Chem. Res. Toxicol. 1993,6, 662-668
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Fatty Acid 8-Oxidation-DependentBioactivation of Halogenated Thiaalkanoic Acids in Isolated Rat Hepatocytes Michael E. Fitzsimmons and M. W. Anders* Department of Pharmacology, University of Rochester School of Medicine & Dentistry, 601 Elmwood Avenue, Rochester, New York 14642 Received May 20,1993
5,6-Dichloro-4-thia-5-hexenoic acid (DCTH), the desamino analog of the nephrotoxic cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine,is toxic to liver and kidney mitochondria. The mechanism by which DCTH produces mitochondrial dysfunction has not been defined. The objective of the present experiments was to test the hypothesis that DCTH is bioactivated by the mitochondrial fatty acid B-oxidation system to cytotoxic intermediates. Incubation of isolated rat hepatocytes with DCTH produced a time- and concentration-dependent decrease in cell acid was also cytotoxic, viability. The even-chain, elongated analog 7,8-dichloro-6-thia-7-octenoic whereas the odd-chain-length analogs 6,7-dichloro-5-thia-6-heptenoic acid and 8,9-dichloro-7thia-8-nonenoic acid were not. Sodium benzoate reduced the cytotoxicity of DCTH, indicating a role for coenzyme A in the bioactivation of DCTH. DCTH decreased cellular ATP concentrations, the cellular energy charge, and cellular glutathione concentrations; these changes preceded the decrease in cell viability, indicating that mitochondrial dysfunction may be an acid and early event in DCTH-induced cytotoxicity. 6-Chloro-5,5,6-trifluoro-4-thiahexanoic 5,6,7,8,8-pentachloro-4-thia-5,7-octadienoic acid were also cytotoxic in isolated hepatocytes, acid was not. These data are consistent with the whereas 4-(2-benzothiazolyl)-4-thiabutanoic hypothesis that the mitochondrial fatty acid @-oxidationsystem is involved in the bioactivation of DCTH and that mitochondria may be important cellular targets in DCTH-induced cytotoxicity. Introduction Previous studies on the bioactivation mechanism of the nephrotoxic S-conjugate S-(1,2-dichlorovinyl)-L-cysteine showed that the decarboxylated analog S-(1,2-dichloroviny1)cysteamine was not toxic, whereas the desamino acid [DCTH: Figure analog 5,6-dichloro-4-thia-5-hexenoic 1;also named S-(1,2-dichlorovinyl)-3-mercaptopropionic acid] was highly toxic (1). DCTH is a potent inhibitor of rat liver and kidney mitochondrial respiration and inhibits 2-oxoacid dehydrogenases (2, 3). Although the bioactivation mechanism of S-(1,2-dichlorovinyl)-L-cysteinehas been elucidated (4-3, the bioactivation mechanism of DCTH has not been defined. The objective of the present study was to elucidate the bioactivation mechanism of DCTH. The hypothesis tested was that DCTH is bioactivated by enzymes of the fatty acid @-oxidationsystem to cytotoxic intermediates (Figure 2). Initial coenzyme A thioester formation with DCTH (1) may be catalyzed by fatty acid acyl-CoA synthetase (EC 6.2.1.3) to afford 5,6-dichloro-4-thia-5-hexenoyl-CoA (2). CoA thioester 2 may be a substrate for the fatty acid medium-chainacyl-CoA dehydrogenase (EC 1.3.99.3), the first enzyme in the fatty acid @-oxidationsystem, to give 5,6-dichloro-4-thia-2,5-hexadienoyl-CoA (3), which may be biotransformed by enoyl-CoA hydratase (EC 4.2.1.17) to the thiohemiacetal 5,6-dichloro-4-thia-3-hydroxy-5hexenoyl-CoA (4). Thiohemiacetal 4 would be expected to eliminate malonylsemialdehyde-CoA 6 and 1,2-dichlo-
* To whom correspondence and reprint requests should be addressed. Phone: (716)275-1681;Fax: (716)244-9283. * Abstract published in Advance ACS Abstracts, September 1,1993. 1 Abbreviation: DCTH,5,6-dichloro-4-thia-5-hexenoic acid.
CI 6,7-DicHom-5-thiab-heptemicacid
7.6-DichbroG-thia-7omicacid
ci
CI
5,6,7,8,S-Pentac~m-4-ttia-5,7-cctadiemic acid
4-(2-Benzottiazo31)-4-ttiabutamicacid
HO
CI
8,9-DicMm-7-thiaB-m~mic acid
Figure 1. Structures of thiaalkanoic acids.
roethenethiol 5, whose formation is associated with the toxicity of S-(1,2-dichlorovinyl)-L-cysteine(5, 8). The fatty acid @-oxidationsystem is involved in the biotransformation of various xenobiotics. For example, even-chain-length w-fluorocarboxylic acids are biotransformed to fluoroacetic acid by the fatty acid @-oxidation system (9). Fluoroacetic acid, when converted to fluorocitric acid, is a potent inhibitor of aconitase (10). 4-Thiaand 4-oxaoctanoic acid are biotransformed by mediumchain acyl-CoA dehydrogenase and enoyl-CoA hydratase to butanethiol and butanol, respectively, and to malonylsemialdehyde-CoA (11, 12).
0893-228~/93/2706-0662$04.00/00 1993 American Chemical Society
Fatty Acid j3-Oxidation of 4-Thiaalkanoic Acids
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50 mL of methanol, and the mixture was heated at reflux for 30 min. The methanol was removed in uacuo, and 50 mL of ice-cold W S A O H water was added. The reaction mixture was adjusted to pH 5 I CI 1 with 1N NaOH and was then extracted with ether. The ether CoASH layer was dried with anhydrous sodium sulfate, and the ether AcyCCoA Synthetase was removed in uacuo. Methyl 4-bromobutanoate was used without further purification. Methyl 6-Phenyl-5-thiahexanoate. Methyl 4-bromoG S . A S C o A butanoate (3.2 g, 18 mmol) was added to a suspension of lithium CI 2 hydride (0.14 g, 18mmol) in 75 mL of dry tetrahydrofuran with stirring at room temperature. Benzyl mercaptan (2.1 mL, 18 Acyl-CoA Dehydrogenase mmol), in 10 mL of dry tetrahydrofuran, was slowly added to the FADH2 reaction mixture, and the reaction mixture was stirred at room CI 0 temperature until HOevolution ceased. The reaction mixture was filtered to remove unreacted lithium hydride, and the solvent H " S 4 S C o A was removed in uacuo. The resulting oil was dissolved in ether, 61 3 the ether was extracted with dilute NaOH, and the phases were separated. The ether was removed in uacuo. Methyl 6-phenylEnoyl-CoA Hydratase 5-thiahexanoatewas purified by silica gel column chromatography (hexane/ethyl acetate, 9:l). The purified product gave a single spot by TLC (hexane/ethyl acetate, 9:l). 6-Phenyl-5-thiahexanoic Acid. Methyl 6-phenyl-5-thiahexanoate was hydrolyzed with 0.5 M NaOH under reflux conditions for 20 min. The aqueous solution was acidified to pH 1 with concentrated HC1 and was extracted with ether. The phases were separated, the ether layer was dried with anhydrous sodium sulfate, and the ether was removed in uacuo. The product thus obtained was used without further purification. 6,7-Dichloro-5-thia-6-heptenoic Acid (Figure 1). 6-PhenylCI 5-thiahexanoic acid (0.6 g, 3 mmol) was dissolved in 200 mL of 5 6 liquid ammonia at -80 "C, and sodium metal was added until the Figure 2. Proposed bioactivation mechanism of 5,bdichlorodeep blue color persisted. Trichloroethene (0.9 mL, 10 mmol) 4-thiad-hexenoic acid. 1,5,6-dichlor0-4-thia-5-hexenoic acid; 2, was added, and the reaction was allowed to warm to room 5,6-dichloro-4-thia-5-hexenoyl-CoA; 3, 5,6-dichloro-4-thia-2,5hexadienoyl-CoA; 4, 5,6-dichloro-4-thia-3-hydroxy-5-hexenoyl- temperature with stirring overnight. The ammonia was evaporated at room temperature, 50 mL of water was added, and the CoA; 5, 1,2-dichloroethenethiol; 6, malonylsemialdehyde-CoA. resulting mixture was acidified to pH 1 with 1 N HC1. The Data are presented herein indicating that DCTH and solution was extracted with ether, the ether layer was dried over other halogenated 4-thiaalkanoic acids are metabolized anhydrous sodium sulfate, and the ether was removed in uacuo. The resulting oil was purified by silica gel chromatography by the enzymes of the fatty acid j3-oxidation system and (hexane/ethyl acetate/acetic acid, 90:103) to yield 150 mg (15% that bioactivation by this metabolic pathway may account of theoretical from the initial methyl 4-bromobutanoate synthetic for their observed cytotoxicity. step) of 6,7-dichloro-5-thia-6-heptenoic acid 1H NMR (CDCl3) 6 1.9 (m, 2 H), 2.5 (t, 2 H), 3.0 (t, 2 H), 6.5 (s, 1 H). A sample Experimental Procedures of 6,7-dichloro-5-thia-6-heptenoic acid was treated with diazomethane to give the methyl ester; GUMS analysis gave an M+ Materials. Male Long-Evansrata (150-300 g) were purchased ion at m/z 228 and a chlorine isotope pattern consistent with the from Charles River Laboratories (Wilmington, MA). Mercapproposed structure. topropionic acid, trichloroethene, 4-bromobutanoic acid, 5-bro(C) 7,8-Dichloro-6-thia-7-octenoicAcid and 8,9-Dichloromopentanoic acid, 6-bromohexanoicacid, benzyl mercaptan, and 7-thiad-nonenoic Acid (Figure 1). These chain-elongated 2-chlorobenzothiazole were purchased from Aldrich Chemical analogs were synthesized with 5-bromopentanoic acid and Co. (Milwaukee, WI). Sodium benzoate, octanoic acid, ATP, 6-bromohexanoic acid as starting materials, rather than 4-broADP, and AMP were purchased from Sigma Chemical Co. (St. mobutanoic acid. The yield was 200 mg (20.4%) and 340 mg Louis, MO). Chlorotrifluoroethene was purchased from PCR (33.8% ) for 7,8-dichloro-6-thia-7-octenoic acid and 8,9-dichloroInc. (Gainesville, FL). 7-thia-8-nonenoic acid, respectively. 7,8-Dichloro-6-thia-7Instrumental Analyses. lH NMR spectra were acquired octenoic acid lH NMR (CDCl3) 6 1.8 (multiplet due to the with a Bruker WP270 spectrometer operating at 270.13 MHz overlapping chemical shifts of the methylene groups, 4 H), 2.4 and are reported in ppm downfield from tetramethylsilane. Mass (t, 2 H), 2.9 (t, 2 H), 6.4 ( 8 , 1 H). A sample of 7,8-dichloro-6spectra were acquired with Hewlett-Packard 5880A gas chrothia-7-octenoic acid was treated with diazomethane to give the matograph, which was fitted with a 250 X 0.02 cm (i.d.) fusedmethyl ester; GUMS analysis gave an M+ ion at m/z 242 and a silica capillary column coated with cross-linked methyl silicone, chlorine isotope pattern consistent with the proposed structure. coupled to a Hewlett-Packard 5970 mass-selective detector. The 8,9-Dichloro-7-thia-8-nonenoic acid: 'H NMR (CDCl3) 6 1.5 (m, carrier gas (helium) flow rate was 1mL/min. The initial column 2 H), 1.7 (m, 4 H), 2.4 (t,2 H), 2.9 (t,2 H), 6.4 (s, 1H). A sample temperature was held at 50 "C for 3 min and was then increased of 8,9-dichloro-7-thia-8-nonenoic acid was treated with diazat 10 OC/min to 250 OC; the column temperature was held at 250 omethane to give the methyl ester; GC/MS analysis gave an M+ "C for 10 min. HPLC analyses were performed with Gilson 305/ ion at m/z 256 and a chlorine isotope pattern consistent with the 306 pump system and a Perkin-Elmer LC-235 diode-array proposed structure. detector. (D) 6-Chloro-5,5,6-trifluoro-4-thiahexanoic Acid (Figure Syntheses. (A) 5,6-Dichloro-4-thia-5-hexenoic Acid (Fig1). 3-Mercaptopropanoic acid (20 g, 0.19 mol) was dissolved in ure 1). This compound was prepared as described by McKinney 200 mL of 75% ethanol/water, and the reaction mixture was et al. (13). adjusted to pH 10 with concentrated ammonium hydroxide. The (B) 6,7-Dichloro-5-thia-6-heptenoic Acid (Figure 1). Methreaction flask,fitted with a balloon, was filled with chlorotriyl 4-bromobutanoate was prepared by mixing 4-bromobutanoic fluoroethene, and the reaction mixture was stirred at room acid (5.0 g, 30 mmol) and concentrated sulfuric acid (0.5 mL) in
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Fitzsimmons and Anders
Chem. Res. Toxicol., Vol. 6, No. 5, 1993
temperature for 6 h. Concentrated ammonium hydroxide was added periodically to maintain a pH of 10-11. Chlorotrifluoroethene was added as needed throughout the course of the reaction through a rubber septum. When no more chlorotrifluoroethene was consumed, the ethanol was removed in uucuo, and the resulting aqueous solution was adjusted to pH 1with 1 N HCl. The aqueous layer was extracted with ether, and the ether layer was dried over anhydrous sodium sulfate. The ether was removed in uucuo to yield 4.8 g (11%)of 6-chloro-5,5,6trifluoro-4-thiahexanoicacid. 'H NMR (CDCl3) 6 2.8 (t,2 H), 3.1 (t, 2 H), 6.2 (m, 1 H). A sample of 6-chloro-5,5,6-trifluoro4-thiahexanoic acid was treated with diazomethane to give the methyl ester; GC/MS analysis gave a single peak corresponding to methyl 6-chloro-5,5,6-trifluoro-4-thiahexanoate with an M+ ion at m/z 236. (E) 4-(2-Benzothiazolyl)-4-thiabutanoicAcid (Figure 1). 3-Mercaptopropionic acid (5.3 g, 50 mmol) was added dropwise to a suspension of lithium hydride (0.8 g, 100 mmol) in 150 mL of dry dimethylformamide with stirring at room temperature. When Hz evolution ceased, 2-chlorobenzothiazole (6.5 mL, 50 mmol) was added, and the reaction mixture was stirred overnight at room temperature. Water (200 mL) was added to the reaction mixture, and the aqueous layer was adjusted to pH 2 with 1 M KHSO,. The mixture was extracted with ether, and the ether layer was separated and then extracted with several portions of water. The ether was dried over anhydrous sodium sulfate and concentrated in uucuo to yield 1.73 g (14%) of 442-benzothiazolyl)-4-thiabutanoic acid: mp 148-149 "C; lH NMR (CDCls) 6 4.2 (t, 2 H), 4.9 (t, 2 H), 9.1 (m, 4 H). A sample of 442benzothiazolyl)-4-thiabutanoic acid was treated with diazomethane to give the methyl ester; GUMS analysis gave a single peak corresponding to methyl 4-(2-benzothiazolyl)-4-thiabutanoate with an M+ ion at m/z 253. (F)5,6,7,8,8-Pentachloro-4-t hia-5,7-octadienoicAcid (Figure 1). 3-Mercaptopropionic acid (1.0 g, 9.4 mmol) waa added dropwise to a suspension of lithium hydride (150mg, 18.9 mmol) in 10 mL of dry dimethylformamide with stirring at room temperature. When Hz evolution ceased, hexachlorobutadiene (5.0 mL, 31.5 mmol) was added, and the reaction mixture was stirred overnight at room temperature. Water was added to the reaction mixture, and the aqueous layer was adjusted to acidic pH with 1 N HC1. The mixture was extracted with ether, and the ether layer was separated and then extracted with several portions of water. The ether was dried over anhydrous sodium sulfate and concentrated in uucuo to yield 73 mg (2.4%) of 5,6,7,8,8pentachloro4-thia-5,7-&dienoic acid lH NMR (CDCq) 6 2.7 (t, 2 H), 3.2 (t, 2 H). A sample of 5,6,7,8,8-pentachloro4-thia-5,7-octadienoicacid was treated with diazomethane to give the methyl ester; GUMS analysis gave a single peak correspondingto methyl 5,6,7,8,8-pentachloro-4-thia-5,7-octadienoate with an M+ ion at m/z 342 and an isotopic cluster pattern indicating the presence of 5 chlorine atoms. Hepatocyte Isolation and Treatment. Hepatocytes were isolated from male, Long-Evans rata (150-300 g) by the collagenase perfusion method of Moldbus et al. (14). Hepatocyte viability was determined by trypan blue exclusion. Hepatocyte suspensions with a viability greater than 90% were diluted to 1.0 X 108cells/mL with Krebs-Henseleit buffer (118mM NaC1,4.80 mM KC1,0.95 mM KHzPOr, 1.20 mM MgS04.7Hz0, 23.8 mM NaHC03, and 3.40 mM CaC12, adjusted to pH 7.3-7.35 by bubbling with 95% 02/5% C02) supplemented with 25 mM 442-hydroxyethy1)-1-piperazineethanesulfonic acid and 2 % bovine serum albumin. All incubations were performed in an atmosphere of 95 % 02/5%COz at 37 "C in 25-mL Erlenmeyer flasks fitted with rubber septa. All compounds except 4-(2-benzothiazolyl)-4thiabutanoic acid were dissolved in Krebs-Henseleit buffer, the pH was adjusted to 7.0 with 5 N NaOH, and the compounds were added to the incubation mixtures through the rubber septa. 442Benzothiazolyl)-4-thiabutanoicacid was dissolved in dimethyl sulfoxide and was added to the incubation mixtures; the final concentration of dimethyl sulfoxide was 1% (v/v).
0
1
2
3
Time (h)
Figure 3. Concentration and time dependence of 5,6-dichloro4-thia-5-hexenoic acid (DCTH)-induced cytotoxicity in isolated hepatocytes. Hepatocytes (1 X 10s/mL) were incubated with 0
(O),50(+),100(r),or200pM(m)DCTH.Viabilitywaameaaured
*
by trypan blue exclusion. Results are shown as means SD, n = 3. 'Statistically significant (p
