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Communication Vol. 266, No. 28, THE Issue of October 5, pp ... · PDF file Communication Vol. 266, No. 28, Issue of October 5, pp. 18415-18418,1991 Printed in U.S.A. THE JOURNAL OF

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  • Communication Vol. 266, No. 28, Issue of October 5, pp. 18415-18418,1991 Printed in U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY

    Detection of Cysteine Conjugate Metabolite Adduct Formation with Specific Mitochondrial Proteins Using Antibodies Raised against Halothane Metabolite Adducts*

    (Received for publication, June 19, 1991) Patrick J. Hayden, Takaharu Ichimura, Denis J. McCann, Lance R. PohlS, and James L. Stevens4 From the W. Alton Jones Cell Science Center, Lake Placid, New York 12946 and the $Laboratory of Chemical Pharmacology, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892

    Antibodies raised against halothane metabolite ad- ducts cross-react with S-(l,l,2,2-tetrafluoroethyl)-~- cysteine (TFEC) andS-(2-chloro-l,l,2-trifluoroethyl)- L-cysteine metabolite adducts. Using these antibodies in immunohistochemical experiments, metabolite bind- ing was localized to the damaged areas of the proximal tubule after treatment of male rats with TFEC. Im- munoblot analysis of subcellular fractions of rat kidney tissue after in vivo treatment with TFEC revealed a high specificity for binding of metabolites to proteins of the mitochondrial fraction. These proteins may rep- resent target molecules which play a role in cysteine conjugate induced nephrotoxicity.

    The nephrotoxic cysteine conjugates S-(1,1,2,2-tetrafluo- roethy1)-L-cysteine (TFEC)’ and S-(2-chloro-1,1,2-trifluoroe- thy1)-L-cysteine (CTFC) are metabolized by renal &lyase enzymes to thionoacylating metabolites (Commandeur et al., 1989; Dekant et al., 1987). Renal cysteine conjugate P-lyase (@-lyase) activity is primarily due to glutamine transaminase K, which catalyses the interconversion of amino acids and a- keto acids (Stevens et d., 1986). For cysteine conjugates with electronegative &substituents ( i e . good leaving groups), this enzyme also catalyzes a @-elimination reaction which pro- duces pyruvate, ammonia, and a sulfur-containing species. The thionoacylating species resulting from the @-lyase reac-

    * These studies were supported by Grants DK38925 and CA38579 from the National Institutes of Health (to J. L. S). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    § To whom correspondence should be addressed. The abbreviations used are: TFEC, S-(1,1,2,2-tetrafluoroethyl)-

    L-cysteine; CTFC, S-(2-chloro-1,1,2-trifluoroethyl)-~-cysteine; PCBC, S-(1,2,3,4,4-pentachlorobutadienyl)-~-cysteine; DFTAL, N-difluoro- thionoacetyl-N“-acetyl-L-lysine; DFTA-TFEC, S-(l,l,Z,Z-tetrafluo- roethy1)-N-difluorothionoacetyl-L-cysteine; SDS, sodium dodecyl sul- fate; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; DFTA-BSA, difluoro- thionoacetyl-bovine serum albumin; DCVC, S-(1,2-dichlorovinyl)-~- cysteine; TFE, tetrafluoroethylene; CTFE, chlorotrifluoroethylene; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; HEPES, N - 2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid.

    tion form stable thioamide adducts with protein lysyl residues (Hayden et al., 1991)* and phosphatidylethanolamine (Welsh et al., 1991).3 This adduct formation with cellular molecules is presumed to initiate a cascade of events which eventually leads to cell death.

    The mitochondrion has been considered to be a primary target for cysteine conjugate metabolite binding and toxicity. Mitochondria contain @-lyase enzymes (Stonard and Parker, 1971b; Stevens, 1985; Lash et al., 1986; Stevens et al., 1988; Hayden and Stevens, 1990), and metabolism of cysteine con- jugates has been shown to result in inhibition of respiration (Parker, 1965; Stonard and Parker, 1971a, 1971b; Jones et al., 1986; Lash and Anders, 1986,1987; Lash et al., 1986; Hayden and Stevens, 1990), uncoupling of oxidative phosphorylation (PCBC only) (Jones et al., 1986; Schnellmann et al., 1987, 1989; Hayden et al., 1990), inhibition of 2-oxoacid dehydro- genases (Stonard and Parker, 1971a), succinate:cytochrome c and isocitrate dehydrogenases (Lash and Anders, 1987) inhi- bition of lipoyl dehydrogenase (Lock and Schnellmann, 1990), collapse of membrane potential and release of sequestered calcium (Jones et al., 1986; Lash and Anders, 1987; Wallin et al., 1987) and binding to mitochondrial protein (Hayden and Stevens, 1990) and lipids (Welsh et al., 1991).3 However, the specific molecular targets for binding and the mechanisms which couple binding to cell death remain unknown (see Dekant and Vamvakas (1989), Stevens and Jones (1989), and Commandeur and Vermeulen (1990) for recent reviews).

    The inhalation anesthetic halothane is oxidatively metab- olized by hepatic microsomal enzymes to yield a metabolite which is very similar to those of TFEC and CTFC: trifluo- roacetyl chloride (Kenna et al., 1988). This trifluoroacylating metabolite forms stable trifluoroacetyllysine adducts with a high specificity for several microsomal proteins (Kenna et al., 1987, 1988.) An immune-mediated response to these altered proteins is thought to be involved in the “halothane hepatitis” experienced by some patients who have received this anes- thetic (for recent review see Pohl et al. (1989, 1990)).

    Because of the structural similarities between the protein adducts produced by halothane (trifluoroacetamides) and pro- tein adducts produced by TFEC and CTFC (difluoro-, or chlorofluorothioacetamides), we investigated the possible cross-reactivity of antibodies raised against halothane metab- olite adducts with TFEC- and CTFC-derived adducts. We now report that antibodies raised against halothane adducts do cross-react with TFEC and CTFC adducts. Using these antibodies in immunohistochemical experiments, metabolite binding was localized to the damaged areas of the proximal tubule after treatment of male rats with TFEC. Additionally, immunoblot analysis of subcellular fractions of kidney tissue treated in vivo with TFEC revealed a high specificity for binding of metabolites to proteins of the mitochondrial frac- tion. The modified proteins detected in these experiments may represent important molecular targets involved in cys- teine conjugate induced nephrotoxicity.

    * After P-lyase cleavage, CTFC forms stable thioamides with lysine analogous to those reported for TFEC (P. J. Hayden and J. L. Stevens, unpublished results).

    ‘P. J. Hayden, C. J . Welsh, Y. Yang, W. H. Schaeffer, A. J. I. Ward, and J. L. Stevens, submitted for publication.

    18415

  • 18416 Cysteine Conjugate Metabolite Adduct Formation

    EXPERIMENTAL PROCEDURES

    Materiaki-Cysteine conjugates and "S-labeled cysteine conjugates were synthesized as previously reported (Hayden and Stevens, 1990). N-Difluorothionoacetyl-N"-acetyl-L-lysine (DFTAL) and N-difluo- rothionoacetyl-S-( 1,1,2,2-tetrafluoroethyl)-~-cysteine (DFTA- TFEC) were synthesized as previously reported (Hayden et al., 1991). Specific anti-trifluoroacetyllysyl serum was obtained as previously described (Satoh et al., 1985).

    Enzyme Purification-Cysteine conjugate @-lyase was purified to a specific activity of 6.01 prnol/lO min-mg (10 mM L-phenylalanine and 5 mM a-keto-y-methiolbutyrate as substrates) according to the procedure of Cooper and Meister (1981) as modified by Stevens et al. (1986).

    In Vitro Generation of Protein Adducts-Difluorothionoacetyl-bo- vine serum albumin (DFTA-BSA) was prepared by incubating 2.0 pmol of TFEC, 173 pg of &lyase and 1.0 pmol of a-keto-y-methiol- butyrate in the presence of 2.0 mg of BSA in a total volume of 700 p1 of 50 mM potassium phosphate buffer (pH 7.5) for 1 h at 37 "C. BSA was precipitated by addition of 2 volumes of ice-cold acetone and 10 pl of glacial acetic acid. After centrifugation for 10 min at 16,000 X g, the BSA pellet was washed twice by dissolving it in 400 p1 of 50 mM potassium phosphate buffer (pH 7.5) and repeating the precipi- tation and centrifugation steps.

    For immunoblot (dot blot) experiments, 1.0 pmol of TFEC, CTFC, or DCVC or 0.1 pmol of PCBC was incubated with 100 p1 of rat kidney cytosol (12.0 mg/ml) (see "Preparation of Tissue Samples after in Vivo Treatment") for 1 h at 37 "C. Buffer (400 pl) containing 2% sodium dodecyl sulfate (SDS) and 50 pl/ml 2-mercaptoethanol were then added, and the samples were heated at 95 "C for 5 min. After cooling to room temperature, various amounts of protein were applied to nitrocellulose paper using a Minifold I microsample filtra- tion manifold (Schleicher & Schuell, Keene, NH). After washing three times with 100 p1 of buffer, proteins bound to the nitrocellulose paper were assayed for immunoreactivity with anti-trifluoroacetyl serum as described below for immunoblotting. Adduct formation was quantitated in parallel experiments with 35S-conjugates. Cytosolic protein was precipitated by addition of 2.0 ml of 10% trichloroacetic acid. After 1 h at 4 "C, precipitated protein was collected onto What- man GF/C glass fiber filters and washed twice with 5 ml of 5% trichloroacetic acid and then twice with 5 ml of ice-cold 95% ethanol. Incorporation of 35S into protein was then quantitated by scintillation counting.

    Preparation of Tissue Samples after in Vivo Treatment-Male Sprague-Dawley rats (300-350 g) were housed in plastic cages, had free access to food and water, and were exposed to 12-h cycles of light and darkness. TFEC was administered int

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