Chem. Res. Toxicol. 1993,6, 223-230
223
Formation, Characterization, and Immunoreactivity of Lysine Thioamide Adducts from Fluorinated Nephrotoxic Cysteine Conjugates in Vitro and in Vivo Michael B. Fisher,t Patrick J. Hayden,$ Sam A. Bruschi,t Deanne M. Dulik,s Yun Yang,ll Anthony J. I. Ward,il and James L. Stevens*9t The W.Alton Jones Cell Science Center, Old Barn Road, Lake Placid, New York 12946, Department of Chemistry, Clarkson University, Potsdam, New York 13699-5548, Drug Metabolism and Pharmacokinetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, and Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received September 9, 1992
Fluorinated nephrotoxic cysteine conjugates undergo bioactivation via the @-lyasepathway to thionoacetyl fluorides (TAF), the putative reactive intermediates. The TAF derived from S-(1,1,2,2-tetrafluoroethyl)-~-cysteine (TFEC) difluorothionoacetylatesamine nucleophiles found in proteins and lipids. A specific antisera, raised against (trifluoroacetamido)lysine adducts formed in vivo after halothane treatment, has previously been used to localize TFEC-derived protein adducts immunohistochemically,and a good correlation between adduction and toxicity was demonstrated. Interestingly, thioamide formation is facilitated by acyl-transfer catalysts such as imidazoles and phenols. However, although putative lysine adducts have been reported to be formed from the related TAF derived from S-(2-chloro-1,1,2-trifluoroethyl)-~-cysteine (CTFC),protein adducts derived from CTFC metabolism have not been completely characterized. In the present investigation we characterize (chlorofluorothionoacetamido)lysine(CFTAL) adduct formation during S-(2-chloro-1,1,2-trifluoroethyl)-~-cysteine (CTFC) metabolism, both in vitro and in vivo. Our data indicate that formation of CTFC-derived lysine thioamides was not as dependent on nucleophilic catalysis as observed for TFEC, and this appears to be due to an apparent greater reactivity of the TAF resulting in a higher trapping efficiency in the absence of catalyst. Also, qualitative and quantitative differences in the structures and time course of CTFC versus TFEC adduct breakdown were observed. Antibodies raised against the halothane metabolite protein adduct (trifluoroacetamido)lysine cross-react with specific mitochondrial proteins from the kidneys of TFEC-treated rats. Using this antibody, we have found that the pattern of adducted proteins from TFEC- and CTFC-treated Fischer rats was similar, but the intensity was considerably lower after treatment with equimolar concentrations of CTFC in vivo.
Introduction Mercapturic acid biosynthesis is an important detoxification pathway for many lipophilic, electrophilic xenobiotics. Electrophiles may preferentially react with glutathione rather than with other cellular molecules, resulting in deactivation. The GSH conjugate is enzymatically cleaved to the cysteine conjugate which is then N-acetylated and subsequently excreted in the urine. However, in recent years a number of molecules, such as the halogenated ethylenes, have been found to produce nephrotoxicity via this glutathione conjugation pathway (1-5). It has been demonstrated that the cysteine conjugates (Figure 1, 11) of halogenated ethylenes are substrates for renal cysteine conjugate 0-lyase (@-lyase)'(EC 4.4.1.13). This enzyme catalyzes a &elimination reaction from the conjugate, yielding pyruvate, ammonia, and a reactive mercaptan (111). The mercaptan, possessing both a thiol and halogen substituents, spontaneously rearranges to a reactive electrophile which covalently binds to biological macromolecules, resulting in toxicity (6-1 0).
* T o whom correspondence should be addressed. + f
The W. Alton Jones Cell Science Center. National Institute of Environmental Health Sciences.
5 SmithKline Beecham Pharmaceuticals. 11 Clarkson University.
Structural elucidation of these reactive intermediates has been the goal of a number of studies involving the nephrotoxic cysteine conjugates. It has been demonstrated that the mercaptans resulting from 0-elimination of vinylic chlorinated conjugates, such as S-(1,2-dichlorovinyl)-~cysteine (DCVC)and S-(1,2,3,4,4-pentachlorobutadienyl)L-cysteine (PCBC),rearrange to thioketenes, under certain conditions (II), and they have been shown to be thionoacylating agents (12). However, the bioactivation of the aliphatic fluorinated conjugates has been the more thoroughly studied. It has been shown that some of the fluorinated ethylene-derived mercaptans can rearrange to thionoacetyl fluorides (13, 14). Dekant et al. have trapped the free nucleophilic thiol (111)and the electrophilic acyl halide (IV) from S-(2-chloro-1,1,2-trifluoroAbbreviations: BSA, bovineserum albumin; CTFC, S-(2-chloro-1,1,2trifluoroethy1)-L-cysteine; TFEC, S-(1,1,2,2-tetrafluoroethyl)-~-cysteine;
DCVC, S-(1,2-dichlorovinyl)-~-cysteine; CTFA-CTFC, N-(chlorofluorothioacetamido)-S-(2-chloro-1,1,2-trifluor~thyl)-~-~steine; CFTAL,N(ch1orofluorothioacetamido)-Nm-acetyl-L-lysine; DFTAL, N-(difluorothioacetamido)-N*-ace~l-tlysine;PLP, pyridoxal 5'-phosphate; a-TFAL, anti-(trifluoroacetamido)lysine;Cu-PLP, mixture of CuSO4 and PLP; &lyase, cysteine conjugate 8-lyase; DNPH, dinitrophenylhydrazine; FAB, fast atom PCBC, S-(1,2,3,4,4-pentachlorobutadienyl)-~-cysteine; bombardment; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NAHis, N"-acetyl-L-histidine; NATyr, M-acetyl-L-tyrosine; DMAP, 4-(NJV-dimethylamino)pyridine;TFA, trifluoroacetate.
0 1993 American Chemical Society 0893-228~/93/2706-0223$04.00/0
224 Chem. Res. Toxicol., Vol. 6,No.2, 1993 F
F + GSH
:C=C'
X
'F
4 :
Acyl Transfer
06
:Nu
7 s
,Nu
Fisher et al.
treated with TFEC are localized to specific molecular weight proteins of the mitochondrial fraction (19). Although putative thioamide adducts from CTFC metabolism have been reported in vitro with protein by Harris et al. (18)and with lipids by Hayden et al. (16),the biological adducts derived from CTFC and the role of nucleophilic catalysis have not been completely characterized. Therefore, we have fully characterized the N f (chlorofluorothioacetamido)-N"-acetyl-L-lysine (CFTAL) adduct formed by CTFC cleavage and have compared the characteristics of this compound with adducts formed with bovine serum albumin (BSA) invitro and in kidney protein after administration of CTFC in vivo. We have confirmed reports that CTFC is metabolized to a chlorofluorothionoacetyl fluoride intermediate. We also show that chlorofluorothioamide adducts form with protein lysines,both in vitro and in vivo. This adduct formation is less dependent on acyl-transfer catalysis than TFEC due to an apparent increased reactivity of the intermediate. Also, anti-(trifluoroacetamido)lysine (a-TFAL) antiserum crossreacts with specific mitochondrial proteins from the kidneys of CTFC-treated rats. However, there were interesting and significant differences in the pattern and intensity of adduction compared to TFEC. Finally, we demonstrate that protein and lysine adducts of CTFC metabolites are unstable and degrade to unidentified defluorinated species. The instability of the chlorofluorothioacetamido adduct may account for the defluorination detected by others (14).
Experimental Section
ethyl)-L-cysteine(CTFC) metabolism in vitro (15). However, Commandeur et al. (14)have suggested that there is a correlation between leaving group ability at the 2-carbon (F < C1< Br) and a tendency to rearrange to a thiirane intermediate via intramolecular halogen displacement. They proposed that the thiirane from CTFC could hydrolyze, forming fully defluorinated species. However, this pathway has never been conclusively demonstrated or disproven. Since it is the reactive intermediates which are apparently responsiblefor the nephrotoxicity of these conjugates, through adduct formation with macromolecules, identification of these macromolecular adducts is an important step toward understanding the mechanisms of toxicity. The fluorinated conjugate S-(1,1,2,2-tetrafluoroethyl)-~cysteine (TFEC) has been shown to form difluorothioacetamido-lipid adducts with phosphatidylethanolamine in vitro (16) and (difluoroacetamido)lysine adducts with protein in vitro and in vivo (17,18).Moreover, nucleophilic catalysis may also play a role in adduct formation (17). Studies with anti-adduct antibodies showed that the vast majority of the adducts present in the kidneys of rats
Synthesis of Conjugates and Conjugate Adducts. [W]Labeled and unlabeled CTFC and TFEC were synthesized as previously reported (20). lgF-NMRanalysis of CTFC (data not shown) revealed a splitting pattern consistent with previous reports for the glutathione conjugate (21). The conjugates have been fully characterized previously by 'H-NMR, mass spectrometry, and elemental analysis; the data are reported elsewhere (20). N-(Chlorofluorothioacetamido)-S-(2-chloro-l,l,2-trifluoroethy1)-L-cysteine (CFTA-CTFC), the so called CTFC selfadduct (17) or pseudo-mercapturate (13) (Figure 1, VII), was synthesized as previously reported for the TFEC self-adduct ( I 7). CFTAL, the thioamide adduct of lysine derived from reaction of CTFC metabolites with N"-acetyl-L-lysine, was synthesized as previously reported for the TFEC adduct ( I 7). Physical data for CFTAL and CFTA-CTFC appear in Table I. Instrumentation. lgF-NMRspectrawererecordedonan IBM NRi250 FT-NMR spectrometer operating at 235.33 MHz and were referenced to trifluoroacetate (0 ppm). I3C-NMR (proton decoupled and non-decoupled) spectra were recorded on the same instrument operating a t 62.90 MHz and were referenced to tetramethylsilane (0 ppm). Samples were dissolved in 50 mM potassium phosphate buffer (pH 7.4) when appropriate. Analytical and preparative-scale HPLC were performed on a Rainin Rabbit HP dual-pump system. Detection was with a Gilson Model 116 dual-wavelength UV detector. The mobile phase was water/O.l% trifluoroacetate (TFA) (solvent A) vs acetonitrilei0.09 % trifluoroacetate (TFA) (solventB). Analytical samples were assayed with a Waters pBondapak C18column (300 X 3.9 mm) operating at a flow rate of 1.0 mL/min. Preparative samples were purified on a Waters pBondapak C18 column (300 X 7.8 mm) operating a t a flow rate of 3.0 mlimin. Flow-Fast atom bombardment (Flow-FAB)mass spectra were obtained on a Finnigan TSQ70 mass spectrometer (Finnigan MAT, San Jose, CA) equipped with a Finnigan continuous-flow FAB interface. Samples were dissolved in methanoliwater (1:l) and loop injected into a Hewlett-Packard 1090 liquid chromatograph. The mobile phase consisted of solvent 1 , O . l M ammonium
Chem. Res. Toxicol., Val. 6, No.2, 1993 225
S-(2-Chloro-l,l,2-trifluoroethyl)-~-cysteine Thioamides acetate (adjusted to pH 5.0 with glacial acetic acid) containing 5 % glycerol, and solvent 2,acetonitrile/water (60:40)containing 5% glycerol. The flow rate was 0.25 mL/min; eluent was split a t an approximate 40:l ratio into the mass spectrometer. Spectra were acquired in alternating positive/negative ion scan mode. Probe-FAB mass spectra were acquired on a VG 7070 EHF instrument operated with an accelerating potential of 6 kV using FAB ionization. Fast xenon atoms were generatedusing a saddlefield fast atom gun operated a t 8 kV, 1 mA. Samples were ionized from a liquid matrix of glycerol. Spectra were obtained in continuous scan mode with the magnet scanning a t a rate of 20 sidecade from 1300 to 100 amu. Analysis and Trapping of 8-Cleavage Products. In catalysis experiments, a partially purified @-lyase(22) was used [specific activity = 1.43 pmol/(lO min-mg) with 10 mM L-phenylalanine and 5 mM a-keto-y(methy1thio)butyrate as substrates]. Conjugate (200nmol), @-lyase(1.56pg), a-keto-y-(methy1thio)butyrate (200 nmol), and Nm-acetyl-L-lysine(20 pmol) were dissolved in 50 mM potassium phosphate buffer (1mL, pH 7.4) and were incubated for 30 min a t 37 OC with or without nucleophilic catalyst (16 pmol). For quantitation of adduct formation, the incubation was acidified with 1 mL of 2 N HC1 and extracted with 2 mL of ethyl acetate. The organic layer containing the product was removed, and the solvent was evaporated. The product was taken up in 200 pL of potassium phosphate buffer (pH 7.4),and 50 pL was analyzed by HPLC with detection at 280 nm. The amount of adduct (nmolil-mL incubation) was calculated by comparing the integrated peaks in unknowns with a standard curve constructed with authentic N'-thioacetamido-N'-acetyl-L-lysine adduct and correcting for extraction efficiency. Conditions for the separation of lysine and self-adducts of each conjugate are similar to those already reported for TFEC metabolites (17). To determine the trapping efficiency, adduct formation was normalized to total metabolism as determined by pyruvate production. Pyruvate was trapped as the dinitrophenylhydrazone by adding 125 p L of 0.1 % DNPH in 2 N HC1 to 0.5 mL of incubation. After 10 min, 50 pL was analyzed by HPLC. Gradients typically were 30-60% solvent B over 18 min, and detection was at 360 nm. The pyruvate derivative eluted as two unequal peaks (22)(cis and trans isomers) which were always formed in the same ratio. The amount of pyruvate was determined by comparing the area of the major peak (11min) with a series of pyruvate standards derivatized as described above. For structural analysis of protein or lysine adducts, a pyridoxal 5'-phosphate (PLP) and Cu2+system (Cu-PLP) similar to those previously reported (14, 17,23) was used to generate sufficient quantities of @-eliminationproducts from cysteine conjugates. Conjugate (20pmol), PLP (4pmol), and CuS04 (2pmol) were incubated with either BSA (20mg) or N"-acetyl-L-lysine (400 pmol) in 20 mL of potassium phosphate buffer (pH 7.4)a t 37 "C for 1 h. Lysine adducts were purified as described (1 7). BSA was precipitated by the addition of 20 mL of 20% ice-cold trichloroacetic acid. After 1 h a t 4 OC, the suspension was centrifuged a t 2500g for 5 min and the pellet was washed by repeated precipitation. The pellet was then resuspended in 6 mL of phosphate buffer (pH 7.4)and digested with three 25-mg portions of proteinase K (2h a t 37 "Ciportion) to ensure maximal digestion. The suspension was then centrifuged a t llOOg and the supernatant was analyzed by 19F-NMR. The final concentrations of CTFC and TFEC adducts (based on 3%-radiolabel incorporation) were 16.5 and 5.6 mol of adduct/mol of BSA, respectively. Identification of CTFC Protein Adducts Formed in Vivo. For in vivo experiments, Fischer rats were injected ip with TFEC or CTFC a t a dose of 30 or 300 mg/kg body weight and were sacrificed a t 6 h for the 30 mg/kg dose and a t 1 h for the 300 mg/kg dose. Kidneys were removed, homogenized, and mitochondria were isolated by the method of Schnaitman and Greenwalt (24). An aliquot of each was removed for Western blotting, with the remainder used for NMR analysis. Mitochondrial and postmitochondrial protein were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) on 8.5 % gels and transferred to nitrocellulose. Adducted proteins from either CTFC- or TFEC-treated animals were detected as previously described (19)usingantiseraraisedagainst (trifluoroacety1)lysine (25),averygenerous gift of Dr. Lance Pohl (NIH). This antibody, which has been previously chmacterized, recognizes protein containing both TFEC and CTFC adducts (19).
For NMR of mitochondrial fractions, protein was precipitated by the addition of 2 volumes of ice-cold 1% glacial acetic acid in acetone (samples typically contained 10-30 mg/mL protein). After 1 h at 4 "C, samples were centrifuged at llOOg for 5 min, and the pellet was washed by repeated precipitation. The pellet was then resuspended in 50 mM ammonium bicarbonate buffer (pH 7.8) and incubated with 10 mg of proteinase K a t 50 "C. After 3 h, an additional 10 mg was added and digestion was continued overnight. Finally, samples were removed and analyzed by NMR. Statistics. A one-way ANOVA was used to determine significant differences. Individual means were compared using a Neuman-Keulsmultiple comparison test. Values with the same superscript letter designation are not different, while values with different letter designations are significantly different, p < 0.01.
Results In order to generate large amounts of @-elimination products from CTFC, we used an in vitro metabolite generating system (CuSO4 and the @-lyase catalytic cofactor PLP) as reported previously (17,23).l9F NMR analysis of CTFC @-eliminationproducts showed that halofluoroacetate, halofluorothioacetate, and self-adducts were the only metabolites detected (data not shown), consistent with previous data for fluoroethyl-S-conjugates such as TFEC and CTFC (14,17).These products are consistent with acyl fluoride formation from @-elimination as the major metabolic path. Using this system, we generated metabolites from CTFC in the presence of BSA as a model protein to determine the nature of the protein adduct formed. After proteinase K digestion of adducted BSA, 19FNMR analysis detected a doublet at -56.94 ppm with a coupling constant ( 2 J=~51.4 ~ Hz) consistent with geminal H-F coupling expected for a thioamide (17,18) (Figure 2a). The doublet was shifted upfield from that observed for TFEC-derived thioamides by about 15 ppm due to the presence of slightly less deshielding from a chlorine vs fluorine substitution. Metabolites of TFEC form thioamides with lysine residues of BSA (17)and CTFC models have been shown to yield thioacetamido-like compounds with protein (18). In order to determine if e(thioacetamid0)lysine adducts of CTFC metabolites are formed in proteins, we synthesized the CFTAL adduct by generating CTFC metabolites with the @-lyasemimetic system, this time in the presence of Na-acetyl-L-lysine. Products from the reaction were extracted under acidic conditions and analyzed by reversephase HPLC (Figure 3). Two peaks were observed, only one of which was dependent on the presence of Na-acetylL-lysine (peak 1). l9F-NMR analysis of peak 1 showed a doublet with chemical shift and coupling constant (Figure 4a) identical to those of the BSA adduct, signals which are characteristic of a thioamide. Proton-decoupled 13CNMR analysis yielded 10 peaks, all assignable to CFTAL (Table I). Mass spectral analysis of peak 1 confirmed the structural assignments made from NMR data. The negative ion probe FAB mass spectrum of peak 1 (Table I) contained a molecular ion (M - H)- at mlz 297,299 with an isotope
226
Chem. Res. Toxicol., Vol. 6, No.2, 1993
Fisher et al.
a.
H CFTAL
I -20
-70
-60
-50
-40
-30
b.
I
I -15
I
I
-20
-25
I
I
-30 -35
I
I
I
-40
-45
-50
I -55
I
I
-60
-65
PPM
a. Odays
Figure 2. Identification of CTFC metabolite protein adducts
using 19FNMR. l9F-NMR spectra of protein adducts derived from CTFC metabolism. (a) Bovine serum albumin: 20 pmol of CTFC and 1 mg/mL BSA were incubated with a &lyase mimetic system consisting of pyridoxal 5/-phosphate (PLP, 2 pmol) and CuSO4 (1pmol) in 5 mL of phosphate buffer (pH 7.4) at 37 "C = 51.4 Hz. (b) Rat kidney mitochondrial for 3 h; (d) 'JHF protein: Kidney mitochondrial protein from rats was prepared as described (7)1 h after they had received 300 mg/kg CTFC; (d) ~ J H =F 51.4 Hz. Prior to analysis, both BSA and kidney mitochondrial protein were first washed by precipitation as described in the Experimental Section and then resuspended in the appropriate buffer and digested with proteinase K (5 mgi mL, 37 OC overnight). The NMR resonance due to fluoride was shifted slightly in the spectrum b due to the presence of ammonium bicarbonate buffer (pH 7.8, see Experimental Section).
distribution characteristic of the presence of one chlorine atom and one sulfur atom (2971299ratio = 100137). A trifluoroacetate (TFA) adduct of the molecular ion (M + TFA - H)-, due to the TFA in the HPLC solvent system, was alsodetectedat mlz 411,413.Positive ion continuousflow FAB mass spectral analysis (Figure 5 and Table I) confirmed the molecular weight and gave further diagnostic structural information. The molecular ion (M + H)+ at mlz 299 had an associated peak at mlz 301 which was again consistent with the presence of one chlorine atom. The fragments at mlz 279 and 281 are due to loss of HF and retain the chlorine isotopic distribution. Fragments at mlz 265, 264,and 263 are due to reductive dehalogenation, radical cation formation due to loss of Cl', and loss of HC1, respectively. The chlorine-containing peak at mlz 243 is due to loss of HF from the 263 fragment. The ion at mlz 194 is an unidentified fragment. Peak 2 (Figure 3)was present in incubations containing only CTFC and Cu-PLP. Both the mass spectral and NMR analyses were consistent with the formation of CFTA-CTFC (VII) produced by the reaction of the thionoacyl halide intermediate with the free amine on the
1 b. 4days
I1
I
I
I
I
I
-42
-44
-46
-48
I -50
I -52
1 -54
I
I
-56
-58
PPM
Figure 4. l9F-NMR of N-(chlorofluorothioacetamido)-Naacetyl-L-lysine (CFTAL) degradation. CFTAL was synthesized using the Cu2+ and pyridoxal phosphate mimetic system as described in the legend to Figure 3. After purification by reversephase HPLC, peak I (CFTAL) was analyzed by l9F-NMR immediately (a) and after 4 days (b) CFTAL (d) -56.94 ppm; 'JHF = 51.4 Hz. Fluoride ion: ( 8 ) -44 ppm. cysteine of CTFC (Table I). The proton-decoupled 13CNMR of CFTA-CTFC was somewhat complex due to overlapping multiplets. The doublet of triplets centered at 97.63ppm and the triplet of doublets centered at 125.54 ppm are from the two halogenated carbons of the parent CTFC. The presence of two doublets in the 13C (97.78 and 98.41 ppm) and 19F NMR (-57.48 and -56.39 ppm) of CFTA-CTFC correspond to the chiral chlorofluorocarbon moiety of CTFC from which the thionoacyl halide is derived (14,21).This pattern probably depends on a conformational effect due to intramolecular hydrogen
S-(2-Chlor0-1,1,2-trifluoroethyl)-~-cysteine Thioamides
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 227
TFEC for 6 h and prepared samples as above. A doublet was detected at -42 ppm ( 2 J=~55~ Hz), indicative of NMR (Intensity, Multiplicity) (proton decoupled) Ne-(difluorothioacetamido)-Na-L-lysine (DFTAL) (I 7,18). compound 19F (ppm) (PPd Since it appeared that our CTFC dose had been too low, 29.13 ( 8 ) ; 60.47 ( 8 ) ; 97.78 CFTA-CTFC -72.94 (m); -57.48 we increased from 30 to 300 mg/kg CTFC. l9F-NMR (d, 'JHF = 51.4 Hz); (d, 'JCF = analysis of kidney protein after a 300 mglkg dose showed 252.71 (Hz);98.41 (d, =F -56.39 (d, ~ J H a doublet at -56.9 ppm ( 2 J =~51.4 ~ Hz), identical with VCF= 257.41 Hz); 51.4 Hz); -43.83 ( 8 ) ; synthetic CFTAL (Figure 2b). The data are entirely -10.52 (m) 97.63 (dt, 'JCF 252.10 Hz, 2 J c = ~ 34.5 Hz); consistent with the l9F-NMR signal from the BSA adduct 125.54 (td, 'JCF= 283.95 and of synthetic CFTAL. Hz, *JCF= 29.2 Hz); Previous studies have suggested that thioamide adducts 173.91 (9); 193.93 (d, 'JCF = 18.6 Hz) are converted to the amide upon aging (18). This is 22.29 ( 8 ) ; 22.82 (9); 26.44 ( 8 ) ; CFTAL -56.94 (d, VHF = accompanied by an upfield shift in the l9F-NMRresonance 51.4 Hz) 31.49 ( 8 ) ; 45.70 ( 8 ) ; by approximately 8 ppm with retention of the geminal 55.39 (a); 98.44 (d, ~ J C=F H-F coupling. However, in these studies no such shift 257.4 Hz); 173.87 ( 8 ) ; 179.59 ( 8 ) ; 192.65 was observed for CFTAL, either for protein or for the ~ Hz) (d, 2 J =~21.2 synthetic adduct. On the other hand, the presence of FAB-MS (Ion) fluoride (-41 ppm) was always detected in the protein negative ion (mlz) compound positive ion (mlz) samples from CTFC-treated rats (Figure 2b). In order to CFTA-CTFC ND" (M - H)- 346; (MH determine if aging of thioamide adducts from CTFC might - C1)'- 312; result in defluorination, we prepared CTFAL in phosphate (CHClFCF2S)buffer (pH 7.4) and compared l9F-NMR spectra imme149 diately after preparation and after 4 days. The data clearly CFTAL (M + H)+ 299; (M + NH4)+ (M - H)- 297; 316; (M - HF + H)+ 279; (M + TFA - H)show that CFTAL degrades to fluoride (-44 ppm) and 411; (M (M - C1+ H)+ 265; (MH unidentified defluorinated products upon aging (Figure - C1)'- 264; (M - HCl + HF - H)- 277; 4). Acidic conditions prevented the breakdown, while basic (MH - C1)*-262 H)+ 263;(MH - C1conditions resulted in complete loss of the doublet centered HF)+ 243 -56.9 ppm and appearance of fluoride at -44 ppm (data at ND = not determined. not shown). The difference in the chemical shift of the 100 resonance due to fluoride can be attributed to the exquisite [M+H]+ 299 sensitivity of the fluoride anion to the composition and pH of the buffers used (data not shown). It has been reported that DFTAL formation can be significantly increased in the presence of imidazole and phenol by formation of acyl-transfer intermediates, Le., 3 nucleophilic catalysis (17). Since all our evidence had -2ul suggested that the reactive intermediate derived from a, @-eliminationof CTFC rearranges to a chlorofluorothionoacetyl fluoride, we generated this intermediate with partially purified @-lyasein the presence of Na-acetyl-Llysine and various catalysts to determine if acyl transfer I 263 plays a role in CFTAL formation. The effect of various catalysts on total metabolism were corrected for by normalizing the amount of adduct formed to pyruvate production, thus yielding trapping efficiency. On com0 paring the results to identical incubations with TFEC as 200 250 300 350 400 m/z a positive control, we found that CFTAL formation was Figure 5. Positive ion continuous-flow FAB mass spectral also prone to catalysis, especially with tyrosine (Table 11). analysis of W-(chlorofluorothioacetamido)-N~-acetyl-L-lysine. However, catalysis occurred to a much lesser extent CFTAL was synthesized using the Cu2+and pyridoxal phosphate compared to DFTAL formation, which was increased 4445% mimetic system as described in the legend to Figure 3. After with imidazole. Also, CTFC yielded a much higher purification by reverse-phase HPLC, CFTAL was analyzed by FAB-MS. trapping efficiency under all conditions. In order to determine the effect of target concentration on catalysis, bonding since in the presence of aprotic hydrophobic lysine concentration was varied in the presence of 16 mM solventsthese doublets merge (datanot shown). This "selfimidazole, @-lyase,and either 200 pM CTFC or 200 pM adduct" or "pseudo-mercapturicacid" has been previously TFEC (Figure 6). Although the degree of catalysis reported for CTFC (14)and for TFEC (13,17). increased with decreasing lysine concentration for TFEC, Figure 6 shows that CFTAL formation is much less Since we had shown that CTFC forms thioamide adducta dependent on acyl-transfer catalysis than DFTAL. These in vitro with free and protein lysines, we sought to detect results strongly suggest that acyl-transfer catalysis plays these adducts in kidney protein from CTFC-treated rats. a much greater role in the formation of lysine adducts Protein from the kidneys of rats treated with 30 mglkg from TFEC metabolism because the reactive intermediate CTFC for 6 h yielded no l9F-NMRsignal after subcellular from CTFC @-elimination is more reactive than the fractionation and proteinase K digestion. Therefore, as corresponding intermediate from TFEC. a positive control (18),we dosed animals with 30 mglkg Table I. Summary of Physical Data
0
d .
I
Fisher et al.
228 Chem. Res. Toxicol., Vol. 6, No.2,1993 Table 11. Catalysis of TFEC and CTFC Metabolite Lysine Adduct Formation.
catalyst
bindingnmol of adduct f SD % control f SD
control NAHis imidazole NATyr DMAP
0.25 i O.Olb 0.70 f 0.04c 1.11 f O.O!jd 0.51 i 0.04e 0.47 f 0.03e
metabolism nmol of product f SD % control f SD TFEC
100 i 4b 280 f 16' 444 f 2od 204 f 16e 188 f 12e
23.6 i 2.3b 20.1 f 2.0b*c 22.6 f 0.4b*c 17.2 f 2.2c 20.9 f 0 . 7 ' ~ ~
100 i l o b 85 f 8b*c 96 f 26*c 73 9e 89 f 36*c
*
trannina w - ~ y y a - a b
efficiency (% ) 1.1 3.5 4.9 3.0 2.2
CTFC 5.0 100 i 7b 13.9 f 0.6b 100 f 4b control 0.70 f 0.0!j6 10.2 NAHis 1.25 f 0.03c 179 f 4c 12.3 f 0.3b 88 f 2b 10.1 93 f 76 imidazole 1.30 f O.llc 186 16e 12.9 f 0.g6 15.1 NATyr 1.47 f O.OEid 210 f 7d 9.7 f 1.3c 70 f 9c 4.9 14.2 f 0.7b 102 f 56 99 f 36 DMAP 0.69 i 0.02b a 200 nmol of conjugate, 200 nmol of a-keto-y-(methylthio)butyrate,20 pmol of Nu-acetyl-L-lysine, and 1.56 pg of &lyase with or without 16 pmol of catalyst in 50 mM phosphate buffer, pH 7.4, at 37 "C for 30 min. Produds were quantitated for either adduction or metabolism as described in the Experimental Section. Adduction was normalized to metabolism to determine trapping efficiency (n = 3). Values with the same superscript letter designation are not different, while values with different letter designations are significantly different, p < 0.01. NAHis = Nt-acetyl-L-histidine. NATyr = W-acetyl-L-tyrosine. DMAP = 4-(N&-dimethylamino)pyridine.
105
-aa L
E
-al I-
74 300,
46
m
g
200.
01 0
I
I
1
1
10
20
30
40
Lyslne (mM)
Figure 6. Dependence of nucleophilic catalysis by imidazole on lysine concentration. 200 nmol of conjugate, 200 nmol of a-ketoy-(methylthio)butyrate, 16 pmol of imidazole, and 1.56 pg of @-lyasewere incubated with various amounts of Na-acetyl-L-lysine in 1mL of 50 mM phosphate buffer (pH 7.4) a t 37 "C for 30 min. Pyruvate and CTFAL formation were quantitated as described in the Experimental Section. The trapping efficiency was determined by normalizing CFTAL formation to pyruvate [ (CFTAL/pyruvate) X 100 = trapping efficiency]. The percent increase in trapping efficiency was determined by comparing catalyzed to uncatalyzed trapping a t each lysine concentration. Thus, a 100% increase represents a doubling of the uncatalyzed trapping efficiency.
Halothane metabolites form (trifluoroacety1)lysine adducts with several hepatic microsomal proteins (26,27); antisera against these adducts have been shown to crossreact with TFEC- and CTFC-treated rat kidney cytosol (19).Specific mitochondrial protein from the kidneys of TFEC-treated rats also cross-reacted with this antisera. Therefore, as an additional assay for in vivo (thioacetamido)lysineadduct formation, we treated rats with 30,60, 120,and 300 mg/kg CTFC and 30mg/kg TFEC as a positive control (19)and looked for subcellularadduct distribution with anti(trifluoroacety1)lysine antisera. The resulting immunoblotshowed a pattern of CTFC-adductedproteins, all in the mitochondria, which was very similar to the pattern for TFEC adduction (approximately 98,84,66, 52,48 kDa) (Figure 7). a-TFAL antisera did not crossreact with either mitochondrial or nonmitochondrial protein from the kidneys of untreated rats (data not presented). However, the intensity of staining for CTFC treatment was dose dependent and was decreased com-
Figure 7. Western blot of kidney fractions from TFEC- and CTFC-treated rats. Protein samples were prepared for SDSPAGE and western blotting as described in the Experimental Section. Each lane contains 25 pg of protein except lane H which contained only 6.25 pg. Lane A, protein from rats treated with 30 mg/kg TFEC. Lanes B-I contain proteins from rats treated with various concentrations of CTFC. Lanes A, B, D, F, and H are samples from mitochondrial fractions of rat kidneys, while lanes C, E, G, and I are postmitochondrial fractions from kidneys of the same samples. CTFC doses were as follows: lanes B and C,30 mg/kg; lanes D q d E, 60 mg/kg; lanes F and G, 120mg/kg, and lanes H and I, 300 mg/kg.
pared with TFEC a t equimolar doses, especially the staining of the 98-, 84-, and 48-kDa proteins. This difference could be attributed to different intrinsic antigenicities of the two conjugate adducts or to the relative instability of CFTAL compared to DFTAL, or indicative of the relative degree of protein adduction between the two conjugates.
Discussion Since the relationship between trichloroethylene-induced outbreaks of fatal aplastic anemia in cows and formation of DCVC was recognized (I),the mechanisms underlying halogenated ethylene metabolism and toxicity have been sought. Derr and Schultze (28)first described the binding of cysteine conjugatemetabolitesto biological macromolecules. Further work by this group clearly demonstrated the enzymatic cleavageof DCVC by a PLPlike enzyme and. the elimination of a reactive thiol containing species as a @-eliminationproduct (29). The enzyme was later named cysteine conjugate p-lyase and has been purified from a variety of sources (22,30). A number of studies have linked cysteine conjugate cleavage by @-lyasewith the appearance of cytotoxicity (6-10,31, 32). However, despite the detailed knowledge of the
S-(2-Chloro-1,1,2-trifluoroethyl)-~-cysteine Thioamides
toxicology,enzymology,and chemistry of the nephrotoxic cysteine conjugates, little work has been done to characterize the structure of the adducts which these metabolites form with biological macromolecules. Difluorothioamide formation with proteins by TFEC metabolites has been previously described (17, 18). Although some work with in vitro systems suggests similarities to TFEC, CTFC protein adduct formation has not been fully characterized. Harris et al. (18) detected an apparent protein thioamide adduct in vitro using a 2-nitrophenyl disulfide CTFC metabolite generating system, but failed to show a thioamide doublet by l9F-NMR after in vivo CTFC administration. Utilizing either partially purified cysteine conjugate &lyase or a 6-lyase mimetic model, copper(I1) sulfate with the catalytic cofactor PLP (14,17),we generated 6-elimination-derived reactive intermediates from two related cysteine conjugates, TFEC and CTFC, investigated their binding to BSA or N*-acetyl-L-lysine,and compared these adducts to those found in vivo. Physical characterizationby lgF-NMR,13CNMR, and mass spectrometry of synthetic N*-acetyl-Llysine adduct derived from CTFC confirmed the structure as CFTAL. l9F-NMRchemical shift and coupling constant of kidney mitochondrial protein from rats treated with CTFC were identical to in vitro modified BSA and synthetic CFTAL, We also confirmed previous reports (14) that a CTFC “self-adduct” forms due to the attack of the thionoacyl halide on the free amine of CTFC and that a difluorothioamide on lysineforms in vivo from TFEC treatment (17, 18). Furthermore, anti-(trifluoroacety1)lysine antibody cross-reacted with kidney mitochondrial proteins from CTFC-treated rats. Therefore, we suggest that the primary amino acid acylated in vivo by CTFC metabolites is lysine, and we demonstrate formation of (ch1orofluorothioacetamido)lysine adducts in vivo from CTFC treatment. A nucleophilic catalysis mechanism similar to that observed with TFEC also operates with CTFC. However, there were interesting differences between catalysis with CTFC vs TFEC metabolites. The greater trapping efficiency (nmol of adduct/nmol of reactive intermediate produced) from CTFC metabolites under all conditions in addition to the greater catalysis seen for TFEC metabolites strongly suggests that the reactive intermediate derived from CTFC is more reactive under the conditions used. However, Hayden et al. (20) have reported equal metabolism of [35Sl-bindingof TFEC and CTFC to protein in isolated mitochondria. Furthermore, the loss of fluoride from CFTAL and not from DFTAL indicates that CTFC-derived adducts are less stable than the corresponding TFEC adducts. Therefore, it appears that if CTFC yields similar numbers of adducts as TFEC initially, the instability of CTFC-derived adducts could contribute to the difficulty in detecting CTFC adducts in vivo. It is difficult to evaluate the role of nucleophilic catalysis in adduct formation in vivo. Tyrosine and histidine residues in proteins could stabilize these reactive acyl groups from attack by a common nucleophile, such as hydroxide ion, until reaction occurs with lysine residues (i.e., increase target trapping efficiency) to form stable thioamide adducts. Because the extent of nucleophilic catalysis is greater a t low target density (Figure 61, it may be that catalysis of lysine adduct formation by adjacent residues assumes greater importance under physiological
Chem. Res. Toxicol., Vol. 6, No. 2,1993 229
conditions. Consequently, areas on a protein which have tyrosines and histidines nearby to lysine or other targets could be predisposed to adduct formation by the conjugate metabolite. Although the data reported here demonstrate thioamide adduct formation from CTFC and TFEC metabolites, other adducts may play a role in fluorinated conjugate induced toxicity. Thiirane formation from CTFC has been postulated previously. A structure/activity study by Commandeur (14) showed that 2-chloro- and (2-bromo1,l-difluoroethy1)cysteineconjugates, including CTFC, yielded little or no detectable haloacetic acids compared to TFEC, indicative of a different bioactivation mechanism. Ab initio calculations suggested that only TFEC would be predicted to form thionoacyl halides, with the others forming thiiranes derived from intramolecular chlorine or bromine displacement. However, there was no indication in our studies for any species other than thionoacyl halide formation. Some evidence for formation of dithioester adducts in vitro was found by Hayden et al. (17), but due to their lability under acidic conditions such as protein precipitation and HPLC solvent systems, they could not be isolated and characterized. Therefore, the contribution of uncharacterized adducts to toxicity remains unclear. A considerable amount of experimental work has been directed toward elucidation of the targets for halogenated ethylene induced nephrotoxicity. The damage is relatively specific for the S3 segment of the proximal tubule, and a variety of studies implicate the mitochondrion as a target organelle (33-36). Themajorityof [35Sl-bindingfrom[35SlTFEC to rat kidney mitochondria in vitro occurs in the protein fraction, although significant binding to lipids is also observed (16). Recently, Hayden et al. (19)reported that specifickidney proteins from 30 mg/kg TFEC-treated rats form thioacetamidolysine adducts, and these adducts are localized exclusively to the mitochondrial fraction. However, in our studies with CTFC, some staining was seen in the postmitochondrial supernatant (Figure 7). Hargus et al. (37)and Harris et al. (18)also found evidence for conjugate adducts in cytosolic as well as mitochondrial fractions, but this may have been due to the extremely high dose used in those studies (1 mmol/kg) which will cause mitochondrial damage and leakage into the cytosol as we observed at 300 mg/kg but not at 30 mg/kg CTFC. It is possible that differences in both adduct structure and target specificity may account for differences in the toxicity of chlorinated and fluorinated cysteine conjugates. Identification of target proteins or other macromolecular targets for cysteine conjugate metabolite binding will be an important step in elucidating the mechanism of cysteine conjugate induced cytotoxicity. Further experimentation will be necessary before these events can be linked together to reveal the molecular and cellular mechanisms leading to cysteine conjugate induced nephrotoxicity. Acknowledgment. We thank Dr. Lance Pohl (NIH) for generously providing a-TFAL antisera and for helpful discussions, William Schaefer for providing probe FAB mass spectra, and Marilyn Hauer for her help in preparation of the tables. This work was supported by Grant DK38925 (to J.L.S.) from the National Institute for Diabetes, Digestive and Kidney Diseases.
References (1) McKinney, L. L., Weakley, F. B., Eldridge, A. C., Campbell, R. E., Cowan, J . C., Picken, J. C., Jr., and Biester, H . E. (1957)
230 Chem. Res. Toxicol., Vol. 6, No. 2, 1993 S-(Dichloroviny1)-L-cysteine:An agent causing fatal aplastic anemia in calves. J . Am. Chem. SOC. 79, 3932-3933. (2) Lash, L. H., and Anders, M. W. (1986) Cytotoxicity of S-(1,2dichloroviny1)glutathione and S-(1,2-dichlorovinyl)-~-cysteine in isolated rat kidney cells. J . Biol. Chem. 261, 13076-13081. (3) Nash, J. A., King, L. J., Lock, E. A., and Green, T. (1984) The metabolism and disposition of hexachloro-1,3-butadienein the rat and its relevance to nephrotoxicity. Toxicol. Appl. Pharmacol. 73, 124-137. (4) Terracini, B., and Parker, V. H. (1965) A pathological study on the toxicity of S-dichlorovinyl-L-cysteine.Food Cosmet. Toxicol. 3,6774. (5) Green, T., and Odom, J. (1985) Structureiactivity studies of the nephrotoxic and mutagenic action of cysteine conjugates of chloroand fluoroalkenes. Chem.-Bid. Interact. 54, 15-31. (6) Stevens, J. L., and Jones, D. P. (1989) The mercapturic acid pathway: Biosynthesis, intermediary metabolism and physiological disposition. In Glutathione: Chemical, Biochemical, and Medical Aspects, Part B (Dolphin, D., Poulson, R., and Avramovic, O., Eds.) pp 45-84, Wiley, New York. ( 7 ) Stevens, J. L., and Wallin, A. (1990) Is the toxicity of cysteine conjugates formed during mercapturic acid biosynthesis relevant to the toxicity of covalently bound drug residues? Drug Metab. Rev. 22,617-635. (8) Koob, M., and Dekant, W. (1991) Bioactivation of xenobiotics by formation of toxic glutathione conjugates. Chem-Bid.Interact. 77, 107-136. (9) Anders, M. W., Dekant, W., Elfarra, A. A., and Dohn, D. R. (1988) Biosynthesis and biotransformation of glutathione S-conjugates to toxic metabolites. CRC Crit. Rev. Toxicol. 18, 311-341. (10) Commandeur, J. N. M., and Vermeulen, P. E. (1990) Molecular and biochemical mechanisms of chemically induced nephrotoxicity: A review. Chem. Res. Toxicol3, 171-194. (11) Dekant, W., Urban, G., Gorsmann, C., and Anders, M. W. (1991) Thioketene formation from a-haloalkenyl2-nitrophenyldisulfides: Models for biological reactive intermediates of cytotoxic S-conjugates. J. Am. Chem. SOC. 113, 5120-5122. (12) Dekant, W., Berthold, K., Vamvakas, S., Henschler, D., and Anders, M. W. (1988) Thioacylating intermediates as metabolites of S-(1,2dichloroviny1)-L-cysteine and S-(1,2,2-trichlorovinyl)-~-cysteine formed by cysteine conjugate 0-lyase. Chem. Res. Toxicol. 1,175178. (13) Commandeur, J. N. M., De Kanter, F. J. J., and Vermeulen, N. P. E. (1989) Bioactivation of the cysteine-S-conjugate and mercapturic acid of tetrafluoroethylene to acylating reactive intermediates in the rat: Dependence of activation and deactivation activities on acetyl coenzyme A availability. Mol. Pharmacol. 36, 654-663. (14) Commandeur, J. N. M. (1991) Molecular Mechanisms of Chemically Induced NeDhrotoxicitv. ” . Ph.D. Thesis. Universitv of Leiden. The Netherlands. (15) Dekant. W.. Lash. L. H.. and Anders. M. W. (1987) Bioactivation mechanism of the cytotoxic and nephrotoxic S-conjugate S-(Z-chloro1,1,2-trifluoroethy1)-L-cysteine.Proc. Natl. Acad. Sci. U.S.A. 84, 7443-7447. (16) Hayden, P. J., Welsh, C. J., Yang, Y., Schaefer, W. H., Ward, A. J. I., and Stevens, J. L. (1992) Formationofmitochondrialphospholipid adducts by nephrotoxic cysteine conjugate metabolites. Chem.Res. T O X ~ C5,O231-237. ~. (17) Hayden, P. J., Yang, Y., Ward, A. J. I., Dulik, D. M., McCann, D. J., and Stevens, J. L. (1991) Formation of difluorothionoacetylprotein adducts by S-(1,1,2,2-tetrafluoroethyl)-~-cysteine metabolites: Nucleophilic catalysis of stable lysyl adduct formation by histidine and tyrosine. Biochemistry 30, 5935-5943. (18) Harris, J. W.,Dekant, W.,andAnders,M. W. (1992)Invivodetection and characterization of protein adducts resulting from bioactivation of haloethene cysteine S-conjugates by I9F NMR: Chlorotrifluoroethene and tetrafluoroethene. Chem. Res. Toxzcol. 5 , 34-41. (19) Hayden, P. J., Ichimura, T., McCann, D. J., Pohl, L. R., and Stevens, J. L. (1991) Detection of cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies
Fisher et al. raised against halothane metabolite adducts. J. Bid. Chem. 266, 18415-18418. (20) Hayden, P. J., and Stevens, J. L. (1990) Cysteine conjugate toxicity, metabolism, and binding to macromolecules in isolated rat kidney mitochondria. Mol. Pharmacol. 37,468-476. (21) Dohn, D. R., Quebbeman, A. J., Borch, R. F., and Anders, M. W. (1985) The enzymatic reaction of CTFE with glutathione: 19F Evidence for stereochemical control of the reaction. Biochemistry 24,5137-5143. (22) Stevens, J. L., Robbins, J. D., and Byrd, R. A. (1986) A Purified cysteine conjugate 0-lyase from rat kidney cytosol. J. Bid. Chem. 261, 15529-15537. (23) Metzler, D. E., Ikawa. M.. and Snell. E. E. (1954) A General mechanism for vitamin B6-catalyzed reactions. J. Am. Chem. SOC. 76, 648-652. (24) Schnaitman, C., and Greenwalt, J. W. (1968) Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J. Cell Bioi. 38, 158-175. (25) Satoh, H., Fukuda, Y., Anderson, D. K., Ferrans, V. J., Gillette, J. R., and Pohl, L. R. (1985) Immunological studies on the mechanism of halothane-induced hepatoxicity: Immunohistochemical evidence of trifluoroacetylated hepatocytes. J. Pharmacol. Exp. Ther. 233, 857-862. (26) Kenna, J. G., Neuberger, J., and Williams, R. (1987) Identification by immunoblotting of three halothane-induced liver microsomal polypeptide antigens recognized by antibodies in sera from patients with halothane-associated hepatitis. J. Pharmacol. Exp. Ther.242, 733-740. (27) Kenna, J. G.,Satoh, H., Christ,D. D., andPohl,L. R. (1988)Metabolic basis for a drug hypersensitivity-Antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothane. J. Pharmacol. Exp. Ther. 245, 1103-1109. (28) Derr, R. F., and Schultze, M. 0. (1963) The Metabolism of W(l,Z-dichlorovinyl)-L-cysteine in the rat. Biochem. Pharmacol. 12, 465-474. (29) Anderson, P. M., and Schultze, M. 0. (1965) Interaction of S-(1,2dichloroviny1)-L-cysteinewith proteins. Arch. Biochem. Biophys. 109,615-621. (30) Bhattacharya, R. K.,andSchultze, M. 0.(1967) Enzymesfrom bovine and turkey kidneys which cleave S-(l,Z-dichlorovinyl)-L-cysteine. Comp. Biochem. Physiol. 22, 723-735. (31) Wolfgang, G. H. I., Gandolfi, A. J., Stevens, J. L., and Brendel, K. (1989) N-Acetyl&( 1,2-dichloroviny1)-L-cysteine produces a similar toxicity to S-(1,2-dichlorovinyl)-~-cysteine in rabbit renal slices: Differential transport and metabolism. Toxicol. Appl. Pharmacol. 101, 205-219. (32) Stevens, J. L., Hayden, P., and Taylor, G. (1986) The Role of glutathione conjugate metabolism and cysteine conjugate @-lyasein the mechanism of S-cysteine conjugate toxicity in LLC-PK1 cells. J. Bid. Chem. 261, 3325-3332. (33) Wallin, A., Jones, T. W., Vercesi, A. E., Cotgreave, I., Ormstad, K., and Orrenius, S. (1987) Toxicity of S-pentachlorobutadieny1-Lcysteine studied with isolated rat renal cortical mitochondria. Arch. Biochem. Biophys. 258, 365-372. (34) Jones, T. W., Wallin, A., Thor, H., Gerdes, R. G., Ormstad, K., and Orrenius, S. (1986) The Mechanism of pentachlorobutadienylglutathione nephrotoxicity studied with isolated rat renal epithelial cells. Arch. Biochem. Biophys. 251, 504-513. (35) Stonard, M. D., and Parker, V. H. (1971) 2-Oxoacid dehydrogenases of rat liver mitochondria as the site of action of S-(l,Z-dichlorovinyl)L-cysteine and S-(1,2-dichlorovinyl)-3-mercaptopropionic acid. Biochem. Pharmacol. 20, 2417-2427. (36) Schnellmann, R. G., Lock, E. A., and Mandel, L. J. (1987) A Mechanism of S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-~-cysteine toxicity to rabbit renal proximal tubules. Toxicol. Appl. Pharmacol. 90, 513-521. (37) Hargus, S. J., and Anders, M. W. (1991) Immunochemical detection of covalently modified kidney proteins in S-(1,1,2,2-tetrafluoroethyl)L-cysteine-treated rats. Biochem. Pharmacol. 42, 17-20.
