Chem. Res. Toxicol. 1992,5, 193-201 Chem. SOC., Perkin Trans. 1 (lo), 1204-1211. (12) Bergman, J., Hogberg, S., and Lindstrom, J.-0. (1970) Macrocyclic condensation products of indole and simple aldehydes. Tetrahedron 26, 3347-3352. (13) Amat-Guerri, F., Martinez-Utrilla, R., and Pascual, C. (1984) Condensation of 3-hydroxymethylindoles with 3-substituted indoles. Formation of 2,3'-methylenediindole derivatives. J. Chem. Res., Miniprint, 1578-1586. (14) Bergman, J. (1970) Condensation of indole and formaldehyde in the presence of air and sensitizers. Tetrahedron 26,3353-3355. (15) Amat-Guerri, F., Lbpez-Gonzilez, M. M. C., and MartinezUtrilla, R. (1983) Dye-sensitized photooxidation of l-methylindolyl-3-acetic acid. Tetrahedron Lett. 24, 3749-3752. (16) Rannug, A., Rannug, U., Rosenkranz, H. S., Winqvist, L., Westerholm, R., Agurell, E., and Grafstrom, A.-K. (1987) Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal
193
substances. J. B i d . Chem. 262, 15422-15427. (17) Braude, E. A., Jackman, L. M., and Linstead, R. P. (1954) Hydrogen transfer. Part 11. The dehydrogenation of 1,4-dihydronaphthalene by quinones. Kinetics and mechanism. J. Chem. Soc., 3548-3556. (18) BeMiller, J. N., and Colilla, W. (1972) Mechanism of corn indole-3-acetic acid oxidase in vitro. Phytochemistry 11, 3393-3402. (19) Suzuki, Y., and Kawarada, A. (1978) Products of peroxidase catalyzed oxidation of indolyl-3-acetic acid. Agric. Biol. Chem. 42, 1315-1321. (20) Casnati, G., Dossena, A., and Pochini, A. (1972) Electrophilic substitution in indoles: Direct attack at the 2-position of 3-alkylindoles. Tetrahedron Lett. 52, 5277-5280. (21) Biswas, K. M., and Jackson, A. H. (1969) Electrophilic substitution in indoles-V. Indolenines as intermediates in the benzylation of 3-substituted indoles. Tetrahedron 25, 227-241.
Flavin-Containing Monooxygenase-Dependent Stereoselective S-Oxygenation and Cytotoxicity of Cysteine S-Conjugates and Mercapturates Sang Bum Park,?John D. Osterloh,l Spyridon Vamvakas,§ Mazzaz Hashmi," M. W. Anders," and John R. Cashman**+ Department of Pharmaceutical Chemistry and Liver Center and Department of Laboratory Medicine, University of California, Sun Francisco, California 94143, Institute fur Toxicologie, Universitiit Wurzburg, Versbacher Strasse 9, Wurzburg, Federal Republic of Germany, and Department of Pharmacology, University of Rochester, Rochester, New York 14642 Received October 21, 1991
The metabolism of cysteine S-conjugates of both cis- and trans-1,3-dichloropropenein the presence of rat kidney microsomes and purified flavin-containing monooxygenase from hog liver was investigated in vitro. Preliminary studies with isolated rat kidney cells demonstrated that cysteine S-conjugates were quite toxic to the cells in a process which was consistent with a role of the flavin-containing monooxygenase in the bioactivation of the nephrotoxins. Putative S-oxide metabolites of cysteine S-conjugates were chemically synthesized, and diastereomers were separated and identified by spedrmopic means. The metabolic products of cysteine S-conjugates were identified by comparing the chemical properties of the metabolites with authentic synthetic cysteine S-conjugate S-oxides. Surprisingly, S-conjugate S-oxygenase activity was not observed with rat kidney microsomes but was present when cysteine S-conjugates were incubated with the highly purified flavin-containing monooxygenase from hog liver. The kinetic parameters indicated that considerable S-oxygenase stereoselectivity and structural selectivity was observed cis cysteine S-conjugates were preferred substrates and N-acetylation of cysteine S-conjugates decreased substrate activity. S-Oxygenation was considerably diastereoselective and diastereoselectivity was much greater for cysteine S-conjugates with higher V,, values. Cysteine S-conjugate S-oxides were not indefinitely stable, and under certain conditions, the S-oxides underwent a [2,3]-sigmatropic rearrangement to acrolein. Formation of acrolein or other electrophilic products from S-(chloropropeny1)cysteineconjugate S-oxides may contribute to the renal effects observed for S-(chloropropeny1)cysteine conjugates. Thus, cytotoxicity studies with isolated rat proximal tubular cells or LLC-PK1 cells treated with cysteine S-conjugates showed a time- and dose-dependent decrease in cell viability. Reduction of renal cytotoxicity of cysteine S-conjugates in the presence of methimazole, a n alternate substrate competitive inhibitor of the flavin-containing monooxygenase, suggested that this enzyme may contribute to the renal effects of 1,3-dichloropropene.
I ntroductlon Although glutathione S-conjugate formation is an important detoxication process, reports of haloalkane and
* To whom correspondence should be addressed.
t Department of Pharmaceutical Chemistry and Liver Center, University of California. Department of Laboratory Medicine, University of California. 8 Universitat Wiirzburg. 11 University of Rochester.
*
haloalkene bioactivation via glutathione S-conjugate formation have appeared (for reviews, see refs 1and 2). The glutathione-dependent bioactivation of vicinal dihaloalkanes involves the intermediate formation of half-sulfur mustards that give rise to reactive episulfonium ions, which are mutagenic and nephrotoxic (3). A range of nephrotoxic haloalkenes are bioactivated via glutathione S-conjugate formation, metabolism of the glutathione S-conjugates to cysteine S-conjugates, translocation to the kidney, and renal bioactivation by cysteine conjugate @-lyase(4).
QS93-228~/92/2705-Ql93$03.QQ/Q 0 1992 American Chemical Society
194 Chem. Res. Toxicol., Vol. 5,No.2, 1992
Park et al.
Chart I. Summary of the Chemical Structures of Cysteine S-Conjugates 1-3, Mercapturate S-Conjugates 4-6, and Their Corresponding S-Oxides, 7-9 and 10-12, Respectively
R'
H
R2
S A C O O H NHR3
1, cis-CPC: R1= H, R2= Cl, R3= H 2 , tram-CPC: R'= C1, R2= H, R3= H 4, cis-CPNAc: R1= H, R2= C1, R3= COCH3
5 , trans-CPNAc: R'= C1, R2= H, R3= COCH3
&
H S
7, cis-CPC SO: R'= H, R2= C1. R3= H 8, trams-CPC SO: R1= C1, R2= H, R3= H 10, cis-CPNAc SO: R1= H, R2=Cl,R3= COCH3 11, trans-CPNAc SO: R1= C1, R2= H, R3= COCH3 0
COOH NHR3
3, SBC: R3= H 6 , SBCNAC: R3= COCH3
Q-/ +
I A C O O H NHR3
9, SBC SO: R3= H 12, SBCNAc SO: R3= COCH3
Although mercapturate S-oxides are known as metabolites of xenobiotics (5, 6 ) and cysteine S-conjugates are extensively S-oxidized in vivo (7-ll), S-oxygenation-dependent bioactivation has apparently not been described. Although the molecular basis for the formation of cysteine S-conjugates and mercapturate S-oxides has yet to be completely defined, recent reports indicate that cysteine S-conjugates may undergo S-oxygenation by a flavin-containing monooxygenase present in rat liver and kidney microsomes. The metabolism of trichloroethylene along this putative pathway results in S-(1,2-dichlorovinyl)-~cysteine sulfoxide, which has been shown to be nephrotoxic (12, 13). cis- and trans-1,3-dichloropropene(DCP)' isomers are major components of a soil fumigant widely used in the fields of California and elsewhere for the control of nematodes. DCP has a relatively low toxicity in mammals (e.g., rat oral LDm = 250-500 mg/kg) (14),but has shown mild effects on the kidney in animals and man (14-17).DCP is metabolized to the glutathione S-conjugate, which is eliminated in the bile, or to the mercapturate S43chloro-2-propenyl)-N-acetyl-~-cysteine, which is eliminated in the urine in man and animals (15, 18, 19). In this study, we investigated the metabolic basis for the S-oxygenationof S-(cis-3-chloro-2-propenyl)-~-cysteine and S-(trans-3-chloro-2-propenyl)cysteineand their corresponding mercapturates. In addition, we studied the Soxygenation of S-benzyl-L-cysteineand its mercapturate. The data reported herein demonstrate that the flavincontaining monooxygenase (FMO) from hog liver catalyzes the stereoselective conversion of a number of cysteine S-conjugates to their corresponding S-oxides (Chart I). That cysteine S-conjugate S-oxides of DCP were not indefinitely stable and slowly underwent an apparent [2,3]-sigmatropicrearrangement to acrolein suggests that it is possible that electrophilic rearrangement products such as acrolein or other materials may contribute, at least in part, to the nephrotoxicity observed for cysteine Sconjugates of dichloropropene. Our studies were spurred Abbreviations: FMO, flavin-containing monooxygenase;EI, electron impact; LSIMS,liquid secondary ion mase spectrometry;TLC, thin-layer chromatography;HPLC,high-pressure liquid chromatography;SDS, aodium dodecyl sulfate; HEPES, N-(2-hydrosyethyl)piperazine-N'-2ethaneeulfonicacid; DMEM, Dulbecco's modified Eagle's medium;EBSS, Eagle's balanced salt solution; DCP, 1,3-dichloropropene; CPC, (3chloro-2-propenyl)-~-cysteine; SBC, S-benzyl-L-cysteine;CPNAc, (3chloro-2-propenyl)-N-acetyl-~-cysteine; SBCNAc, N-acetyl-S-benzyl-Lcysteine.
H
by the pioneering observations of Casida and others (20), who have shown that electrophilic rearrangement products from biological and chemical oxidations could contribute to the toxicity of pesticides, herbicides, and other xenobiotics. We also studied the cytotoxicity of S-(cis-3-chloro-2propenyl)-N-acetyl-L-cysteine,S-(trans-3-chloro-2propenyl)-N-acetyl-L-cysteine,and S(cis-3-chloro-2propenyl)-L-cysteine in isolated rat renal proximal tubular cells and in the pig kidney-derived LLC-PK1 cell line. The mercapturates and cysteine S-conjugate were cytotoxic, and the cytotoxicity was blocked by the FMO inhibitor methimazole, indicating a role for FMO-dependent biotransformation in the observed cytotoxicity.
Experimental Section Chemicals. Chemicals used in this study were of the highest purity available from commercial sources. S-Benzyl-L-cysteine (SBCNAc),' N-acetyl+ (SBC),' N-acetyl-S-benzyl-L-cysteine cysteine, and L-cysteine were purchased from Sigma Chemical Co. (St. Louis, MO) and Schweizerhall, Inc. (South Plainfield, were NJ), respectively. cis- and trans-1,3-dichloropr~pene~ purchased from Columbia Organic Chemical Co., Camden, SC. S-(cis-3-Chloro-2-propenyl)-N-acetyl-~-cysteine and S-(trans-3chloro-2-propenyl)-N-acetyl-~-cysteine were prepared by the method of Climie et al. (21). The 'H NMFt spectra were consistent with reported values (22). S-(cis-3-Chlorc~2-propenyl)-~-cysteine and S-(trans-3-chloro-2-propenyl)-~-cysteine were prepared in 31% and 51% yield, respectively, by the reaction of cis- and trans-1,3-dichloropropenewith L-cysteine under the conditions described by Climie et al. (21). For S-(cis-3-chloro-2propenyl)-L-cysteine, the 'H NMR (270 MHZ, DzO)was as follows: 6 2.8 (dd, 2 H),3.2 (dd, 2 H),3.7 (t, 1 H), 5.75 (t, 1 H), and 6.1 the 'H (d, 1 H). For S-(trans-3-chloro-2-propenyl)-~-cysteine, NMR (270 MHz, DzO) was as follows: 6 2.7 (dd, 2 H), 3.0 (dd, 2 H), 3.65 (t, 1 H), 5.75 (t, 1 H), and 6.05 (d, 1 H). A previous synthesis has been described for cis- and trans-CPNAc (23). Aminobenzotriazole was a gift of Professor Paul Ortiz de Montellano, University of California, San Francisco. All of the compounds of the NADPH-generating system were obtained from Sigma Chemical Co. AU other reagents and buffers were obtained from a commercial source with the highest quality possible. Instrument Analysis. Unless otherwise indicated, 'H NMR spectra were recorded with a General Electric spectrometer operating at 300 MHz. 'H chemical shifts are expressed in ppm Caution! 1,3-Dichloropropene causes irritation to skin, eyes, and mucous membrane and h e r and kidney injury. Great care should be exercised in all experimental procedures to minimize exposure. All experiments with DCP haue to be carried out in a filter-equipped hood.
Stereoselective S-Oxygenation of Cysteine S-Conjugates
compound cis-CPC S-oxide (7)
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 195
Table I. Spectral Characteristics of S-Cysteine Conjugate S-Oxides 'H NMR LSIMS m / z (relative intensity, %) (in D20) 6 3.11 (m, 1 H, H1&,3.32 (m, 1 H, H2& 3.74-4.05 (m, 3 212 (a), 234/235 (63/26), 256/258 (100/37), 159 (loo), 131 (41) Hs, H,' and HJ, 6.10 (m, 1 H, Hb),6.69 (d, 1 H, J = 7.2 Hz, H -1
(in-D20) 6 3.10-3.38 (m, 2 Hs, Hs), 3.58-4.02 (m, 3 Hs, Ha and HJ, 6.10 (m, 1 H, Ha), 6.50 (d, 1 H, J = 14.4 Hz, H,) (in D20) 6 3.20 (m, 2 Hs, HB),3.92 (m, 1 H, Ha), 4.17 (m, 1 H, SBC S-oxide (9) H1 ), 4.37 (m, 1 H, H2,), 7.39-7.49 (m, 5 Hs, PhH) (in &,OD) 6 2.02 (8, 3 Hs, CH,CO), 3.16-3.52 (m, 2 Hds, H.&, cis-CPNAc S-oxide (10) 3.66-4.10 (m, 2 Hs, HJ, 4.93 (m, 1 H, He), 6.06 (m, 1 H, Hs), 6.56 (d, 1 H, J = 7.5 Hz, He) trans-CPNAc S-oxide (11) (in CD,OD) 6 2.03 (8, 3 Hs, CH,CO), 3.10-3.52 (m, 2 Hs, HB), 3.78 (m, 1 H, HlJ, 3.63 (m, 1 H, HZ,), 4.82 (m, 1 H, HJ, 6.10 (m, 1 H, Ha),6.56 (d, 1 H, J = 16.8 Hz, H,) (in CD,OD) 6 2.02 (m, 3 Hs, CH,CO), 3.06-3.52 (m, 2 Hs, H&, SBCNAc S-oxide (12) 4.05-4.36 (m, 2 Hs, H A 4.82 (m, 1 H, Ha), 7.41 (m, 5 He, PhH) trans-CPC S-oxide (8)
Table 11. HPLC Properties of Cysteine 5-Conjugates and Cysteine S-Conjugate S-Oxide Diastereomers retention wavelength volume, solvent monitored, compound mL system' nm A 220 22.6 cis-CPC (1) 24.7 A 220 trans-CPC (2) 12.6 B 220 SBC (3) C 220 22.7 cis-CPNAc (4) 27.7 C 220 trans-CPNAc (5) D 220 12.9 SBCNAc (6) A 220 5.4; 5.6' cis-CPC S-oxide (7) A 220 trans-CPC S-oxide (8) 5.4, 5.6 B 220 5.1, 5.4 SBC S-oxide (9) C 220 cis-CPNAc S-oxide (10) 4.7, 4.9 C 220 trans-CPNAc S-oxide (11) 4.8, 5.1 D 220 4.1, 4.3 SBCNAc S-oxide (12) 'Solvent systems: A, 5% MeOH/H,O/O.l% TFA; B, 10% CH,CN/H,O; C, 20% MeOH/H,O/O.l% TFA; D, 40% MeOH/ HzO/O.l% TFA. *Minor diasteromer. Major diastereomer. downfield from tetramethylsilane. Electron impact (EI) mass spectra were recorded with a VG70S spectrometer at 6 kV and source temperature of 60 "C. Liquid secondary ion mass spectrometry (LSIMS) was recorded with a Kratos MS50 set at 8 kV and equipped with a cesium ion gun. UV spectra were recorded with a Perkin-Elmer 559A spectrometer. Synthesis of Amino Acid S-Oxides. Hydrogen peroxide (30% technical grade, 0.07 mL, 0.6 "01) was added to a stirred solution of 1,2, or 3 (0.5 mmol) (see Chart I) in water (4.5 mL) containing sodium hydroxide (0.2 mL, 2 N) at 0 "C. The rmulting mixture was stirred for 24 h a t 25 OC. Analysis of the reaction by TLC with an eluant of CH2C12/CH30H/glacialacetic acid (69301 v/v) showed that no starting material remained. Dimethyl sulfoxide (20 mg, 0.26 mmol) was added to the crude reaction mixture to consume any excess H202,and bicarbonate was added to basicify the reaction (pH 7.5). The reaction mixture was evaporated to dryness to give the amino acid S-oxide in quantitative yield. HPLC of the product indicated that the purity was greater than 97%. The spectral properties of the corresponding mercapturate S-oxides 7, 8, and 9 are listed in Table I. Synthesis of Mercapturate S-Oxides. Hydrogen peroxide (30% technical grade, 0.07 mL, 0.6 nmol) was added to a stirred solution of 4,5, or 6 (0.5 mmol) in methanol/water (5.0 mL) (1:l v/v) at 0 "C. The resulting mixtures were stirred for 24 h at 25 "C. Analysis of the reaction by TLC with an eluant of CH2C12/CH30H/trifluoroaceticacid (94:5:1 v/v) showed that no starting material remained. Dimethyl sulfoxide (20 mg, 0.26 mmol) was added to the crude reaction mixture to consume any excess H202. The reaction mixture was evaporated to dryness to give the mercapturate S-oxide in quantitative yield. HPLC of the product indicated that the purity was greater than 95% (Table 11). The spectral properties of the corresponding mercapturate S-oxides 10, 11, and 12 are listed in Table I. Liver Preparations. Microsome fractions were isolated according to the method described previously from the livers of hogs
212 (9), 234/236 (34/16), 256/258 (54/20),
159 (67), 131 (100) 228 (37), 250 (loo), 272 (87), 91 (92) 254/256 (13/5), 276/278 (62/22), 298/300 (74/27), 130 (15), 115 (100) 254/256 (100/40), 276/278 (17/6), 238/240 (5/2), 162 (27), 130 (74) 270 (26), 292 (25), 254 (4), 130 (21), 115 (29), 91 (100)
Table 111. S-Oxygenation of cis-CPC, trans-CPC, and SBC by Highly Purified Hog Liver Flavin-Containing Monooxygenase S-oxide formation, nmol/ (minamg substrate descriDtion of motein) cis-CPC (1) completea** 93.9 f 16.5 -NADPH ND' ND -protein +aminobenzotriazole (1.0 mM) 97.1 f 9.4 +n-octylamine (5.0 mM) 80.2 10.3 ND +heat inactivation trans-CPC (2) completea 62.9 f 11.7 -NADPH ND -protein ND SBC (3) complete" 79.5 f 7.8 -NADPH ND -protein ND ~~
*
'The complete system contained 50 mM phosphate buffer (pH 8.4), the NADPH-generating system, 125 nmol of substrate, and 40 pg of protein incubated for 10 min at 37 "C. Data represent the mean of 4 determinations ASD. *Under similar conditions as above [i.e., 50 mM phosphate buffer (pH 8.41, the NADPH-generating system, 150 nmol of substrate and 44 fig of protein] the highly purified FMO from hog liver catalyzed the N-oxygenation of dimethylaniline with a rate of 270 nmol/(min.mg of protein) as determined by HPLC (see Experimental Section). cND, not detectable. or from the kidneys of male rats (24). The flavin-containing monooxygenase (FMO) was isolated and purified from hog liver microsomes by a modification of a procedure previously described (25). To minimize inactivation of FMO activity, all steps were carried out as quickly as possible at 4 "C. The hog liver FMO exhibited characteristically high S-oxygenase activities and was judged to be homogeneous by SDS-polyacrylamide gel electrophoresis. Dimethylaniline N-oxidase activity was determined by incubating 44 pg of highly purified hog liver FMO with 0.5 mM dimethylaniline a t pH 8.4 (as described below). The products of the reaction were quantified by HPLC (26) after extraction with dichloromethane. The results are listed in Table 111. Rat kidney microsomes possessed significant FMO activity [i.e., Long-Evans and Sprague-Dawley rat kidney microsomes formed 1.2 and 0.9 nmol of dimethylaniline N-oxide/(minmg of protein), respectively] as determined by HPLC (26).The cytochrome P-450 content of the rat kidney microsome preparations that was determined spectrophotometrically (27) was 0.1 nmol of P-450/mg of protein, and detectable amounts [i.e., 0.1-0.3 nmol/(min.mg of protein)] of dimethylaniline N-demethylase activity were observed (26). Enzyme Assays. The incubation medium for assays with highly purified FMO and microsomes from hog liver contained 0.05 M potassium phosphate (pH 8.4),0.4 mM NADP+, 0.4 mM glucose 6-phosphate, and 1 IU of glucose-6-phosphate dehydrogenase. In incubations with rat kidney microsomes, the
196 Chem. Res. Toxicol., Vol. 5, No. 2, 1992
Park et al.
CPC S-oxides
cofactor generating system was the same as above but the pH was 7.2. In incubations with purified FMO, 25-40 pg of protein was used, and for incubations with microsomes, 1-2 mg of protein was used, in a total volume of 0.25 mL. The reaction was initiated by the addition of substrate and incubated at 31 OC with constant shaking. At various time intervals, the reaction was stopped by the addition of 0.7 mL of cold CH2C12/2-propanol(1:2 v/v). After saturation of the aqueous phase with approximately 2 mg of anhydrous sodium carbonate, the mixture was vigorously mixed and the organic layer was separated from the aqueous layer by a brief centrifugation. After filtration through a 0.45-pm nylon filter and evaporation, the extract was taken up in methanol. The profile of metabolites after incubation of 1-6 was determined by HPLC. The metabolic products from the extract were eluted from a Dynamax Microsorb C-18 (7.7 mm X 25 cm) analytical reverse-phase column and quantified by an IBM Model 9533 HPLC interfaced to a HP Model 3396 integrator with a W detector set at 220 nm. The mobile phase consisted of an isocratic system of methanol/water/trfluoroacetic acid as shown in Table 11. This system efficiently separated amino acids or mercapturates from their corresponding S-oxides (Figure 1). Metabolites were quantified by comparing the metabolite and substrate peak areas of the chromatogram. The recovery of material as judged by HPLC was more than 65%, and greater than 95% of the material observed was either the substrate or the corresponding S-oxide. Other Analytical Methods. Heat inactivation of micrcrsomes was accomplished as previously described (28). The concentration of protein used was determined by the Pierce BCA method (Rockford, IL). Cytotoxicity Studies. Rat renal proximal tubular cells were isolated from male Fischer 344 rats (200-250 g; Charles River Laboratories, W-n, MA) by the method of Jones et al. (29). Cell concentrations and viability were determined in a hemocytometer in the presence of 0.2% trypan blue. Cell viability was expressed as the percent of cells excluding trypan blue. When methimazole was used as an alternate substrate competitive inhibitor of FMO, the cells were incubated with methimazole for 15 min before addition of CPNAc. LLC-PK1 cells (passage 192-212; American Type Culture Collection) were grown in DMEM supplemented with 20 mM HEPES, 10% fetal bovine serum albumin, penicilin (100 units/mL), streptomycin (10 pg/mL), glucose (3 g/L), sodium bicarbonate (1.7g/L), and 2 mM glutamine. Cells (1.2 X lo5) were plated in 35-mm wells (6 wells per plate); 1 day later, monolayers in exponential growth were washed twice with EBSS and were exposed in triplicate to the additions indicated in the figures and tables. In some experiments, the monolayers were exposed to methimazole (0.5 mM) or (aminooxy)acetic acid (0.5 mM) for 1 h before addition of the Sconjugates, the inhibitors were present throughout the experiment. Cell viability was determined by measuring release of lactate dehydrogenase activity into the medium (29).
Results Chemistry. The chemical oxidation of (cis-or (transchloropropeny1)cysteineor S-benzylcysteine (i.e., cis-CPC, trans-CPC, or SBC, respectively), or their N-acetyl derivatives, to the corresponding S-oxide was readily accomplished with sodium metaperiodate (data not shown). To mimic more closely the biological catalysts involved in hepatic S-oxygenation, we also synthesized the S-oxides in essentially quantitative yield with hydrogen peroxide and completely characterized the products spectrally. Interestingly, attempts to stereoselectively synthesize diastereometrically enriched S-oxides of 3 and 6 with the modified Sharpless oxidation procedure (30) or by oxidation with sodium metaperiodate in the presence of bovine serum albumin (31) resulted only in formation of racemic S-oxides. Apparently, the relative conformationalmobility of 3 and 6 did not allow diastereoselective oxidation of the sulfide sulfur atom. The S-oxides (7-12) were relatively stable in protic solvents and in water. Thus, 7-12 could be readily extracted from biological matrices and analyzed by HPLC.
A
CPC
i U Figure 1. Typical chromatograms of authentic cysteine S-con-
jugates and their diaatereometric S-oxides (A) and substrate and its metabolic products (B).
However, allylic S-oxides such as 7,8, or 10 and 11, were not indefinitely stable and underwent apparent [2,3]-sigmatropic rearrangement. This rearrangement was markedly accelerated by the presence of thiophiles such as phosphines and phosphites. Thus, in the presence of trimethyl phosphite, 10 was completely consumed, with an approximate half-life of 5-6 h as evidenced by an increase in the aldehyde proton resonance observed at 9.78 ppm or the proton resonances in the vinyl region as monitored by lH NMR a t 70 O C . In the presence of trimethyl phosphite, changes in the vinyl proton resonances of 7 were obtained by NMR at 80 "C but no aldehyde peak was observed. The results indicated that rearrangement of 7 was considerably slower than rearrangement of 10 (data not shown). We tentatively concluded that [2,3]sigmatropic rearrangement produced acrolein which also
Stereoselective S-Oxygenation of Cysteine S-Conjugates
1
Cysteine S-conjugate P-lyase
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 197 Table IV. Stereoselective S-Oxygenation of S-Cysteine Conjugates by Hog Liver Flavin-Containing Monooxygenaeeo minor S-oxide, major S-oxide diastereonmol/(min.mg nmol/(min.mg meric excess? % substrate of motein). (%) of orotein). (%) . . . . cis-CPC (1) 10.6 h 6.8 (11.2) 85.4 h 17.9 (88.8) 77.9 tram-CPC (2) 7.7 h 3.3 (12.3) 55.3 9.1 (87.6) 75.6 78.1 SBC (3) 8.3 h 1.9 (11.1) 67.7 4.9 (88.9) cis-CPNAC (4) 12.2 h 3.3 (31.2) 26.8 h 0.7 (68.8) 37.6 tram-CPNAc (5) 18.9 f 8.1 (44.9) 23.3 10.0 (55.1) 10.4 SBCNAc (6) 14.8 0.4 (39.9) 22.3 h 0.1 (60.1) 20.3
*
*
a Purified hog liver FMO (40pg) was incubated with substrate (500 pM) in the presence of an NADPH-generating system in potassium
phosphate buffer (pH 8.4)at 37 O C for 10 min. Each value is the mean of 4 determinations standard deviation. * S-Oxide product diastereoselective excess: [ % major S-oxide - % minor S-oxide/(%major S-oxide + 70 minor S-oxide)].
*
0
" R2 I%
SH +
H3C
A COOH
+
NH3
Figure 2. Possible metabolic transformation of cis- or trans-CPC by the cysteine S-conjugate 8-lyase pathway. cis-CPC (1): R' = H, R2 = C1. trans-CPC (2): R1= C1, R2= H. decomposed or polymerized during the 'H NMR experiment. Metabolic Studies: Kidney and Liver Preparations. Cysteine conjugates of cis- and trans-l,2-dichloropropene formed in vivo are known to be nephrotoxic in various mammalian species (1-4). Because cis- and trans-l,&dichloropropenes were reported to be extensively metabolized to 1 and 2, respectively, and possibly further metabolized (Figure 2), and because renal effects have been reported after animal and human exposure (14-19, we examined the S-oxygenation of 1 and 2 with different rat kidney microsome preparations. We were unable to detect formation of S-oxides 7 and 8, 10, or 11, and in contrast to a previous report (12), we were also unable to detect formation of 9 or 12 from the corresponding sulfides in rat kidney microsomes. Thus, kidney microsomes prepared from Long-Evans or Sprague-Dawley rata pretreated with phenobarbital or arochlor, or untreated, were all equally inactive toward NADPH-dependent S-oxygenation of 1-6. The rat kidney microsomes that we used possessed good cytochrome P-450 and FMO activity (i.e., see Experimental Section). Therefore, the lack of amino acid or mercapturate S-oxidase activity was not due to the absence of kidney monooxygenase activity. The only product detected in extracts of reactions catalyzed by microsomes from rat kidney did not elute with retention volume similar with that of authentic S-oxide upon separation by HPLC, but no attempt was made to characterize the unknown metabolite. As a model monooxygenase system, we examined the S-oxygenation of 1-6 by the highly purified FMO from hog liver. The S-oxygenation of 1-6 was linearly dependent on time (0-15 min) and protein concentration (0-50pg of highly purified hog liver FMO). As shown in Table 111, S-oxygenation of 1-3 was completely dependent on the presence of NADPH and active FMO. The S-oxygenation of 1 was examined in more detail. Heat inactivation of hog liver FMO under conditions which inactivated essentially 100% of FMO activity (28, 32) completely abolished Soxygenation of 1. Aminobenzotriazole,a mechanism-based inactivator of cytochrome P-450 (33), slightly stimulated (i.e., 4%) S-oxygenation, and n-octylamine, a positive effector for hepatic FMO (32), slightly inhibited (Le., 14%) S-oxygenase activity. The effect of inhibitors and cofactors on S-oxygenase activity was nevertheless consistent with a role of FMO and does not indicate any contribution from
Table V. Kinetic Constants for S-Oxygenation of Cysteine 5-Conjugates and Other Derivatives Catalyzed by Purified
hog Liver Flavin-Containing Monooxygenase substrate cis-CPC (1) trans-CPC (2) SBC (3) cis-CPNAC (4) trans-CPNAc (5) SBCNAc (6)
K,." uM 1111 344 769 1538 1250 1429
vm,,
nmol/(min.me of orotein) 555 263 384 153 133 143
"Kinetic constants were calculated from initial velocity measurements by the methods described in Experimental Section at pH 8.4,37 OC, with variable substrate concentrations. nonenzymatic S-oxygenation. To examine this aspect more carefully, the stereoselectivity of S-oxygenation by hog liver FMO was examined. Of the possible diastereoisomeric S-oxide products that could form from cysteine S-conjugates, one S-oxide diastereoisomer was formed to a much greater extent than another (Table IV). Thus, the mean diastereoselective excess for formation of 7-9 was calculated to be 77.2%. In contrast, much less diastereoselectivity was observed for S-oxygenation of 4-6. Thus, the mean diastereoselective excess for formation of 10-12 was calculated to be 22.8%. Kinetic constanta for the S-oxygenation of 1-6 catalyzed by the highly purified FMO from hog liver microsomes were caleulated from the rate of S-oxide formation at variable substrate concentrations by the HPLC procedure described in Experimental Section. As shown by the kinetic constants listed in Table V, compounds 1-6 were substrates for the highly purified FMO from hog liver. The concentration required for half-maximal activity was, however, considerably greater than that of other sulfides for this enzyme (32). The data were consistent with previous studies (32, 34) which indicated that biologically essential or physiologically-important sulfur-containing compounds (i.e., glutathione, cysteine, etc.) would not be preferential substrates for the FMO. However, in contrast to physiologically important sulfur-containing compounds, S-alkylcysteine conjugates are substrates for FMO (Figure 3). The only products detected in dichloromethane/2propanol extracts of reactions catalyzed by the purified hepatic FMO were those eluting with retention volume identical with that of authentic S-oxide diastereomers upon separation by HPLC. Cytotoxicity Studies. Incubation of S-(cis-3-chloro2-propenyl)-N-acetyl-~-cysteine with isolated rat renal proximal tubular cells resulted in time- and concentration-dependent cytotoxicity, as measured by trypan blue exclusion (Figure 4). In addition to S-(cis-3-chloro-2propenyl)-N-acetyl-L-cysteine,S-(trans-3-chloro-2-
198 Chem. Res. Toxicol., Vol. 5, No. 2, 1992
Park et al.
H ClHC
= CHCH2-
J
S
COOH
Oxidation
NH2
(A)
-
-
Y CMC
H
= CHCH2- S +
COOH NH2
c1
1
c
[2,3] -Sigmatropic rearrangement
c5l
H2C
= CHCH-
H
4COOH 3
0- S
-
0 H2C
II
= CHCH
H
+
7
HS &COOH
Reduction
NH2 (C) (D) Figure 3. S-Oxygenation of cis- or trans-CPC (A) and [2,3]-sigmatropic rearrangement to produce the sulfenate (B) and ultimately acrolein (C) and cysteine (D). NH2
(B)
80 u7 4
60 al
4
5> w
40 20
n Time (min)
Figure 4. Time- and concentration-dependent cytotoxicity of
S-(cis-3-chloro-2-propenyl)-N-acetyl-~-cysteine (4)in isolated rat renal proximal tubular cells. Cells (1 X 106/mL) were incubated with 0 ( 0 1 , O . l (A),0.5 ( O ) , 1.0 (m), or 5 (0) mM 4 for the indicated times. Cell viability was quantified by trypan blue exclusion, as indicated in Experimental Section. Results are shown as mean f SE for 4-6 experiments, except for the data for 0.1,0.5, and 5 mM CPNAc, which are the average for 2 experiments. Table VI. Cytotoxicity of S-(cis -3-Chloro-2-propenyl)-N-acetyl-~-cysteine (cis-CPNAc), S - (trams-3-Chloro-2-propenyl)-N-acetyl-~-cysteine (trans-CPNAc), and S-(cis-3-Chloro-2-propenyl)-~-~ysteine in LLC-PK1 Cells" % LDH addition release control 5.5 f 0.6 67.0 f 1.2 cis-CPNAc, 5 mM cis-CPNAc, 1 mM 60.5 f 2.5 54.0 f 4.5 cis-CPNAc, 0.5 mM 28.0 f 3.0 cis-CPNAc, 0.1 mM 6.2 f 0.5 (aminooxy)acetic acid, 0.5 mM cis-CPNAc, 1 mM, plus (aminooxy)acetic acid, 62.0 f 3.0 0.5 mM 7.9 f 0.5 methimazole, 0.5 mM cis-CPNAc, 1 mM, plus methimazole, 0.5 mM 16.2 f 1.0 62.0 f 2.0 trans-CPNAc, 1 mM trans-CPNAc, 1 mM, plus methimazole, 0.5 mM 15.5 f 0.6 61.0 f 4.0 CPC, 1 mM CPC, 1 mM, plus methimazole, 0.5 mM 14.3 f 0.9 "LLC-PK1 cells were cultured and incubated with the indicated additions, as described in the Experimental Section; the release of lactate dehydrogenase (LDH) activity into the medium was determined as described in the Experimental Section. Data are shown as means f SE, n I 3.
Figure 5. Effect of methimazole on the cytotoxicity of S-(cis3-chloro-2-propenyl)-N-acetyl-~-cysteine (4),S-(trans-3-chloro2-propenyl)-N-acetyl-~-cysteine (5), and S-(cis-3-chloro-2propenyl)+ teine (1) in isolated rat renal proximal tubular cells. Cells (1 X 10T /mL) were incubated with 1.0 mM 1,4, or 5 for 120 min in the absence and presence of 0.5 mM methimazole (cross-hatched bars). Methimazole was added 15 min before addition of S-conjugates and was present throughout the experiments. Cell viability was quantified by trypan blue exclusion, as indicated in Experimental Section. Data are presented as mean & SE for 3 experiments.
propenyl)-N-acetyl-L-cysteineand S-(cis-3-chloro-2propenyl)-L-cysteine were cytotoxic in isolated rat renal proximal tubular cells. Moreover, the cytotoxicity of these compounds was blocked by the FMO alternate substrate competitive inhibitor methimazole (Figure 5 ) (32).Similar results were obtained in the pig kidney-derived LLC-PK1 cell line: S-(cis-3-chloro-2-propenyl)-N-acetyl-~-cysteine, S-(trans-3-chloro-2-propenyl)-N-acetyl-~-cysteine, and S-(cis-3-chloro-2-propenyl)-~-cysteine were cytotoxic in LLC-PK1 cells, and their cytotoxicity was blocked by methimazole (Table VI). The cysteine conjugate P-lyase inhibitor (aminooxy)acetic acid (35)failed to block mercapturate-induced cytotoxicity.
Dlscussion Chemical Aspects. Haloalkanes such as 1,3-dichloropropene are efficiently converted to S-glutathione and S-cysteine conjugates. Once formed, the conjugates may be further biotransformed and/or excreted in the bile or excreted in urine. As shown in Figure 2, metabolism of cis- or trans-CPC by the P-lyase metabolic pathway would
Stereoselective S-Oxygenation of Cysteine S-Conjugates
produce an allylic thiol (Le., 3-chloropropenthiol) which is anticipated to be nontoxic in itself or lead to nontoxic metabolites. It is not likely that other routes of chemical transformation of 1 or 2 would lead to highly electrophilic metabolites (i.e., episulfonium ions). Renal effects of DCP have been shown in animals and man. A previous experiment showed that by pretreating animals with (aminooxy)acetic acid, a @-lyaseinhibitor, renal injury from DCP was decreased (17). However, (aminooxy)aceticacid may nonspecifically block renal uptake of mercapturates and cysteine S-conjugates, thus explaining this apparent incongruity. Additional mechanisms must be involved for the bioactivation of compounds such as 1 or 2 to reactive electrophilic metabolites capable of covalent binding, which may lead to cell necrosis and ultimately to cell death. The evidence reported herein points to a role for a new oxidative mechanism for the conversion of 1 or 2 to reactive metabolites. As a model system for kidney or hepatic monooxygenase transformations, we examined the hydrogen peroxidemediated S-oxygenation of 1-6. Synthesis of 7-12 with hydrogen peroxide was readily accomplished. Attempts to diastereoselectively synthesize 7-12 with the modified Sharpless reagent (30)produced racemic S-oxides. Further studies employing sodium metaperiodate oxidation in a chiral matrix (i.e., aqueous bovine serum albumin, 10 mg/mL) (31)also did not produce any diastereoselective S-oxygenation. The allylic sulfoxides of 1,2,4, and 5 were not indefinitely stable, however, and ‘H NMR experiments clearly showed conversion of the allylic sulfoxides to transient aldehyde-containing materials. The anticipated product, acrolein, is known to polymerize and decompose, and no attempt was made to characterize further the products arising from rearrangement and degradation of 1, 2, 4, and 5. We postulate that acrolein arises via [2,3]-sigmatropic rearrangement of the allylic sulfoxide after reduction of the sulfenate (Figure 3). Because the rearrangement requires elevated temperatures and prolonged reaction time, it is possible that formation of acrolein in vivo contributes to the observed nephrotoxicity only if the rearrangement is facilitated by enzymatic or biological assistance (36). The [2,3]-sigmatropic rearrangement of allylic sulfoxides has been previously identified as a possible contributor to the generation of electrophilic metabolites of other chemicals (37). For example, the potent bacterial mutagen 2-chloroacrolein is produced from oxidation of S-(2,3-dichloroallyl) diisopropylthiocarbamate (diallate) by biological or chemical means (20, 37). It is possible that bioactivation of allylic sulfides to allylic sulfoxides represents a novel mechanism for the formation of reactive electrophilic metabolites. Metabolic o r Enzymatic Aspects. Attempts to demonstrate the production of 7-12 in the presence of microsomes from rat kidney were completely unsuccessful. In contrast to a previous report (12),we could not show the conversion of 3 or 6 to 9 or 12 by any of the rat kidney microsome preparations used. We did, however, observe a metabolite of 3 that was formed by rat kidney microsomes. The metabolite was considerably less polar than 9, but we could not isolate sufficient material for structural characterization. Formation of the unknown nonpolar metabolite of SBC was strictly dependent upon active kidney microsomes and NADPH. Interestingly, formation of the unknown metabolite was only slightly inhibited by heat inactivation of the microsomes under conditions which preserve 80% of cytochrome P-450 activity and almost completely abolishes FMO activity (28,32) [Le., the complete system, 2.1 i 0.2 nmol of metabolite/(min-mg
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 199
of protein), was only slightly decreased, 1.6 f 0.4 nmol of metabolite/ (mimmg of protein), after heat inactivation of the microsomes]. Currently, the molecular basis for rat kidney cysteine S-conjugate S-oxygenation is unknown. Some data point to a role for the FMO (12),but the support is not definitive. It is probable that there are multiple forms of FMO in microsomes of rat kidney (38),but it is currently unclear how each enzyme form responds to heat inactivation or what is the substrate specificity for each enzyme form. As a model for rat kidney FMO form I, we examined the S-oxygenation of cysteine S-conjugates by microsomes and FMO from hog liver. Studies of compounds 1-6 showed that hog liver microsomes supplemented with NADPH catalyzed the formation of 7-12, respectively. Formation of the sulfoxides appeared to be dependent upon the presence of microsomal FMO. However, an impurity that coeluted by HPLC near the S-oxide metabolites precluded a definitive statement on this point. Attempts to avoid extraction of this coeluting material by derivatizing the metabolites as their methyl esters or other derivatives and extracting the incubations with a less polar solvent system (i.e., dichloromethane) were not successful. Because microsome preparations gave a coeluting impurity by HPLC, studies with highly purified FMO from hog liver were initiated. Studies showed that highly purified hog liver FMO catalyzed the S-oxygenation of 1-6. The kinetic constants (Table V) showed that S-cysteine and S-mercapturate conjugates were substrates for the purified FMO. The concentration of the amino acids (1-3) required to halfsaturate the enzyme was in the 300-1000 pM range, and the V,, at infinite amino acid concentration varied from 300 to 550 nmol/(min-mg of protein). This is the first report describing the substrate activity of an amino acid for the purified hog liver FMO. Previous reports (34,39) of S-alkyl amino acids (i.e., methionine) or amino acids (i.e., cysteine, glutathione) (34) or peptides (i.e., vasopressin, coenzyme A, pantetheine) showed that all of the above materials were not substrates for the purified hog liver FMO. On the other hand, lipoic acid and other various thiolbenzoates and monothiolalkyl carboxylic acids were substrates for purified hog liver FMO (40). Hepatic FMO apparently discriminates between essential and foreign compounds by “excluding the former rather than selectively binding the latter” (41). The molecular basis for excluding essential nitrogen- and sulfur-containing compounds is currently not known because the three-dimensional structure of FMO is unknown, but stereoselectivity studies of sulfide (42) and tertiary amine (28) substrates have shown that steric considerations influence substrate binding. Also, substrates with negative charges on the substrate distinct from the heteroatom exhibit low or no substrate activity. Considering these two points, it is notable that amino acids 1-3 were substrates for FMO but removal of the zwitterionic nature of the substrate significantly reduces substrate activity (i.e., mercapturates 4-7 were poor Substrates). In keeping with the FMOmediated regioselectivity observed previously (42-45) where oxygenation is preferentially at the least sterically hindered sulfur lone pair, trans-substituted allyl sulfides had greater affinity for FMO (Table V). S-Oxygenation Stereoselectivity Considerations. Because highly purified hog liver FMO works in a highly stereoselective fashion (301,we anticipated that 1-3 would be diastereoaelectivelyconverted to 7-9. In agreement with this hypothesis, we observed considerable diastereoselectivity in the S-oxygenation of 1-3. That we observed low S-oxygenation diastereoselectivityfor S-oxygenation of 4-6
200 Chem. Res. Toxicol., Vol. 5, No. 2, 1992
was in agreement with our conclusion that 4-6 were poor substrates for hog liver FMO. The stereoselectivity data (i.e., high diastereoselectivity for 1-3 albeit low diastereoselectivity for 4-6) also suggest that nonenzymatic oxidation does not significantly contribute to the Soxygenation of 1-6. Hog liver FMO readily catalyzes the formation of tertiary amine N-oxides from monoprotonated amines (40) or the formation of S-oxygenation products from anionic sulfur-containing compounds (39). Introduction of a second charged group (i.e., monoprotonated amine or carboxylic acid) generally decreases substrate specificity. For example, cysteamine, the only known endogenous substrate for FMO (34) (Le., K, = 110 pM), is a much better substrate than cysteine (i.e., no detectable substrate activity) or the substrates reported here for FMO (Table V). It is possible that a negatively charged amino acid near the active site preferentially excludes negatively charged substrates from the active site, but zwitterionic amino acids can be S-oxygenated, albeit at a reduced efficiency. Demonstration that S-cysteine conjugates are substrates for the hog liver FMO may provide insight into the structure-function requirements for endogenous FMO substrates which may reveal a physiological role for FMO. Toxicological Considerations. While DCP exerts nephrotoxic effects in animals at high doses, its renal effects on exposed human populations are modest (15). However, in vitro toxicity has been demonstrated in cultured renal cells and freshly isolated renal tubular cells at concentrations of cis-CPNAc approaching urinary concentrations in agricultural workers. Although inhibition of FMO activity was protective to those cells, our studies do not show that FMO-catalyzed formation of S-oxides occurs in the kidney preparations examined. While Fisher 344 rat kidney cells were used for the in vitro cytotoxicity studies and Sprague-Dawley or Long-Evans rats were used for metabolite studies, data on FMO content among these species does not suggest this as a possible explanation (38). From our in vitro cytotoxicity studies and from the observation that vinylic thioethers are preferred substrates for @-lyase,kidney cell injury mediated by @-lyaseseems unlikely. Previous in vivo experiments employing rats using (amino0xy)aceticacid to block @-lyaseshowed slight protective effects, but these might be explained by the ability of (amino0xy)acetic acid to block cysteine S-conjugate uptake by renal cells. At this point, no clear mechanism is available to explain renal effects of low DCP doses. At very high doses of DCP, glutathione conjugation and oxidative metabolism of S-cysteine conjugates may play a major role (19). Acknowledgment. This work was supported in part by NIH Grant GM36426 to J.R.C., NIEHS Grant E504705 to J.D.O., NIEHS Grant ES03127 to M.W.A., and Deutsche Forschungsgemeinschaft SFB172 to S.V. The generous help of the University of California, San Francisco, Bioorganic Biomedical Mass Spectrometry Resource (A. L. Burlingame, Director) is gratefully acknowledged (supported by NIH Division of Research Resources Grant RRD 1614). We thank Gloria Dela Cruz for her excellent typing. Registry No. 1, 138876-21-8; 2, 138876-22-9; 3, 3054-01-1; 4, 72072-41-4; 5, 107077-73-6; 6, 19542-77-9; 7, 138816-04-3; 8, 138816-05-4; 9,60668-81-7; 10, 138816-06-5; 11, 138816-07-6; 12, 72286-22-7; monooxygenase, 9038-14-6.
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