Metabolism of Tetrachloroethene in Rats: Identification of N. epsilon

Gerhard Birner, Carsten Richling, Dietrich Henschler, M. W. Anders, and Wolfgang Dekant. Chem. Res. Toxicol. , 1994, 7 (6), pp 724–732. DOI: 10.1021...
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Chem. Res. Toxicol. 1994, 7, 724-732

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Articles Metabolism of Tetrachloroethene in Rats: Identification of Nc-(Dichloroacetyl)-L-lysine and Nc-(Trichloroacetyl)-L-lysineas Protein Adducts Gerhard Birner,' Carsten Richling,?Dietrich Henschler, M. W. Anders,* and Wolfgang Dekant*lf Znstitut fur Toxikologie, Universitat Wiirzburg, Versbacherstrasse 9, 97078 Wiirzburg, FRG, and Department of Pharmacology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642 Received June 21, 1994@

Tetrachloroethene causes renal tumors in male rats after inhalation exposure. Tetrachloroethene is metabolized by cytochrome P-450 and by glutathione conjugation. Cytochrome P-450-dependent oxidation results in the formation of trichloroacetyl chloride, which may acylate cellular nucleophiles; glutathione conjugation results in the formation of S-(1,2,2trichlorovinyl)glutathione,which is metabolized to the corresponding cysteine S-conjugate. S-(1,2,2-Trichlorovinyl)-~-cysteine is activated by renal cysteine conjugate P-lyase to give dichlorothioketene. Covalent binding of this electrophile is presumably responsible for the renal toxicity of tetrachloroethene. In this report, we demonstrate the formation of protein adducts formed from tetrachloroethene using SDS-PAGE and immunochemical detection with rabbit anti-trifluoroacetyl serum. This serum recognizes dichloroacetylated rabbit serum albumin prepared by chemical modification of rabbit serum albumin with S-ethyl dichlorothioacetate and exhibited a high specificity for Ne-(dichloroacety1)-L-lysineresidues in proteins as shown by competitive ELISA. In the liver of [14Cltetrachloroethene-treatedrats, the antibody recognized several modified proteins in microsomes. A protein adduct in rat liver identified by G C N S after hydrolysis was Ne-(trichloroacetyl)-L-lysine. Western blots of renal fractions from rats treated with [l4C1tetrachloroethene (200mgkg) or S-(1,2,2-trichlorovinyl)-~-cysteine (40 pmolkg, iv) suggested the presence of modified mitochondrial and cytosolic proteins; no modified proteins were detected in microsomes. Proteins of identical molecular weight were modified by tetrachloroethene and by S-(1,2,2-trichlorovinyl)-~-cysteine in vivo. Radioactivity derived from [l4C1tetrachloroethene comigrated in SDS-PAGE with the proteins in kidney mitochondria and cytosol recognized by the antibody. Incubation of renal mitochondria and cytosol with S-(1,2,2-trichlorovinyl)-~-cysteine in vitro also gave modified proteins; no modified proteins were found in incubations containing (aminooxy)acetic acid, an inhibitor of ,&lyase. In renal mitochondria incubated with S-(1,2,2-trichlorovinyl)-~-cysteine, N'-(dichloroacetyl)L-lysine was identified as a modified amino acid by GCNS/SIM after protein hydrolysis. Ne(Dichloroacety1)-L-lysinewas predominantly present in renal proteins after administration of tetrachloroethene to rats. The detection of dichloroacetylated proteins in kidney after tetrachloroethene exposure further supports a role for /3-lyase in the chronic renal toxicity of tetrachloroethene.

Introduction Tetrachloroethene is a widely used solvent and, due to its volatility and resistance to degradation, a widely distributed environmental contaminant. Tetrachloroethene shows only low acute toxicity. Long-term bioassays of tetrachloroethene for carcinogenicity have revealed increases in liver tumors in mice and a small, but significant, increase in renal carcinoma formation in male rats ( I -3). Tetrachloroethene toxicity is due to metabolic activation reactions (Figure 1). Two different bioactiva-

* Please address correspondence to this author at the Institut fiir Toxikologie, Universitat Wiirzburg, Versbacher Strasse 9, 97078 Wiirzburg, FRG. + Universitat Wurzburg. University of Rochester. Abstract published in Aduance ACS Abstracts, September 15,1994.

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tion mechanisms have been elucidated (4- 7). Tetrachloroethene is oxidized by cytochrome P-450 to give the intermediate trichloroacetyl chloride, which is hydrolyzed to yield trichloroacetic acid, the major metabolite excreted in urine ( 4 9 ) . Trichloroacetyl chloride reacts with amino groups in macromolecules to form trichloroacetamides. The second pathway, catalyzed by glutathione S-transferases, yields S-(1,2,2-trichlorovinyl)glutathioneas initial product (Figure 1). Glutathione conjugation of tetrachloroethene represents a bioactivation reaction and has been implicated in the nephrotoxicity and possible renal carcinogenicity of tetrachloroethene (6, 7, 10). S-(1,2,2-Trichlorovinyl)glutathione formed in the liver is excreted with bile (11) and further metabolized by y-glutamyltransferase and dipeptidases to S-(1,2,2trichloroviny1)-L-cysteine, which is acetylated t o give

0893-228x/94/2707-0724$04.50/0 0 1994 American Chemical Society

Chem. Res. Toxicol., Vol. 7,No. 6, 1994 725

Protein Adducts of Tetrachloroethene in Rats

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P-450

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Lysine In proteins

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Figure 1. Bioactivation of tetrachloroethene (1) by cytochrome P-450 and by glutathione conjugation. Formation of reactive intermediates by cysteine conjugate B-lyase: 2, S-(1,2,2-trichlorovinyl)glutathione; 3, S-(1,2,2-trichlorovinyl)-~-cysteine; 4, dichlo6,Ne-(dichloroacety1)-L-lysinein proteins; 7 , trichloroacetyl chloride; 8, rothioketene; 5, N-acetyl-&( 1,2,2-trichlorovinyl)-~-cysteine; trichloroacetic acid; 9, Nf-(trichloroacetyl)-L-lysinein proteins.

N-acetyl-S-( 1,2,2-trichlorovinyl)-~-cysteine, a minor urinary metabolite of tetrachloroethene in rats (6). SConjugates are accumulated in the kidney and transformed to S-(1,2,2-trichlorovinyl)-~-cysteine. S-(1,2,2Trichloroviny1)-L-cysteineis mutagenic in several strains of Salmonella typhimurium and is toxic to rat kidney cells (11,12). It is cleaved by cysteine conjugate P-lyase to yield pyruvate, ammonia, and dichlorothioketene, which is presumed to be the ultimate metabolite responsible for the mutagenic and nephrotoxic effects (13). Dichlorothioketene may react with DNA and proteins. Protein modifications in the kidney have not been described with tetrachloroethene or S-(1,2,2-trichloroviny1)-L-cysteine. However, protein binding of the structural analogues S-(2-chloro-1,1,2-trifluoroethyl)-~-cysteine and S-(1,1,2,2-tetrafluoroethyl)-~-cysteine has been studied with a polyclonal antibody developed to detect trifluoroacetylated proteins formed from halothane (1416). We used this antibody to characterize the extent and the localization of protein modifications in rat liver and kidney after giving tetrachloroethene or its metabolite S-(1,2,2-trichlorovinyl)-~-cysteine in vivo.

Materials and Methods Chemicals. [14ClTetrachloroethene (46 mCUmmo1) with a radiochemical purity of 98% was purchased from Du Pont (Bad was synHomburg, FRG). S-(1,2,2-Trichlorovinyl)-~-cysteine thesized as described (12). W-(Trifluoroacetyl)-L-lysine and reagents for electrophoresis and immunoblots were purchased from Sigma (St. Louis, MO). Anti-trifluoroacetyl serum (antiTFA) was a gift from L. R. Pohl (NIH, Bethesda, MD). W-(Chloroacety1)-L-lysine(Nf-MCA)and N