition of lung p-nitrophenol (PNP) hydroxylation, a catalytic activity associated with CYP2E1. The loss of PNP hydroxylase activity coincided with a decrease in immunodetectable lung microsomal CYP2E1 protein. Hydroxylation of chlorzoxazone, a prototypic substrate for the CYP2E1 enzyme, was decreased by 60% in lung microsomes from mice treated with DASO₂ (100 mg/kg p.o.) (Simmonds et al., 2004a). The mechanism of CYP2E1 inactivation has been ascribed to formation of an epoxide (1,2-epoxypropyl-3,3'-sulfonyl-1'-propene) from oxidation of DASO₂, leading, in part, to production of the heme adduct N-alkylprotoporphyrin IX in incubations of lung microsomes or recombinant rat CYP2E1 (Forkert et al., 2000; Black et al., 2006). These findings implicated CYP2E1 in the metabolism of DASO₂, resulting in CYP2E1 inactivation and inhibition of the metabolism of CYP2E1 substrates. Here, we have carried out inhibitory studies using DASO₂ to verify that CYP2E1 catalyzes the formation of dAS under biological conditions. The results showed significant inhibition of dAS formation in incubations of VC (0.25–3.0 mM) with lung microsomes from DASO₂-treated (100 mg/kg p.o.) mice (Fig. 7). Irrespective of the amounts of VC used in the incubations, the extent of inhibition was approximately 70% of the corresponding control levels, suggesting that this was the maximum extent inhibitable by DASO₂. Taken together, these data suggested that CYP2E1 is responsible, in large measure, for the production of dAS in the lung microsomal incubations.

Although our CYP2E1 inhibition studies with DASO₂ supported the role of this P450 in catalyzing the formation of VCO and subsequently dAS, the specificity of the inhibitory effect of DASO₂ on P450 enzymes must also be addressed. We have previously determined the effects of DASO₂ on the rates of PNP hydroxylation by rCYP2E1 and recombinant goat CYP2F3 (rCYP2F3). The results showed that incubation of rCYP2E1 and rCYP2F3 with DASO₂ decreased hydroxylase activities by 90 and 70%, respectively (Simmonds et al., 2004a). Moreover, incubation of DASO₂ with rCYP2E1 and rCYP2F3 decreased hydroxylation of chlorzoxazone by 90 and 77%, respectively. These findings demonstrated that DASO₂ inhibited both CYP2E1 and CYP2F3, and additionally suggested the possibility that the CYP2F enzyme, in addition to CYP2E1, may be involved in VC metabolism. In light of these data and our current observation that 30% of the activity remained after maximal inhibition with DASO₂ (Fig. 7), we carried out preliminary experiments to determine whether recombinant mouse CYP2F2 or recombinant rat CYP2F4 could also be involved in VC metabolism, leading to dAS formation. The CYP2F enzyme is expressed selectively in the lung, with relatively little expression in the liver (Hakkola et al., 1994; Shultz et al., 1999). We have incubated VC (1.0 mM) and dAS (12.5 mM) with amounts of recombinant mouse CYP2F2 and recombinant rat CYP2F4 ranging from 0.25 to 40 pmol, using procedures described previously (Simmonds et al., 2004b). Our chromatographic and mass spectral analysis failed to detect formation of the dAS adduct by either the rCYP2F2 or the rCYP2F4 enzyme. Taken together, these findings suggested that CYP2E1 plays a major role in the production of dAS in the lung microsomal incubations. These results do not, however, preclude the participation of other P450 isoforms, as yet unidentified, in VC metabolism.

The human relevance of the studies with VC and EC has a historical perspective. EC was used as a cosolvent for analgesic and sedative drugs in Japan between 1950 and 1975, and it was estimated that the total dose of EC administered to a 60-kg patient was approximately 0.6 to 3.0 g (Nomura, 1975). This period represented 25 years during which millions of humans were administered "the largest doses of a pure carcinogen that is on record" (Miller, 1991, p. 1323). Today, human exposures occur inadvertently via the consumption of fermented foods and alcoholic beverages (Ough 1976; Battaglia et al., 1990). In this study, the results from incubations of lung microsomes with VC revealed that the rate of dAS production was not fully saturated at a concentration of 3.0 mM (Fig. 5C). In the case of the incubations of rCYP2E1 with VC, saturation was achieved at a concentration of 2.5 mM. These findings suggested that formation of dAS occurs at a slow rate and at relatively high VC concentrations, suggesting that VC may not pose a carcinogenic risk in humans because exposures are predictably at extremely low levels, especially when VC exposure is via generation from EC. However, the potential adverse effects of VC exposure in human lung remain to be investigated and characterized. Nonetheless, in reference to EC, Miller and Miller (1983, p. 13) have reiterated that "it is possible that even low life-time intakes of so-called `weak' carcinogens could induce cancers in some humans with especially predisposing genetic backgrounds and dietary habits". Ethyl carbamate has exhibited moderate to weak carcinogenic activity in experimental animals.

The results of this investigation have demonstrated the ability of lung microsomes to catalyze the oxidation of VC via CYP2E1, resulting in the production of dAS. This finding is consistent with data from previous in vivo studies that identified dAS and 3,N⁴-ethenodeoxycytidine in liver and lung DNA of mice treated with EC or VC (Fernando et al., 1996; Titis and Forkert, 2001). Both carbamate compounds also formed 7-(2'-oxoethyl)deoxyguanosine and dAS in liver DNA of rodents (Ribovich et al., 1982; Miller and Miller, 1983; Scherer et al., 1986). Although 7-(2'-oxoethyl)deoxyguanosine comprises 98% of total adducts in hepatic DNA, the minor lesion affecting dAS has been regarded as significant because of its ability to miscode in transcription of DNA (Barbin and Bartsch, 1986; Basu et al., 1993). The propensity for the miscoding in DNA transcription is associated with base changes such as A G and A T in mammalian cells (Pandya and Moriya, 1996). Our finding of dAS formation in the lung has provided a vital link to data from our recent studies showing high frequencies of A:T G:C and A:T T:A mutations in the lungs of VC-treated mice (Hernandez and Forkert, 2007). Although similar types of mutations were generated by VC and EC, the incidences of mutant frequencies induced by VC required an EC dose that was 17-fold higher, thus confirming the greater carcinogenic potential of VC versus EC. In this regard, VC has been found not only to elicit a high frequency of mutations, but also to be active in inducing micronuclei and sister chromatid exchange (Dahl et al., 1978, 1980; Allen et al., 1982; Wild, 1991; Hernandez and Forkert, 2007). In summary, our results have shown formation of dAS as a result of VC oxidation by CYP2E1 and supported the contention that such a mechanism is the underlying molecular event for the DNA damage sustained by animals treated with VC that ultimately results in lung tumor development.

	Acknowledgments

 
We thank Dr. Francoise Sauriol for the 600 MHz ¹H NMR spectrum. We also thank Drs. Alan Buckpitt, Michael Shultz, and Michael Baldwin for contribution of recombinant CYP2F2 and recombinant CYP2F4 for our preliminary studies.

	Footnotes

 
This study was funded by the Canadian Cancer Society through Grant 014061 from the National Cancer Institute of Canada (to P.G.F.). Funding for the mass spectrometer (LC-MS/MS) and for the Service Contract on the LC-MS/MS was provided by Grant MA-9475 and Grant MMA-69106, respectively, from the Canadian Institutes of Health Research (to G.J.). M.K. was supported by an Ontario Graduate Scholarship. A.S. was supported by postdoctoral fellowships from the Cancer Research Society and Queen's University Principal's Development Fund.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.013805.

ABBREVIATIONS: VC, vinyl carbamate; dAS, 2'-deoxyadenosine; DDAS, 2'-deoxy-2-deuteroethenoadenosine; DASO₂, diallyl sulfone; dAS, 1,N⁶-ethenodeoxyadenosine; EC, ethyl carbamate; HPLC, high performance liquid chromatography; LC-MS, high performance liquid chromatography-mass spectrometry; LC-MS/MS, high performance liquid chromatography-tandem mass spectrometry; mCPBA, m-chloroperoxybenzoic acid; MRM, multiple reaction monitoring; P450, cytochrome P450; PNP, p-nitrophenol; r, recombinant; VCO, vinyl carbamate epoxide.

The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

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

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