Chem. Res. Toricol. 1990, 3, 212-218
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Identification of N-Acetyl(2,2-dichlorovinyl)- and N-Acetyl( 1,2-dichlorovinyl)-~-cysteineas Two Regioisomeric Mercapturic Acids of Trichloroethylene in the Rat J. N. M. Commandeur and N. P. E. Vermeulenh Department of Pharmacochemistry, Division of Molecular Toxicology, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Received November 3, 1989
T h e regioselectivity of glutathione conjugation to trichloroethylene (TRI) and the metabolism of its cysteine and N-acetyl-L-cysteine conjugates were investigated in the rat. Intraperitoneal (ip) administration of TRI t o rats a t a dose of 400 mg/kg resulted in excretion in urine of small amounts of the two distinct regioisomers N-acetyl-S-(1,Zdichlorovinyl)- cysteine (1,2-DCV-NAC) and N-acetyl-S-(2,2-dichlorovinyl)-~-cysteine (2,2-DCV-NAC). T h e vicinal (vic) isomer was excreted in a 2 times higher amount (16 nmol) than the geminal (gem) isomer (8 nmol). Intraperitoneal administration of a 1:l mixture (2.5 Fmollkg each) of the two regioisomers of S-(dichloroviny1)-L-cysteine(DCVC) t o the rat resulted in excretion of the corresponding mercapturic acids in urine, the main fractions being excreted within 8 h after administration. T h e gem-dichlorovinyl isomer appeared to be acetylated to a higher extend than the 1,2-dichlorovinyl isomer; 73 % vs 50 % of the dose administered. Intraperitoneal administration of a 1:l mixture (12.5 pmol/kg each) of the two regioisomers of N-(trideuterioacety1)-S-(dichloroviny1)-L-cysteine (DCV-NAC-d3) resulted in excretion of both deuterium-labeled and unlabeled mercapturic acids in urine. T h e vic isomer was excreted unchanged a t a significantly higher percentage, 34% of dose (Le., still deuterium labeled), than the gem isomer, 17% of the dose. This suggests less efficient metabolism of the vic isomer when compared to the gem isomer. Both regioisomers of DCV-NAC-d3 were excreted in urine unlabeled a t 40% of the dose, which indicates that for both isomers deacetylation, followed by reacetylation (resulting in unlabeled DCV-NAC), is an important metabolic pathway. T h e present data are indicative for different rates of in vivo metabolism of two regioisomers of DCVC and DCV-NAC. Experiments with commercial acylase I also showed a pronounced stereoselective deacetylation: 2,2-DCV-NAC was deacetylated a t a 50-fold higher rate than 1,2-DCV-NAC. From the present in vivo data, however, it cannot be concluded which of the enzymes involved in metabolism of cysteine conjugates and mercapturic acids are responsible for the observed stereoselective metabolism in vivo.
Introduction Trichloroethylene (TRI) is widely used as a solvent in metal cleaning. Other industrial applications are drycleaning, printing, production of printing ink, and extraction. Although TRI is readily degradable in the atmosphere (estimated tlI2.= 70 h), this compound appears to be an ubiquitous environmental pollutant due to the large-scale emissions. Toxicological studies demonstrated that TRI is a suspected carcinogen. Mice treated chronically with TRI showed a high incidence of hepatocellular carcinoma (1-3). Data on the metabolism of TRI indicated that TRI is bioactivated by oxidative metabolism by the hepatic mixed-function oxidase system ( 4 , 5). Some of the metabolites, glyoxylate, carbon monoxide, and formic acid, are supposed to be derived from an intermediate electrophilic epoxide (6). Other possible reactive products such as chloral, formyl chloride, and dichloroacetyl chloride, however, probably result from a pre-epoxide transition state involving the binding of TRI to the activated oxygen of cytochrome P-450 (6). In male rats chronically exposed to TRI an increase in kidney tumours was seen, next to a high frequency of toxic
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nephrosis (2,3). In urine of rats exposed to a single dose of TRI very small amounts of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCV-NAC)have been detected (7). The corresponding cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCVC),has been shown to be mutagenic (8,9) and therefore has been associated with the small incidence of renal tumors. Three decades ago, 1,2DCVC already has been identified as the toxic factor in TRI-extracted soybean oil meal, which induced aplastic anemia in cattle (10). Although in principle, different regioisomers of DCVC are possible (Figure l),DCVC as synthesized according to the method of McKinney (10) is generally thought to be in a trans configuration (I, Figure 1). The chemical mechanism of nucleophilic substitution of a thiolate anion with chloroethylenes in dry liquid ammonia is supposed to be an initial base-catalyzed trans dehydrohalogenation reaction to yield an acetylenic intermediate, followed by a nucleophilic addition to the acetylene moiety (19, 20). trans-1,2-Dichloroethylene does not react with thiolates in liquid ammonia, because the trans dehydrohalogenation step is not possible with this compound; however, cis1,2-dichloroethylene reacted very rapidly with thiolate anions, yielding a disubstituted product which is consistent with the proposed mechanism under these conditions (19). Reaction of TRI with an excess of thiolate anions only 0 1990 American Chemical Society
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 213
Mercapturic Acids of Trichloroethylene
(Brussels, Belgium). N-Acetyl-S-(2,2-dichloro-l,l-difluoroethyl)-L-cysteine (DCDFE-NAC) and S-(2,2-dichloro-l,l-difluoroethy1)-L-cysteine (DCDFE-CYS)were prepared as described previously (27). Acylase I was purchased from Sigma Chemical Co. (St. Louis, MO).
Syntheses. N-Acetyl-S-(1,2-dichlorovinyl)-~-cysteine (l,$-DCV-NAC). 1,2-DCV-NACwas synthesized according to the method of McKinney (IO). This involves a reaction between Cl
SR
mans- 1 ,Z-DCV-isomer
0
cis- 1 ,Z-DCV-isomer
0
2.2-DCV-isomer
(m)
Figure 1. Possible regioisomers resulting from nucleophilic substitution of thiol compounds to trichloroethylene: (route A) base-catalyzed elimination/addition mechanism; (route B) addition/elimination mechanism. The trans- and cis-l,2-DCV conjugates are both vicinal (vic) isomers; the 2,2-DCV conjugate is the geminal (gem) isomer.
results in a monosubstituted conjugate. The fact that substitution of a second chlorine only occurred at a very slow rate was suggested to be indicative for the trans configuration of the monoconjugate: only in this regioisomer a trans dehydrohalogenation step would not be possible. However, a decisive proof for the assigned conformation still remains to be given. Nucleophilic substitution of vinyl halides with alkanethiolate anions in aprotic dipolar solvents, such as hexamethylphosphoramide, dimethyl sulfoxide (DMSO), or dimethylformamide, however, does not proceed via an acetylenic intermediate. Instead, it proceeds by an addition/elimination mechanism (21,22). In these solvents, both cis- and trans-1,2-dichioroethylenereact with thiolate anions, yielding S-conjugates with retention of the cis and trans configuration, respectively. In excess of thiolate, all chlorine atoms in TRI are substituted. Therefore, in contrast to the reaction in liquid ammonia, in aprotic solvents the nucleophilic vinylic substitution in TRI by a thiol compound in principle can lead to the three different isomeric monoadducts, shown in Figure 1. Two different methods of synthesis of the N-acetyl+ cysteine conjugate of TRI, DCV-NAC, have been described previously. By Dekant et al. (7), DCV-NAC was synthesized by acetylation of 1,2-DCVC, which was synthesized according to the method of McKinney (IO). Vamvakas et al. (14) synthesized DCV-NAC directly by reacting N-acetyl-L-cysteine with TRI in DMSO in the presence of an organic base. Although in the latter report a t r a n s 1,2-DCV moiety was suggested to occur, the reported electron impact mass spectra of the corresponding methyl ester of this mercapturic acid was significantly different from that described by Dekant et al. (7). The former product had a significant larger molecular ion (21% of the most abundant fragment m / z 88) than the latter (
