Chem. Res. Toxicol. 1996, 9, 41-49
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Sulfoxidation of Mercapturic Acids Derived from Triand Tetrachloroethene by Cytochromes P450 3A: A Bioactivation Reaction in Addition to Deacetylation and Cysteine Conjugate β-Lyase Mediated Cleavage Michael Werner, Gerhard Birner, and Wolfgang Dekant* Institut fu¨ r Toxikologie der Universta¨ t Wu¨ rzburg, Versbacherstrasse 9, 97078 Wu¨ rzburg, FRG Received May 1, 1995X
In the present study we investigated the formation of sulfoxides from N-acetyl-S-(1,2,2trichlorovinyl)-L-cysteine (N-Ac-TCVC), N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (N-Ac-1,2DCVC), and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (N-Ac-2,2-DCVC), which are formed in the glutathione dependent bioactivation of tri- and tetrachloroethene. The first aim was to elucidate the enzymes involved in these oxidation reactions. N-Ac-TCVC, N-Ac-1,2-DCVC, and N-Ac-2,2-DCVC are oxidized to the corresponding sulfoxides mainly, if not exclusively, by cytochrome P450 enzymes in liver microsomes of untreated male rats, since no role for the flavin-containing monooxygenase (FMO) could be demonstrated by heat inactivation experiments and by the use of n-octylamine. The sulfoxidation rates were increased when using liver microsomes of phenobarbital and dexamethasone pretreated male rats as well as liver microsomes of dexamethasone pretreated female rats, while no sulfoxide formation was observed in liver microsomes of untreated female rats, suggesting an involvement of cytochrome P450 3A. Also, troleandomycin, a specific chemical inhibitor for cytochrome P450 3A, drastically reduced sulfoxidation rates. The observed rates of sulfoxidation also correlated well with the rates of oxidation of testosterone at the 6-β-position, a specific marker for P450 3A activity. The second aim of this study was to compare the cytotoxicity of the sulfoxides with the cytotoxicity of the corresponding mercapturic acids in isolated rat renal epithelial cells. Both mercapturic acids and the corresponding sulfoxides were cytotoxic. Cytotoxicity of the mercapturic acids could be blocked by (aminooxy)acetic acid (AOAA), an inhibitor of cysteine conjugate β-lyase, while the cytotoxicity of the sulfoxides was not influenced by this treatment. Moreover, the sulfoxides were significantly more cytotoxic than the corresponding mercapturic acids at equimolar doses. The results show that mercapturic acids derived from TRI and PER are oxidized to sulfoxides by microsomal monooxygenases from rat liver. The cytotoxicity of the produced sulfoxides could not be reduced by AOAA, consistent with a role of the sulfoxides as direct acting electrophiles (i.e., Michael acceptor substrates).
Introduction Tetrachloroethene and trichloroethene are commonly used solvents and, due to their volatility and resistance to degradation, are widely distributed environmental contaminants. Both tetrachloroethene and trichloroethene show only low acute toxicity. Long-term bioassays of tetrachloroethene and trichloroethene for carcinogenicity have revealed increases in liver tumors in mice and a small increase in the incidence of renal carcinoma in male rats (1, 2). Tetrachloroethene and trichloroethene mediated toxicities are due to metabolic activation reactions (Figure 1). Two different pathways of biotransformation have been elucidated (3-10). Tetrachloroethene and trichloroethene are oxidized by cytochrome P450; this pathway results in the formation of trichloroacetic acid, the major metabolite of both tetra- and trichloroethene excreted in urine. The second pathway, catalyzed by glutathione S-transferases, yields S-(1,2,2-trichlorovinyl)glutathione from tetrachloroethene and S-(1,2-dichlorovinyl)glutathione and S-(2,2-dichlorovinyl)glutathione * Address correspondence to this author at the Institut fu¨r Toxikologie, Universita¨t Wu¨rzburg, Versbacherstrasse 9, 97078 Wu¨rzburg, FRG. Tel: +49 (0931) 201 3449; Fax: +49 (0931) 201 3446; E-mail
[email protected]. X Abstract published in Advance ACS Abstracts, November 1, 1995.
0893-228x/96/2709-0041$12.00/0
from trichloroethene as initial products (Figure 1). Glutathione conjugation of tetrachloroethene and trichloroethene represents a bioactivation reaction and has been implicated in the nephrotoxicity and renal tumorigenicity of these haloolefins (11). Glutathione S-conjugates formed in the liver are excreted with bile and further metabolized by γ-glutamyltransferase and dipeptidases to cysteine S-conjugates, which are acetylated to yield mercapturic acids. N-Acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (N-AcTCVC)1 is a minor urinary metabolite of tetrachloroethene, and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAc-1,2-DCVC) and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (N-Ac-2,2-DCVC) are minor metabolites of trichloroethene. These S-conjugates are mutagenic in several strains of Salmonella typhimurium and are toxic to rat kidney cells (12-16). They are cleaved by acylases followed by cysteine conjugate β-lyase to yield pyruvate, ammonia, and chlorothioketenes, which are presumed to 1 Abbreviations: FMO, flavin-containing monooxygenase; HPLC, high performance liquid chromatography; DMSO, dimethyl sulfoxide; DBN, 1,5-diazabicyclo[4.3.0]non-5-ene; N-Ac-TCVC, N-acetyl-S-(1,2,2trichlorovinyl)-L-cysteine; N-Ac-1,2-DCVC, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine; N-Ac-2,2-DCVC, N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine; N-Ac-TCVC-SO, N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide; N-Ac-1,2-DCVC-SO, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide; N-Ac-2,2-DCVC-SO, N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulfoxide.
© 1996 American Chemical Society
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Figure 1. Bioactivation of trichloroethene (R ) H) and tetrachloroethene (R ) Cl) by glutathione conjugation. The formation of the 2,2-isomer of N-acetyl-S-(dichlorovinyl)-Lcysteine is not shown to avoid an overcomplicated figure.
be the ultimate metabolites being responsible for the mutagenic and nephrotoxic effects (17, 18). In vivo studies on the biotransformation of hexachlorobutadiene, another chlorinated alkene, have revealed a novel pathway in the metabolism of halovinyl mercapturic acids, the formation of a sulfoxide (19). Formation of N-acetyl-S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine sulfoxide from hexachlorobutadiene was observed only in male rats. The formation of this vinyl sulfoxide suggests that mercapturic acids biosynthesized from both tetra- and trichloroethene may also be subjected to an oxidation reaction, which may be mediated by cytochrome P450 monooxygenases or the flavin-containing monooxygenases (FMO). Generally, nucleophilic sulfur atoms are oxidized preferentially by the FMO, whereas non-nucleophilic sulfur atoms are preferentially oxidized by the action of cytochrome P450-dependent monooxygenases (20-27). The experiments described here were performed to demonstrate the formation of sulfoxides derived from the mercapturic acids N-Ac-TCVC, N-Ac-1,2-DCVC, and N-Ac-2,2-DCVC, respectively, by microsomal monooxygenases and to compare the cytotoxicity of these mercapturic acids and their corresponding sulfoxides in isolated rat kidney epithelial cells.
Experimental Procedures Chemicals. Trichloroethene, tetrachloroethene, glacial acetic acid, and trifluoroacetic acid were purchased from Merck, Darmstadt, FRG, in the highest purity available. All other chemicals used for synthetic procedures and incubations were obtained from Sigma-Aldrich Chemie GmbH, Deisenhofen, FRG.
Werner et al. Syntheses. N-Ac-TCVC was prepared as described (6). N-Ac-1,2-DCVC was synthesized by the acetylation of S-(1,2dichlorovinyl)-L-cysteine (28) with acetic anhydride (29). N-Ac2,2-DCVC was synthesized by dissolving N-acetyl-L-cysteine (20.0 mmol, 3.26 g) in 40 mL of DMSO in the presence of 1,5diazabicyclo[4.3.0]non-5-ene (DBN) (44.0 mmol, 5.50 mL). After the addition of trichloroethene (22.0 mmol, 1.97 mL), the reaction mixture was stirred for 3 h at room temperature and diluted with water (25 mL). The obtained solution was acidified with hydrochloric acid to pH 1.5 and extracted with ether, and the organic layer was evaporated to dryness. The oily residue, consisting of the two regioisomers N-Ac-2,2-DCVC and N-Ac1,2-DCVC in a ratio of 10:1, was subsequently purified by semipreparative HPLC (250 × 8 mm steel column, filled with Partisil ODS III, linear gradient: 50% H2O/CF3CO2H, in methanol adjusted to pH 2, to 100% methanol over 25 min at a flow rate of 3 mL min-1 with UV detection at 225 nm). This method yielded 1.00 g (19.3%) of N-Ac-2,2-DCVC as a white crystalline solid in a purity >97%. 1H NMR (250 MHz, CD3OD): δ 2.00 (s, 3H, -COCH3), 3.13 (dd, JA-B ) 14.3 Hz, JA-X ) 7.72 Hz, 1H, HA), 3.33 (dd, JB-A ) 14.3 Hz, JB-X ) 4.78 Hz, 1H, HB), 4.62 (dd, JA-X ) 7.72 Hz, JB-X ) 4.78 Hz, 1H, HX), 6.62 (s, 1H, Cl2CdCHS-). 13C NMR (63 MHz, CD3OD): δ 20.8 (q, -COCH3), 34.4 (t, -CH2), 52.7 (d, -CH), 106.4 (s, Cl2CdCHS-), 126.2 (d, Cl2CdCHS-), 165.5 (s, -COCH3), 169.9 (s, -COOH). Electrospray MS: m/z (rel intensity, 35Cl) 290 (M + H + CH3OH, 100), 258 (M + H, 35). The synthesis of the sulfoxides of N-Ac-TCVC, N-Ac-1,2DCVC, and N-Ac-2,2-DCVC was accomplished by dissolving the mercapturic acids (1.00 mmol) in glacial acetic acid or trifluoroacetic acid (10 mL) and adding a 30% solution of H2O2 (112 µL, 1.10 mmol) at 4 °C. The mixtures were stirred for 1 h at 4 °C and afterwards at room temperature. The progress of the reaction was monitored by HPLC (for conditions, see Microsomal Incubations). After all of the starting material was converted to the sulfoxide, the solvent was removed under reduced pressure and the product precipitated by the addition of diethyl ether. The sulfoxides were collected by filtration as colorless solids, yielding a 1:1 mixture of the two possible diastereomers. All products showed a purity >96% as checked by HPLC with UV detection (225 nm). Analytical Characterization of Sulfoxides. N-Acetyl-S(1,2,2-trichlorovinyl)-L-cysteine sulfoxide (N-Ac-TCVC-SO, 1:1 mixture of two diastereomers):1H NMR (250 MHz, (CD3)2SO): δ 1.85 (s, 3H, -COCH3), 1.88 (s, 3H, -COCH3), 3.21-3.77 (m, 2H, -CH2), 4.43 (m, 1H, -CH), 4.50 (m, 1H, -CH), 8.59 (d, J ) 8.06 Hz, 1H, -NHCOCH3), 8.65 (d, J ) 8.06 Hz, 1H, -NHCOCH3). 13C NMR (63 MHz, (CD ) SO): δ 22.3 (q, -COCH ), 46.4 (d, -CH), 3 2 3 46.6 (d, -CH), 53.0 (d, -CH2), 53.5 (d, -CH2), 125.2 (s, Cl2CdCClS), 126.5 (s, Cl2CdCClS-), 135.9 (s, Cl2CdCClS-), 136.4 (s, Cl2CdCClS-), 169.5 (s, -COCH3), 169.9 (s, -COCH3), 170.9 (s, -COOH), 171.1 (s, -COOH). Electrospray MS: m/z (rel intensity, 35Cl) 308 (M + H, 100), 340 (M + H + CH OH, 23). 3 N-Acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulfoxide (N-Ac-2,2DCVC-SO, 1:1 mixture of two diastereomers): 1H NMR (400 MHz, (CD3)2SO): δ 1.85 (s, 3H, -COCH3), 1.88 (s, 3H, -COCH3), 3.15 (dd, JA-B ) 13.0 Hz, JA-X ) 11.5 Hz, 1H, HA), 3.32 (dd, JA-B ) 13.1 Hz, JA-X ) 8.77 Hz, 1H, HA), 3.47 (dd, JB-A ) 13.1 Hz, JB-X ) 5.33 Hz, 1H, HB), 3.52 (dd, JB-A ) 13.1 Hz, JB-X ) 3.35 Hz, 1H, HB), 4.50 (m, 1H, HX), 4.53 (m, 1H, HX), 7.65 (s, 1H, Cl2CdCHS-), 7.50 (s, 1H, Cl2CdCHS-), 8.52 (d, J ) 8.13 Hz, 1H, -NHCOCH3), 8.55 (d, J ) 8.25 Hz, 1H, -NHCOCH3). 13C NMR (100 MHz, (CD3)2SO): δ 22.2 (q, -COCH3), 22.3 (q, -COCH3), 46.1 (d, -CH), 46.5 (d, -CH), 53.8 (t, -CH2), 53.3 (t, -CH2), 130.2 (s, Cl2CdCHS-), 130.6 (s, Cl2CdCHS-), 134.8 (d, Cl2CdCHS-), 134.9 (d, Cl2CdCHS-), 169.2 (s, -COCH3), 169.6 (s, -COCH3), 171.2 (s, -COOH), 171.4 (s, -COOH). Electrospray MS: m/z (rel intensity, 35Cl) 274 (M + H, 100), 306 (M + H + CH3OH, 13). N-Acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (N-Ac-1,2DCVC-SO, 1:1 mixture of two diastereomers): 1H NMR (250 MHz, (CD3)2SO): δ 1.67 (s, 3H, -COCH3), 1.73 (s, 3H, -COCH3), 2.92-3.20 (m, 2H, -CH2), 4.19 (m, 1H, -CH), 4.31 (m, 1H, -CH),
Sulfoxidation of Mercapturic Acids 7.37 (s, 1H, ClHCdCClS-), 7.43 (s, 1H, ClHCdCClS-), 8.43 (d, J ) 7.95 Hz, 1H, -NHCOCH3), 8.48 (d, J ) 8.32 Hz, 1H, -NHCOCH3). 13C NMR (63 MHz, (CD3)2SO): δ 23.1 (q, -COCH3), 47.2 (d, -CH), 47.6 (d, -CH), 53.0 (t, -CH2), 53.6 (t, -CH2), 125.0 (d, ClHCdCClS-), 126.5 (d, ClHCdCClS-), 138.1 (s, ClHCdCClS), 138.6 (s, ClHCdCClS-), 170.1 (s, -COCH3), 170.6 (s, -COCH3), 171.6 (s, -COOH), 171.9 (s, -COOH). Electrospray MS: m/z (rel intensity, 35Cl) 274 (M + H, 100), 306 (M + H + CH3OH, 8). Microsomal Incubations. For the determination of sulfoxide formation, N-Ac-TCVC, N-Ac-1,2-DCVC, and N-Ac-2,2DCVC were incubated with liver and kidney microsomes of male and female rats. Complete incubation systems (final volume 250 µL) contained liver or kidney microsomal protein (2 mg/ mL), 0.1 M phosphate buffer (pH 7.4), 5 mM EDTA, and a NADPH-regenerating system, consisting of 10 mM glucose 6-phosphate, 1 mM NADP+, and 0.5 IU of yeast glucose 6-phosphate dehydrogenase/mL. When kinetic studies were performed, the reaction was started by the addition of the NADPH-regenerating system and terminated after 15 min at 37 °C by transferring the whole incubation mixture to Eppendorf caps, containing 12.5 µL of 70% perchloric acid. Substrate concentrations varied from 0.25 to 3.0 mM. After centrifugation, the supernatants were analyzed by HPLC (250 × 4 mm steel column, filled with Hypersil ODS, 5 µm; metabolites were eluted by applying a linear gradient from 100% H2O/CF3CO2H, adjusted to pH 2, to 100% methanol in 40 min at a flow rate of 1 mL/min). Eluting material was monitored at 225 nm, and sulfoxide formation was quantified by comparison of the peak areas with calibration curves for authentic mercapturic acid sulfoxides. Recovery of synthetic sulfoxides added to enzymatic incubations was >95%. n-Octylamine and the cytochrome P450 3A specific chemical inhibitor troleandomycin were preincubated at 37 °C for 10 min in the presence of microsomes and the regenerating system, and then substrates (0.25-2.0 mM) were added and incubations carried out as described above. To distinguish between cytochromes P450 and FMO, microsomes were heated for 3 min at 48 °C in the absence of the NADPH-regenerating system and then placed on ice, and the regenerating system was added. After a brief preincubation period (3 min, 37 °C), substrates were added and incubations performed as described above. Animals and Treatment. Adult male and female Wistar rats were used for all studies (Zentralinstitut fu¨r Versuchstierkunde, Hannover, FRG); they had free access to water and a standard diet (Altromin). Cytochrome P450 induction experiments were performed according to literature protocols with dexamethasone, phenobarbital (30), and pyridine (31). Twentyfour hours after the last administration of the inducers, animals were sacrificed by cervical dislocation and liver microsomes prepared as described (8, 32, 33). General Methods. Testosterone 6-β-hydroxylation (34, 35), 7-pentoxyresorufin O-dealkylation (36), and chlorzoxazone 6-hydroxylation (37) served as marker enzyme activities for the determination of microsomal cytochrome P450 3A1/2, 2B1/2, and 2E1. Flavin-containing monooxygenase activity was estimated by the conversion of thiobenzamide to the corresponding S-oxide by the procedure of Cashman and Hanzlik as modified by Tynes and Hodgson (38, 39). Total cytochrome P450 content was measured according to the method of Omura and Sato (40), and protein concentrations were determined by the method of Bradford (41) using the Bio-Rad protein kit and bovine γ-globulin as the standard. Instrumental Analyses. HPLC analyses were performed with a Hewlett-Packard HP 1050 series HPLC pump and a Hewlett-Packard 1040 series II diode array detector. 1H and 13C NMR spectra were recorded with a Bruker AC-250 and Bruker W-400 spectrometer. 1H chemical shifts are expressed in ppm downfield from tetramethylsilane. LC/MS analyses were performed with a Finnigan MAT TSQ 7000 mass spectrometer. Samples were introduced via HPLC using two Knauer HPLC pumps 64, equipped with micro pump heads, applying a linear gradient from 100% H2O/CF3CO2H, adjusted to pH 2, to 100% methanol in 15 min at a flow rate of 0.2 mL/min, and using a
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 43 steel column (125 × 2 mm) filled with Supershere RP18 3 µm, (Merck, Darmstadt, FRG). Injection volume was 5 µL. Isolation of Renal Epithelial Cells. Isolated rat renal epithelial cells were prepared from male Wistar rats (Institut fu¨r Versuchstierkunde, Hannover, FRG) weighing 250-300 g as described previously (42). The rats were allowed free access to water and Altromin standard pellets (Lage, FRG). Diazepam (5 mg/kg) and a combination of fentanyl base (0.2 mg/kg) and fluanison (10 mg/kg) were used to achieve a state of neuroleptanalgesia. The obtained cell pellet was gently resuspended in Krebs-Henseleit buffer (pH 7.4), containing 25 mM HEPES, 2.5 mM CaCl2, 15 mM NaHCO3, and 5 mM glucose at a density of approximately 1.2 × 106 cells/mL. All buffers were equilibrated with 95% O2/5% CO2. Cell yield was approximately (2025) × 106 cells/kidney, and typically 85-95% of the freshly prepared cells excluded trypan blue at the beginning of the incubation directly after resuspension of the cell pellet. Viability of control cells did not decrease during 120 min of incubation at 37 °C. Viability was estimated by trypan blue exclusion. At 200 µL aliquot of the cell suspension was mixed with an equal volume of sterile filtered trypan blue solution (0.4%), and the number out of 500 cells that stained was counted immediately. Cytotoxicity of Halovinyl Mercapturic Acids and Their Sulfoxides in Rat Renal Cortex Cells. Cells (106/mL) were incubated at 37 °C in a shaker-water bath with the test compounds dissolved in buffer, and samples were taken after 0, 30, 60, and 90 min for viability determination (42). In experiments with (aminooxy)acetic acid, the cell suspension was preincubated for 10 min with the inhibitor dissolved in 0.9% NaCl before addition of the test compound. The final concentration of (aminooxy)acetic acid was 0.5 mM (42). Mutagenicity of Mercapturic Acids and Their Sulfoxides in the Ames Test. These experiments were performed as described before using Salmonella typhimurium TA100 and TA2638 in presence of kidney cytosol from male rats (43).
Results Synthetic Chemistry. The preparation of the mercapturic acid sulfoxides from the corresponding mercapturic acids using hydrogen peroxide as the oxidizing agent yielded a mixture of two diastereomers, where no significant diastereomeric excess was observed as determined by the comparison of peak areas of the individual isomers in the HPLC chromatogram. These sulfoxides were not characterized before. The diastereomers proved to be configurationally stable under the conditions employed (data not shown). Also, Carren˜o et al. (44) described asymmetric Diels-Alder reactions by the use of optically pure vinyl sulfoxides, which gives additional evidence for the configurational stability of these compounds and indicates that the observed formation of two diastereomers during the synthetic procedure is not simply the result of an epimerization reaction. Identification of Sulfoxide Metabolites. The identification of sulfoxides formed in microsomal incubations was performed by comparison with UV spectra of authentic materials and liquid chromatography/mass spectrometry (LC/MS). Since the UV spectra of the sulfoxides are not very characteristic (not shown), metabolites formed in liver microsomes of untreated and phenobarbital- and dexamethasone-induced male rats were pooled, purified by preparative HPLC, and subsequently analyzed by LC/MS. The biosynthetic formation of N-AcTCVC-SO, N-Ac-1,2-DCVC-SO, and N-Ac-2,2-DCVC-SO was confirmed by electrospray mass spectrometry. Mass spectra showed molecular ions identical to those observed in mass spectra of authentic material and indicated the presence of two (N-Ac-1,2-DCVC-SO and N-Ac-2,2-DCVC-
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Werner et al. Table 1. Effect of Differential Thermal Inactivation, n-Octylamine, and Troleandomycin (TAO) on N-Ac-TCVC, N-Ac-1,2-DCVC, and N-Ac-2,2-DCVC Sulfoxidation in Liver Microsomes of Untreated Male Wistar Rats rates of sulfoxide formationa [pmol/(mg‚min)] incubation conditions
N-AcTCVC-SO
N-Ac-1,2DCVC-SO
N-Ac-2,2DCVC-SO
control -NADPH -protein +n-octylamine (5 mM) heat inactivation +TAO (50 µM) +glutahione (5 mM)
144 ( 40
