Biotransformation, Excretion, and Nephrotoxicity of the

Biotransformation, Excretion, and Nephrotoxicity of the Hexachlorobutadiene Metabolite (E)-N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-l-cysteine Sul...
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Chem. Res. Toxicol. 1998, 11, 750-757

Biotransformation, Excretion, and Nephrotoxicity of the Hexachlorobutadiene Metabolite (E)-N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine Sulfoxide Gerhard Birner, Michael Werner, Elisabeth Rosner, Claudia Mehler, and Wolfgang Dekant* Department of Toxicology, University of Wu¨ rzburg, Versbacher Strasse 9, 97078 Wu¨ rzburg, FRG Received December 5, 1997

Hexachlorobuta-1,3-diene (HCBD) is nephrotoxic in rodents. Its toxicity is based upon a multistep bioactivation pathway. Conjugation with glutathione by glutathione S-transferases to form (E)-S-(1,2,3,4,4-pentachlorobutadienyl)-L-glutathione (PCBG), further processing to the corresponding cysteine S-conjugate, and finally processing to a reactive thioketene are thought to be responsible for the observed nephrotoxic effects. A novel metabolite, identified as (E)N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide (N-AcPCBC-SO), was described after administration of [14C]HCBD to male Wistar rats. This metabolite is formed by sulfoxidation of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (N-AcPCBC) mediated by cytochrome P450 3A and has been found to be cytotoxic to proximal tubular cells in vitro without activation by β-lyase. In rats, given HCBD in vivo, only one diastereomer of the sulfoxide is excreted; however, in rat hepatic microsomes two diastereomers, (R)- and (S)-NAcPCBC-SO, are formed. This study focuses on the mechanisms responsible for this discrepancy and on a possible contribution of N-AcPCBC-SO to the nephrotoxicity of HCBD in vivo. (R,S)N-AcPCBC-SO (1:1 mixture of both diastereomers) and N-acetyl-R-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-D,L-cysteine sulfoxide (R-Me-N-AcPCBC-SO) were administered iv to male and female Wistar rats (20, 40, and 80 µmol/kg of body weight). R-Me-N-AcPCBC-SO cannot be cleaved by cysteine conjugate β-lyase even if R-Me-N-AcPCBC-SO is deacetylated by acylases. Excretion of γ-glutamyltranspeptidase, protein, and glucose in the urine, indicative for kidney damage, and histopathological examination of the kidneys showed marked differences in the renal damage in male and female rats after application of N-AcPCBC-SO and R-Me-N-AcPCBCSO. Necroses of the kidney tubules were only found in male, but not female, rats. Major sex-specific differences were observed in the elimination of sulfoxides; the (R)-isomer was excreted in a 5-10-fold excess to the (S)-isomer after application of (R,S)-N-AcPCBC-SO. After purification, both isomers were administered to male rats resulting in the urinary excretion of (R)-N-AcPCBC-SO after giving the (R)-isomer; treatment with (S)-N-AcPCBC-SO, however, revealed the formation of (S)-N-acetyl-S-(2-glycinylcystein-S-yl-1,3,4,4-tetrachlorobutadienyl)L-cysteine. The results show major sex-specific differences in the nephrotoxic potency of N-AcPCBC-SO and R-Me-N-AcPCBC-SO. However, both N-AcPCBC-SO and R-Me-N-AcPCBCSO are nephrotoxic in males, suggesting the formation of a vinyl sulfoxide as an additional, β-lyase-independent mechanism in HCBD-caused nephrotoxicity.

Introduction Hexachlorobuta-1,3-diene (HCBD)1 is a selective nephrotoxin and a nephrocarcinogen in rats, damaging selectively the pars recta of the proximal tubules (1-4). Its organ-specific toxicity is based upon a multistep bioactivation mechanism involving hepatic and renal enzymes (Scheme 1). Glutathione conjugation appears to be the only bioactivation pathway leading to reactive intermediates (5). HCBD is conjugated with glutathione by hepatic * Author for correspondence: Dr. W. Dekant. Tel: +49-931-201 3449. Fax: +49-931-201 3446. E-mail: [email protected]. 1 Abbreviations: HCBD, hexachlorobuta-1,3-diene; PCBG, S-(1,2,3,4,4pentachlorobutadienyl)-L-glutathione; N-AcPCBC, N-acetyl-S-(1,2,3,4,4pentachlorobutadienyl)-L-cysteine; N-AcPCBC-SO, N-acetyl-S-(1,2,3,4,4pentachlorobutadienyl)-L-cysteine sulfoxide; R-Me-N-AcPCBC-SO, N-acetyl-R-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-D,L-cysteine sulfoxide.

glutathione S-transferases to form S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-glutathione (PCBG). After elimination into the bile and intestinal reabsorption, PCBG and derived S-conjugates are subsequently translocated to the kidney (6, 7) and PCBG is catabolized by renal γ-glutamyltranspeptidase and dipeptidases to S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine (PCBC). PCBC may be acetylated by the action of renal N-acetyltransferases to N-acetyl-S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine (N-AcPCBC). Both PCBC and N-AcPCBC are accumulated in the kidney by the organic anion transport system. While N-AcPCBC is eliminated with the urine, PCBC may be cleaved by renal cysteine conjugate β-lyase yielding a reactive thioketene which is thought to be responsible for nephrotoxic effects observed in rats. A previous study with male and female rats revealed sex-specific differences in the metabolism of HCBD as

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Biotransformation of Halovinyl Sulfoxides Scheme 1. Bioactivation of Hexachlorobutadiene by Glutathione Conjugation

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may not be formed by this pathway. Additionally, after HPLC separation, both diastereomers, (R)-N-AcPCBCSO and (S)-N-AcPCBC-SO, were applied to male rats to check possible differences in their biological activities.

Experimental Procedures

judged from the urinary excretion pattern after oral administration of [14C]HCBD (8). In male rats, a novel metabolite was identified as N-acetyl-S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine sulfoxide (N-AcPCBCSO) which was not detectable in the urine of female rats. This vinyl sulfoxide is formed by sulfoxidation of NAcPCBC, mediated by cytochrome P450 monooxygenases of the 3A family which are expressed solely in adult male, but not female, rats (9). In incubations with isolated renal proximal tubular cells N-AcPCBC-SO was cytotoxic in the presence of the β-lyase inhibitor aminooxyacetic acid (AOAA), suggesting a β-lyase-independent toxicity of this sulfoxide (8). A structurally related sulfoxide derived from S-(1,2-dichlorovinyl)-L-cysteine was also shown to exhibit β-lyase-independent toxicity (10). A discrepancy, however, having not been clarified yet, was observed in both experiments (8, 9, 11): In incubations of N-AcPCBC with rat liver microsomes two diastereomers (R)- and (S)-N-AcPCBC-SO were formed in a nearly 1:1 molar ratio by P450 3A 1/2, but after administration of HCBD to male rats only one diastereomer was found to be excreted with urine. The objectives of this study were (i) to find a molecular basis for the stereoselective formation/excretion of NAcPCBC-SO by checking a possible role of glutathione S-transferases for the disposition of both isomers, (ii) to elucidate the role of N-AcPCBC-SO in the nephrotoxicity of HCBD, and (iii) to clarify the mechanism of toxicity of N-AcPCBC-SO. Therefore (R,S)-N-acetyl-S-(1,2,3,4,4pentachlorobutadienyl)-L-cysteine sulfoxide and N-acetylR-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)- D/L-cysteine sulfoxide (R-Me-N-AcPCBC-SO) were administered to male and female Wistar rats, and kinetics of excretion and parameters indicative of nephrotoxicity were monitored. Because of the lack of a proton in the R-position, R-Me-N-AcPCBC-SO cannot be cleaved by renal cysteine conjugate β-lyase (12-14); thus reactive intermediates

Instrumental Analyses. A HP 1050 series HPLC pump with a HP 1040 series II diode array detector and a HP 1090 diode array system (Hewlett-Packard, Avondale, CA) equipped with Rheodyne injectors were used for the analysis of urine samples. Electrospray mass spectrometry was performed on a Trio 2000 quadrupole mass spectrometer (Fisons, Mainz, FRG) using an ESI interface. CD spectra were determined on a Jasco J-700 spectropolarimeter (Shimadzu, Du¨sseldorf, FRG). Syntheses. All chemicals used for the preparation of synthetic materials were obtained from commercial sources in the highest purity available. N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-l-cysteine. N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (purity > 98% as checked by HPLC) was prepared by the method of Reichert and Schu¨tz and characterized by 1H and 13C NMR and mass spectrometry (9, 15). N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L -cysteine Sulfoxide. N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)L-cysteine sulfoxide was synthesized by stirring N-AcPCBC (1.0 mmol) with 1.05 mmol of H2O2 (30%) in 10 mL of trifluoroacetic acid (9, 16). After 5 h at 4 °C, the solvent was evaporated under reduced pressure and the product was precipitated by the addition of 10 mL of cold diethyl ether. N-AcPCBC-SO was subsequently collected by filtration as a 1:1 mixture of two diastereomers in a yield of 89%. The product was >97% pure as determined by HPLC with UV detection (λ ) 225 nm). Characterization of N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide (1:1 mixture of two diastereomers): mp 167-168 °C dec. 1H NMR (400 MHz, DMSO-d6) (R)isomer: δ 1.86 (s, 3 H, -COCH3), 3.22-3.46 (m, 2 H, -CH2-), 4.48 (m, 1 H, -CH-), 8.57 (d, J ) 7.70 Hz, 1 H, -NHCOCH3). 13C NMR (100 MHz, DMSO-d ) (R)-isomer: δ 22.5 (q, -COCH ), 6 3 46.7 (d, -CH-), 53.2 (t, -CH2-), 122.6 (s, C-4), 126.4 (s, C-2), 126.6 (s, C-3), 142.4 (s, C-1), 169.5 (s, -COCH3), 171.0 (s, -COOH). 1H NMR (400 MHz, DMSO-d6) (S)-isomer: δ 1.90 (s, 3 H, -COCH3), 3.22-3.46 (m, 2 H, -CH2-), 4.49 (m, 1 H, -CH-), 8.66 (d, J ) 7.80 Hz, 1 H, -NHCOCH3). 13C NMR (100 MHz, DMSO-d6) (S)-isomer: δ 22.5 (q, -COCH3), 46.8 (d, -CH), 53.7 (t, -CH2-), 122.7 (s, C-4), 126.5 (s, C-2), 126.6 (s, C-3), 142.6 (s, C-1), 169.6 (s, -COCH3), 171.1 (s, -COOH). Electrospray mass spectrum: m/z 402 [M + H]+, 418 [M + NH3]+. For further characterization the two diastereomers were separated by semipreparative HPLC. Under the conditions applied (see below) the diastereomer of N-AcPCBC-SO with (-)(R)-configuration eluted first at 66.7% methanol (tR 26.7 min) and the (+)(S)-isomer at 70.5% methanol (tR 28.2 min). The configuration of the sulfoxides was assigned by stereoselective oxidation of N-AcPCBC with NaIO4 in the presence of chloroperoxidase. Under these conditions the formation of the (R)-configuration of sulfoxides is preferred (17). A 20% excess of the (R)-diastereomer was obtained in these reactions. [14C]-(R)-N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)L-cysteine Sulfoxide. This was obtained by semipreparative HPLC of urine samples of male rats after treatment with [14C]HCBD (200 mg/kg) from previous experiments (8). The samples were stored at -80 °C. The major urinary metabolite in those samples has been identified as (R)-N-AcPCBC-SO. After preparative HPLC separation of 60 mL of urine from rats treated with [14C]HCBD, 41 mg of [14C]-(R)-N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide was obtained in a purity of >95% based on HPLC with radioactivity detection. (S)-N-Acetyl-S-(2-glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl)-L-cysteine Sulfoxide. The glutathione conjugate of N-AcPCBC-SO was synthesized as described (18) and was

752 Chem. Res. Toxicol., Vol. 11, No. 7, 1998 used for the quantification of the corresponding glycinylcysteine S-conjugate since their electronic spectra are identical. N-Acetyl-r-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)D/L-Cysteine. S-Benzyl-R-methyl-D/L-cysteine was synthesized according to the method of Arnstein by the reaction of benzyl mercaptan (174 mol, 20.9 mL) with 1-chloro-2-propanone (174 mol, 13.9 mL) and sodium metal (194 mmol, 4.50 g) in methanol (100 mL) (19). The colorless product obtained after several workup steps had the same melting point as described by Arnstein (244-246 °C, dec). N-Acetyl-S-benzyl-R-methyl-D/Lcysteine was formed with acetic acid anhydride by a procedure described for the preparation of N-acetyl-S-benzyl-L-cysteine (20). After acidification of the reaction mixture, the precipitated, filtered, and dried compound had a purity of >98% as checked by HPLC with UV detection (λ ) 225 nm) as well as 1H and 13C NMR and was therefore used without further purification steps. For the synthesis of N-Acetyl-R-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-D/L-cysteine, the benzyl moiety of N-acetyl-Sbenzyl-R-methyl-D/L-cysteine was removed in liquid ammonia (21). After evaporation of the solvent, the intermediary N-acetylR-methyl-D/L-cysteine was taken up in methanol in situ and reacted with HCBD and the desired product isolated according to the method of Reichert and Schu¨tz by column chromatography on silica gel with chloroform/acetone/formic acid (65:30:5) as the eluent (15). Characterization of N-acetyl-R-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-D/L-cysteine: mp 173-175 °C dec. 1H NMR (400 MHz, DMSO-d6): δ 1.39 (s, 3 H, R-CH3), 1.83 (s, 3 H, -COCH3), 3.50 (d, JAB ) 14.0 Hz, 1 H, HA), 3.86 (d, JBA ) 14.0 Hz, 1 H, HB), 8.32 (s, 1 H, -NHCOCH3). 13C NMR (100 MHz, DMSOd6): δ 22.2 (q, R-CH3), 22.3 (q, -COCH3), 38.2 (t, -CH2-), 57.5 (s, R-C), 119.2 (s, C-4), 124.6 (s, C-2), 125.2 (s, C-3), 135.1 (s, C-1), 169.3 (s, -COCH3), 173.8 (s, -COOH). Thermospray mass spectrum: m/z (%) ) 400 (45) [M+ + H]. Electrospay mass spectrum: m/z (%) ) 399 (50) [M+]. N-Acetyl-r-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)D/L-cysteine Sulfoxide. N-Acetyl-R-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-D/L-cysteine sulfoxide was obtained by a procedure as described above for N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide. Characterization of N-acetyl-R-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-D/L-cysteine sulfoxide (1:1 mixture of two diastereomers): mp 204-206 °C dec. 1H NMR (400 MHz, DMSOd6) (R)-isomer: δ 1.50 (s, R-CH3), 1.82 (s, 3 H, -COCH3), 3.18 (d, JAB ) 13.4 Hz, 1 H, HA), 3.75 (d, JBA ) 13.4 Hz, 1 H, HB), 8.76 (s, 1 H, -NHCOCH3). 13C NMR (100 MHz, DMSO-d6) (R)-isomer: δ 22.0 (q, R-CH3), 23.3 (q, -COCH3), 55.6 (s, R-C), 57.7 (t, -CH2-), 122.4 (s, C-4), 124.4 (s, C-3), 126.2 (s, C-2), 142.8 (s, C-1), 169.7 (s, -COCH3), 173.5 (s, -COOH). 1H NMR (400 MHz, DMSO-d6) (S)-isomer: δ 1.53 (s, R-CH3), 1.89 (s, 3 H, -COCH3), 3.40 (d, JAB ) 13.4 Hz, 1 H, HA), 3.57 (d, JBA ) 13.4 Hz, 1 H, HB), 8.48 (s, 1 H, -NHCOCH3). 13C NMR (100 MHz, DMSO-d6) (S)-isomer: δ 22.3 (q, R-CH3), 23.7 (q, -COCH3), 56.1 (s, R-C), 58.8 (t, -CH2-), 122.6 (s, C-4), 124.4 (s, C-3), 126.2 (s, C-2), 143.3 (s, C-1), 170.0 (s, -COCH3), 173.5 (s, -COOH). Electrospray mass spectrum: m/z 416 [M + H]+. Animals and Treatment. Adult male (250-300 g) and female (220-260 g) Wistar rats were obtained from the Zentralinstitut fu¨r Versuchstierkunde (Hannover, FRG). N-AcetylS-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine sulfoxide and N-acetyl-R-methyl-S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-D/Lcysteine sulfoxide (20, 40, and 80 µmol/kg of body weight), as 1:1 mixtures of both diastereomers, were dissolved in isotonic saline and given to rats iv via the tail vein. (R)-[14C]-N-AcPCBCSO (40 µmol/kg), (R)-N-AcPCBC-SO, and (S)-N-AcPCBC-SO (20 µmol/kg), respectively, dissolved in isotonic saline were also given iv to male rats. All animals were transferred to all-glass metabolic cages allowing the collection of urine and feces separately. They had free access to a standard diet consisting of Altromin, corn starch, and water. Sampling and Determination of Radioactivity. Urine was collected over dry ice 3, 6, 12, 24, 36, and 48 h after

Birner et al.

Figure 1. Radiochromatogram of urine samples of a male rat treated with [14C]HCBD (200 mg/kg; po); urine was collected for 12 h after administration (A); UV chromatogram of an incubation of N-AcPCBC with liver microsomes of male rats (B). The large peak eluting at 21 min has not been conclusively identified; it likely represents a mercapturic acid derived from the conjugation of N-AcPCBC with glutathione. administration and kept frozen until analysis. Feces samples were taken in 12-h intervals. After the experiment the animals were killed by an overdose of ether; organs were removed and stored at -80 °C. For histopathological examination kidneys were fixed in formalin. Radioactivity in different tissues was determined according to Mahin and Lofberg (22): 500 mg of organs was dissolved in 200 µL of perchloric acid, 400 µL of hydrogen peroxide solution (30%) was added, and 24 h later, after addition of 10 mL of Rotiszint R 2200, the radioactivity was determined by liquid scintillation counting (Tricarb 4000, Packard Instruments, Downers Grove, IL). Separation and Quantification of Metabolites. After filtration (0.45-µm HV filter, Millipore, Eschborn, FRG), aliquots of urine (10-100 µL) were injected directly into HPLC systems with diode array detector (HP 1090 or HP 1040 series II, Hewlett-Packard, Avondale, CA) using steel columns (25 × 0.4 cm) filled with Partisil (Whatman) ODS III or Hypersil (Shandon) ODS, 5 µm, and gradient elution for the separation of metabolites monitoring the eluate at 263 or 254 nm. Solvent A: water, pH 2.0, with trifluoroacetic acid. Solvent B: 100% methanol; linear gradient 0-100% B in 40 min or 30-100% B in 40 min; flow rate 1 mL/min. Quantification was based on comparison of peak areas of standard calibration curves. Limits of detection were 20 nmol/mL. For semipreparative separations steel columns (25 × 0.8 cm) filled with Hypersil ODS, 5 µm (Shandon), and gradient elution were used, monitoring the eluate at 225 and 255 nm. Solvent A: water, pH 2, with trifluoroacetic acid. Solvent B: 100% methanol; linear gradient 0-80% B in 30 min; flow rate 2.5 mL/ min. Incubations of (R,S)-N-AcPCBC-SO. These experiments were performed to investigate a possible reduction of the sulfoxide to the mercapturic acid by thioredoxin. Liver and kidney cytosol was prepared according to the method of Dohn and Anders (23). (R,S)-N-AcPCBC-SO (0.5 mM) was incubated with NADPH (0.5 mM) and liver and kidney cytosol (2.5 mg of protein/mL) of male and female Wistar rats in 500 µL of potassium phosphate buffer (0.1 M; pH 7.4) at 37 °C. After 30 min, perchloric acid (70%; 25 µL) was added, and 50 µL of the supernatant was analyzed by HPLC for formation of N-AcPCBC after centrifugation. Assessment of Nephrotoxicity. Urine Clinical Chemistry: γ-Glutamyltranspeptidase activity (GGT), protein, and

Biotransformation of Halovinyl Sulfoxides

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Figure 2. Urinary excretion of the diastereomers over 24 h after administration of (R,S)-N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)L-cysteine sulfoxide (A and B) and (R,S)-N-acetyl-R-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-D/L-cysteine sulfoxide (C and D) (1:1 mixture of both diastereomers) to male and female rats (doses: 20 µmol/kg, (R)-isomer, (S)-isomer; 80 µmol/kg, (R)-isomer, (S)isomer). glucose in the urine were determined using commercially available test units (Sigma, Deisenhofen; BioRad, Mu¨nchen, FRG). Histopathology: Kidneys were fixed in formalin (12.5%) for 2 days, and routinely processed slices were used for histopathological examination after H & E staining.

Results Elimination of (R,S)-N-AcPCBC-SO in Rats. To obtain additional information about the further processing of (R)- and (S)-N-AcPCBC-SO, (R,S)-N-AcPCBC-SO was given iv to male and female rats. These experiments were performed since the two diastereomers of N-AcPCBCSO are formed in a nearly 1:1 molar ratio in incubations of of N-AcPCBC with rat liver microsomes, whereas after administration of HCBD to male rats, the (R)-diastereomer was found to be the predominant diastereomer excreted with urine (Figure 1). The large peak with a retention time of 21 min likely represents a mercapturic acid formed by conjugation of a sulfoxide metabolite. Its electronic spectrum was identical to that of (S)-N-acetylS-(2-glycinylcystein-S-yl-1,3,4,4-tetrachlorobutadienyl)L-cysteine sulfoxide. After administration of a 1:1 mixture of both diastereomers, less than 10% of the dose was recovered in the urine over 48 h. Most of the sulfoxides were excreted within 6 h after application (Figure 2). No significant sex-specific differences in the excretion of both sulfoxide diastereomers were observed. However, the (R)-diastereomer of N-AcPCBC-SO was excreted in a 5-10-fold excess as compared to the (S)-form after application of a 1:1 mixture of both (Figure 3). The structural assignment of both diastereomers was made after collecting the first eluting isomer and analysis by CD spectroscopy.

The stereoselective processing of both isomers in vivo confirms previous results (8, 9): In incubations of NAcPCBC with rat liver microsomes two diastereomers, (R)- and (S)-N-AcPCBC-SO, were formed in a nearly 1:1 molar ratio by P450 3A 1/2 as shown by HPLC (Figure 1B); in contrast, after administration of HCBD to male rats, only one diastereomer [(R)-N-AcPCBC-SO] was identified in urine (Figure 1A). This discrepancy could be explained by either a stereoselective reduction of the sulfoxide diastereomers or differences in the reactivity of the two isomers toward further biotransformation, e.g., by GSH conjugation. Reduction of Cysteine S-Conjugate Sulfoxides. A stereoselective synthesis of the N-AcPCBC-SO isomers could be excluded based on results by Werner et al. (9). To check for a possible reduction of one or both diastereomers by the NADPH-dependent thioredoxin reductase or other reductases as an explanation for the stereoselective excretion of only one isomer in the urine of male rats after HCBD application, (R,S)-N-AcPCBC-SO (1:1 mixture) was incubated with liver and kidney cytosol of male and female rats in the presence of NADPH and aliquots of the incubation mixtures were analyzed by HPLC (24, 25). Neither a decrease in sulfoxide concentration nor the formation of the corresponding mercapturic acid was observed (data not shown). Elimination of (R)-N-AcPCBC-SO and (S)-NAcPCBC-SO and Identification of (S)-N-Acetyl-S-(2glycinylcystein-S-yl-1,3,4,4-tetrachlorobutadienyl)L-cysteine Sulfoxide as Metabolite of (S)-N-AcPCBCSO. To obtain further information about the mechanism for the stereoselective differences in the urinary excretion, both diastereomers of N-AcPCBC were purified by HPLC and given to male rats under identical conditions.

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Figure 3. HPLC separation of urine of male Wistar rats, excreted 3 h after treatment with (R,S)-N-acetyl-S-(1,2,3,4,4pentachlorobutadienyl)-L-cysteine sulfoxide (80 µmol/kg), UV detection at 263 nm, and the corresponding electronic spectra of both sulfoxide diastereomers. The peaks at tR 21.8 and 22.2 min represent the two diastereomers of N-AcPCBC-SO. (R)-N-AcPCBC-SO is excreted in a 5-10-fold excess after administration of a 1:1 mixture of both diastereomers. Table 1. Disposition of Radioactivity in Male Rats 48 h after iv Application of [14C]-(R)-N-AcPCBC-SO (20 µmol/kg)

Figure 4. HPLC separation of urine of male Wistar rats, excreted 3 h after treatment with 40 µmol/kg (R)-N-acetyl-S(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide (A) and (S)-N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide (B), UV detection at 250 nm, and the corresponding electronic spectra.

Because of the expensive workup, only limited amounts of the isolated isomers were available allowing us only to give a single dose. After application of (R)-N-AcPCBCSO, the urinary excretion of unchanged (R)-N-AcPCBCSO (10.6% of dose within 48 h) was observed (Figure 4A). In contrast, in the urine of rats treated with (S)-NAcPCBC-SO, only traces of unmetabolized sulfoxide were detected. However, HPLC chromatograms of the urine

sample

radioactivity (% of dose)

sample

radioactivity (% of dose)

urine feces liver kidney lung

21.7 12.3 1.8 0.45 0.14

spleen muscle fat plasma erythrocytes

0.3 0.7 3.6 2.7 3.8

samples showed a new metabolite eluting at tR 21.8 min, accounting for 18.3% of the dose applied. After preparative workup, the metabolite was identified as (S)-Nacetyl-S-(2-glycinylcystein-S-yl-1,3,4,4-tetrachlorobutadienyl)-L-cysteine sulfoxide by mass spectrometry and by its electronic spectrum (Figure 4B). The electrospray mass spectrum showed a molecular ion [M + H]+ 544 containing four chlorine atoms; the obtained molecular ion and the electronic spectrum are consistent with (S)N-acetyl-S-(2-glycinylcystein-S-yl-1,3,4,4-tetrachlorobutadienyl)-L-cysteine sulfoxide. GSH conjugates or derived metabolites were not detected in the urine of rats treated with the (R)-diastereomer. Disposition of (R)-N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine Sulfoxide in Male Rats. Only a minor part of the dose applied was found to be excreted with urine. To receive additional data about the disposition of the sulfoxides, [14C]-(R)-N-AcPCBC-SO was given iv to male rats (40 µmol/kg). The excretion of metabolites in urine and feces was monitored and quantified over 48 h. The major radioactive peak in the urine (22% of the dose applied) was found to be unmetabolized (R)-N-AcPCBC-SO. After 48 h the animals were killed, and the disposition of radioactivity in several tissues (Table 1) was determined by LSC according to the method of Mahin and Lofberg (22). There was 6.5% of the dose of [14C]-(R)-N-AcPCBC-SO and its metabolites present in blood and 3.6% in fat; the recovery was 47.5% of the dose applied. Elimination of (R,S)-r-Me-N-AcPCBC-SO in Rats. To clarify the mechanism of toxification of N-AcPCBCSO, R-Me-N-AcPCBC-SO (1:1 mixture of both diastere-

Biotransformation of Halovinyl Sulfoxides

Chem. Res. Toxicol., Vol. 11, No. 7, 1998 755

Table 2. Urinary Excretion of γ-Glutamyltransferase, Glucose, and Protein over 48 h after iv Application of N-AcPCBC-SO to Male and Female Wistar Ratsa male Wistar rats

female Wistar rats

dose (µmol/kg)

γ-glutamyltransferase (103 units/48 h)

glucose (mg/48 h)

protein (mg/48 h)

γ-glutamyltransferase (103 units/48 h)

glucose (mg/48 h)

protein (mg/48 h)

0 20 40 80

0.82 ( 0.38 3.64 ( 0.52+ 19.40 ( 7.17+,* 89.76 ( 14.85+,*

1.95 ( 0.81 2.89 ( 0.48+ 47.84 ( 4.57+,* 143.60 ( 7.97+,*

42 ( 21 73 ( 26+ 215 ( 43+ 321 ( 84+

0.13 ( 0.05 0.10 ( 0.01 0.15 ( 0.09 5.30 ( 3.02*

1.83 ( 0.64 1.37 ( 0.50 2.13 ( 0.45 7.40 ( 0.66*

6(3 6(3 7(3 8(1

a Values are mean ( SD; n ) 5. +Significantly different from female rats (p < 0.01). *Significantly different compared to the lower dose (p < 0.05) using Student’s t test.

Table 3. Urinary Excretion of γ-Glutamyltransferase, Glucose, and Protein over 48 h after iv Application of r-Me-N-AcPCBC-SO to Male and Female Wistar Ratsa male Wistar rats

female Wistar rats

dose (µmol/kg)

γ-glutamyltransferase (103 units/48 h)

glucose (mg/48 h)

protein (mg/48 h)

γ-glutamyltransferase (103 units/48 h)

glucose (mg/48 h)

protein (mg/48 h)

0 20 40 80

0.95 ( 0.48 5.57 ( 1.26+ 16.8 ( 2.10+,* 23.1 ( 4.01+,*

3.10 ( 1.20 4.19 ( 0.46+ 4.87 ( 0.33+ 46.4 ( 18.87+,*

36 ( 16 88 ( 12+ 90 ( 30 194 ( 12+,*

0.24 ( 0.13 1.39 ( 0.86 2.57 ( 0.12* 8.18 ( 1.05*

1.76 ( 0.52 2.68 ( 1.26 3.01 ( 0.77 5.27 ( 0.67*

5(3 7(3 39 ( 26* 31 ( 8

a Values are mean ( SD; n ) 5. +Significantly different from female rats (p < 0.01). *Significantly different compared to the lower dose (p < 0.01) using Student’s t test.

Table 4. Urinary Excretion of γ-Glutamyltransferase, Glucose, and Protein over 48 h after iv Application of (R)-N-AcPCBC-SO and (S)-N-AcPCBC-SO (20 µmol/kg) to Male Wistar Ratsa (R)-N-AcPCBC-SO

(S)-N-AcPCBC-SO

dose (µmol/kg)

γ-glutamyltransferase (103 units/48 h)

glucose (mg/48 h)

protein (mg/48 h)

γ-glutamyltransferase (103 units/48 h)

glucose (mg/48 h)

protein (mg/48 h)

20

2.79 ( 0.85

2.74 ( 0.44

86 ( 13

4.50 ( 2.36

2.69 ( 0.38

84 ( 14

a

Values are mean ( SD; n ) 4.

omers) was also given to male and female rats. R-MeN-AcPCBC-SO cannot be cleaved by renal cysteine conjugate β-lyase (12-14); thus reactive intermediates may not be formed by this pathway, and bioactivation by β-lyase can be excluded. No significant differences in the stereoselective excretion of the diastereomers with urine or in the dose recovered were found in comparison with the data obtained after administration of N-AcPCBCSO (Figure 2). Nephrotoxicity of Sulfoxides. Histopathological examination of the kidneys using H & E staining and urinary clinical chemistry showed marked differences in the renal damage. Urine clinical chemistry showed a marked dose-dependent increase of γ-glutamyltransferase excretion, indicative for damage in the proximal tubules, after administration of N-AcPCBC-SO and R-MeN-AcPCBC-SO to male rats (Tables 2 and 3). The excretion of protein and glucose also increased with the dose administered in male rats. In contrast, the excretion of GGT, protein, and glucose in the urine of female animals was only marginal and not dose-dependent. After separate administration of (R)-N-AcPCBC-SO and (S)-N-AcPCBC-SO (20 µmol/kg), no significant differences were found in the urinary excretion of γ-glutamyltransferase, protein, and glucose between both isomers (Table 4); however, all parameters were significantly increased as compared to control. Histopathology of the kidney after separate administration of N-AcPCBC-SO and R-Me-N-AcPCBC-SO to male rats revealed necroses of the proximal tubular epithelial (data not shown). In comparison, N-AcPCBC-SO revealed a higher nephrotoxic potency than R-Me-N-AcPCBC-SO judged from the extent of the formation of necroses. In contrast, only minor alterations, but no necroses, were observed in

kidney slices of female rats (data not shown). After application of (R)- and (S)-N-AcPCBC-SO (20 µmol/kg), respectively, to male rats, necroses in proximal tubules were detected showing no significant differences in the extent and localization of the necroses between both isomers.

Discussion Previous studies revealed a discrepancy in the formation and/or in the handling of N-AcPCBC-SO in rats: In incubations of N-AcPCBC with liver microsomes two diastereomers, (R)- and (S)-N-AcPCBC-SO, were formed in a 1:1 molar ratio by rat P450 3A 1/2 and human P450 3A 4/5, but after administration of HCBD to male rats only one diastereomer was found to be excreted with urine (8, 9, 11). To elucidate the molecular mechanism for this discrepancy, a 1:1 mixture of (R)- and (S)-NAcPCBC-SO was given to male and female Wistar rats. The (R)-isomer was excreted with urine in a nearly 10fold excess over the (S)-isomer, confirming previous observations about a stereoselective processing of the diastereomers. A stereoselective reduction of the sulfoxide diastereomers by thioredoxin reductase could be excluded by incubating (R,S)-N-AcPCBC-SO with liver cytosol. After isolation by HPLC, (R)- and (S)-N-AcPCBCSO were then given to male rats. In the urine of rats treated with the (S)-isomer a new metabolite was identified as (S)-N-acetyl-S-(2-glycinylcystein-S-yl-1,3,4,4-tetrachlorobutadienyl)-L-cysteine sulfoxide, indicating that the (S)-isomer seems to be conjugated with GSH by glutathione S-transferases (Scheme 2). The formed (S)N-acetyl-S-(2-glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl)-L-cysteine sulfoxide was then cleaved under the loss

756 Chem. Res. Toxicol., Vol. 11, No. 7, 1998 Scheme 2. Formation of (S)-N-acetyl-S-(2glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl)L-cysteine Sulfoxide in Incubations of (S)-N-AcPCBC-SO in Rat Kidneya

a Glutathione conjugation in the kidney was not observed with the (R)-isomer; therefore, this isomer is excreted with urine.

of glutamic acid. In incubations of the two isolated isomers with rat kidney cytosol, only (S)-N-AcPCBC-SO was conjugated with GSH to give (S)-N-acetyl-S-(2glutathion-S-yl-1,3,4,4-tetrachlorobutadienyl)- L -cysteine sulfoxide (18); no reaction of the (R)-isomer with GSH could be observed in renal cytosol. The predominant excretion of the (R)-diastereomer of N-AcPCBC-SO is therefore likely based on a selective conjugation of the (S)-diastereomer in rat kidney resulting in the excretion of a conjugate. The (R)-diastereomer is not subject to this conjugation reaction and thus excreted in much higher concentrations after application of (R,S)-N-AcPCBC-SO (Scheme 2). Differences in the concentration of NAcPCBC-SO, as substrate for the glutathione S-transferases, in the kidney may be responsible for the lack of (S)-N-AcPCBC-SO in the urine after giving HCBD to rats. The nephrotoxicity of HCBD seems to be more pronounced in male than female rats as described in a previous study (9). Differences in the extent of the formation of reactive intermediates by action of β-lyase are unlikely; no significant sex-specific differences in β-lyase activity have been observed (Birner et al., unpublished data). Sex-specific differences, however, were observed in the metabolism of HCBD; N-AcPCBC-SO formed exclusively in male rats showed nephrotoxicity

Birner et al.

in isolated proximal renal tubule cells without involvement of β-lyase. For S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, a structurally related S-conjugate, also a β-lyaseindependent renal toxicity has been demonstrated (10). To check the contribution of N-AcPCBC-SO to the nephrotoxicity of HCBD, (R,S)-N-AcPCBC-SO was given to male and female Wistar rats. To exclude a role of β-lyase activation, R-Me-N-AcPCBC-SO was applied under identical conditions; R-Me-N-AcPCBC-SO may not be cleaved to reactive intermediates by β-lyases because of the methyl group in the R-position (12). No significant sexspecific differences and also no significant differences in the elimination of both compounds, N-AcPCBC-SO and R-Me-N-AcPCBSO, were observed. Both sulfoxides were excreted with urine up to about 15% of the dose applied. In contrast to the renal excretion of sulfoxidessshowing no sex-specific differencessnephrotoxicity was more pronounced in male rats. A dose-dependent increase in renal toxicity was found in male rats as shown by urine parameters indicative for renal toxicity and was in agreement with the results obtained by histopathology. A more pronounced nephrotoxicity in male rats as compared to female rats caused by structurally related cysteine S-conjugates of trichloro- and perchloroethene and hexachlorobutadiene was described recently (26). Uptake and transport mechanisms may also seem to play a role in the renal toxicity of halovinyl sulfoxides. An active renal secretory mechanism for lipophilic organic anions, which is present in female rats (27), may also contribute to sex differences in halovinyl sulfoxideinduced nephrotoxicity in the rat. In summary, the results clearly indicate a contribution of N-AcPCBC-SO to the HCBD-mediated nephrotoxicity in rats. N-AcPCBC-SO and R-Me-N-AcPCBC-SO are nephrotoxic in males suggesting a β-lyase-independent mechanism of nephrotoxicity for these compounds in the rats and a contribution of the sulfoxide to the renal toxicity of HCBD. Both sulfoxides have a chlorinated vinylic substituent on the sulfur and are able to directly react as Michael acceptor substrates with cellular macromolecules to cause renal damage without further bioactivation by β-lyase (28, 29). The formation of a halovinyl sulfoxide thus represents an additional β-lyaseindependent metabolic activation reaction in HCBDmediated toxicity and may explain the increased nephrotoxic potency observed in male rats.

Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 172), Bonn, and the Hauptverband der Berufsgenossenschaften, St. Augustin. The authors gratefully acknowledge the excellent technical assistance of Mrs. H. Keim-Heusler and Ms. N. Wolf.

References (1) Lock, E. A., and Ishmael, J. (1979) The acute toxic effects of hexachloro-1:3-butadiene on the rat kidney. Arch. Toxicol. 43, 4757. (2) Berndt, W. O., and Mehendale, H. M. (1979) Effects of hexachlorobutadiene (HCBD) on renal function and renal organic ion transport in the rat. Toxicology 14, 55-65. (3) Hook, J. B., Rose, M. S., and Lock, E. A. (1982) The nephrotoxicity of hexachloro-1:3-butadiene in the rat: studies of organic anion and cation transport in renal slices and the effect of monooxygenase inducers. Toxicol. Appl. Pharmacol. 65, 373-82. (4) Hook, J. B., Ishmael, J., and Lock, E. A. (1983) Nephrotoxicity of Hexachloro-1:3-butadiene in the rat: the effect of age, sex, and strain. Toxicol. Appl. Pharmacol. 67, 122-31.

Biotransformation of Halovinyl Sulfoxides (5) Dekant, W., Vamvakas, S., and Anders, M. W. (1990) Bioactivation of hexachlorobutadiene by glutathione conjugation. Food Chem. Toxicol. 28, 285-93. (6) Nash, J. A., King, L. J., Lock, E. A., and Green, T. (1984) The metabolism and disposition of hexachloro-1:3-butadiene in the rat and its relevance to nephrotoxicity. Toxicol. Appl. Pharmacol. 73, 124-37. (7) Wolf, C. R., Berry, P. N., Nash, J. A., Green, T., and Lock, E. A. (1984) Role of microsomal and cytosolic glutathione S-transferases in the conjugation of hexachloro-1:3-butadiene and its possible relevance to toxicity. J. Pharmacol. Exp. Ther. 228, 202-8. (8) Birner, G., Werner, M., Ott, M. M., and Dekant, W. (1995) Sex differences in hexachlorobutadiene biotransformation and nephrotoxicity. Toxicol. Appl. Pharmacol. 132, 203-12. (9) Werner, M., Birner, G., and Dekant, W. (1995) The role of cytochrome P4503A1/2 in the sex-specific sulfoxidation of the hexachlorobutadiene metabolite, N-acetyl-S-(pentachlorobutadienyl)-L-cysteine in rats. Drug Metab. Dispos. 23, 861-8. (10) Lash, L. H., Sausen, P. J., Duescher, R. J., Cooley, A. J., and Elfarra, A. A. (1994) Roles of cysteine conjugate beta-lyase and S-oxidase in nephrotoxicity: studies with S-(1,2-dichlorovinyl)L-cysteine and S-(1,2-dichlorovinyl)-L-cysteine sulfoxide. J. Pharmacol. Exp. Ther. 269, 374-83. (11) Werner, M., Guo, Z. Y., Birner, G., Dekant, W., and Guengerich, F. P. (1995) The sulfoxidation of the hexachlorobutadiene metabolite N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine is catalyzed by human cytochrome P450 3A enzymes. Chem. Res. Toxicol. 8, 917-23. (12) Elfarra, A. A., and Anders, M. W. (1984) Renal processing of glutathione conjugates. Role in nephrotoxicity. Biochem. Pharmacol. 33, 3729-32. (13) Elfarra, A. A., Lash, L. H., and Anders, M. W. (1986) Metabolic activation and detoxication of nephrotoxic cysteine and homocysteine S-conjugates. Proc. Natl. Acad. Sci. 83, 2667-71. (14) Vamvakas, S., Elfarra, A. A., Dekant, W., Henschler, D., and Anders, M. W. (1988) Mutagenicity of amino acid and glutathione S-conjugates in the Ames test. Mutat. Res. 206, 83-90. (15) Reichert, D., and Schu¨tz, S. (1986) Mercapturic acid formation is an activation and intermediary step in the metabolism of hexachlorobutadiene. Biochem. Pharmacol. 35, 1271-5. (16) Stoll, A., and Seebeck, E. (1948) U ¨ ber Alliin, die genuine Muttersubstanz des Knoblaucho¨ls. (Alliin, a component in garlic oil.) Helv. Chim. Acta 31, 189-210.

Chem. Res. Toxicol., Vol. 11, No. 7, 1998 757 (17) Colonna, S., Gaggero, N., Manfredi, A., Casella, L., Gullotti, M., Carrea, G., and Pasta, P. (1990) Enantioselective oxidations of sulfides catalyzed by chloroperoxidase. Biochemistry 29, 104658. (18) Rosner, E., Mu¨ller, M., and Dekant, W. (1998) Stereo- and regioselective conjugation of S-halovinylmercapturic acid sulfoxide by glutathione S-transferases. Chem. Res. Toxicol. 11, 12-8. (19) Arnstein, H. R. V. (1958) Synthesis of R-methyl- and β-methylDL-cystine. Biochemistry 68, 333-8. (20) Cash, W. D. (1962) Synthesis of two optically active N-acetyl dipeptides by the p-nitrophenyl ester method. J. Org. Chem. 27, 3329-31. (21) Moore, R. B., and Green, T. (1988) The synthesis of nephrotoxic conjugates of glutathione and cysteine. Toxicol. Environ. Chem. 17, 153-62. (22) Mahin, D. T., and Lofberg, R. T. (1966) A simplified method of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or tissue by liquid scintillation counting. Anal. Biochem. 16, 500-9. (23) Dohn, D. R., and Anders, M. W. (1982) Assay of cysteine conjugate β-lyase activity with S-(2-benzothiazolyl)cysteine as the substrate. Anal. Biochem. 120, 379-86. (24) Anders, M. W., Ratnayake, J. H., Hanna, P. E., and Fuchs, J. A. (1980) Involvement of thioredoxin in sulfoxide reduction by mammalian tissues. Biochem. Biophys. Res. Commun. 97, 84651. (25) Anders, M. W., Ratnayake, J. H., Hanna, P. E., and Fuchs, J. A. (1981) Thioredoxin-dependent sulfoxide reduction by rat renal cytosol. Drug Metab. Dispos. 9, 307-10. (26) Birner, G., Bernauer, U., Werner, M., and Dekant, W. (1997) Biotransformation, excretion and nephrotoxicity of haloalkenederived cysteine S-conjugates. Arch. Toxicol. 72, 1-8. (27) Smith, A. G., and Francis, J. E. (1983) Evidence for the active renal secretion of S-pentachlorophenyl-N-acetyl-L-cysteine by female rats. Biochem. Pharmacol. 32, 3797-801. (28) Kahn, S. D., and Hehre, W. J. (1986) Modeling chemical reactivity. 3. Stereochemistry of Michael additions of vinyl sulfoxides. J. Am. Chem. Soc. 108, 7399-400. (29) Sausen, P. J., and Elfarra, A. A. (1991) Reactivity of cysteine S-conjugate sulfoxides: formation of S-[1-chloro-2-(S-glutathionyl)vinyl]-L-cysteine sulfoxide by the reaction of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide with glutathione. Chem. Res. Toxicol. 4, 655-60.

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