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 intraperitoneally as a 20 mg/ ml solution in water (pH adjusted to 7.4) at a dose of 30 mg/kg. At the appropriate time point, treated animals were sacrificed and their kidneys removed. The kidney tissue was then either cut into sections for immunohistochemistry experiments (see below) or prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) as follows; whole kidney was minced into small pieces before homogenization (10% w/v; 2 strokes; glass homogenizer/motor driven Teflon pestle) in cold buffer containing 70 mM sucrose, 220 mM mannitol, 1.0 mM EGTA, 2.0 mM HEPES (pH 7.4), and 5.0 pg/ml each phenylmethylsulfonyl fluoride, NO-p-tosyl-L-lysine chlorometbyl ketone, leupeptin, antipain, and aprotinin (buffer A). The crude homogenate was centrifuged at 1,000 X g and the pellet discarded. This low speed supernatant (homogenate) was further fractionated by centrifugation at 10,000 X g. The light tan (middle) layer of the pellet containing the mitochondrial fraction was collected and washed by resuspending it in fresh buffer and repeated centrifugation as described by Schnaitman and Greenawalt (1968). The 10,000 X g supernatant was further fractionated by centrifugation at 100,000 x g to produce the cytosol (supernatant) and microsomes (pellet). The pellet was washed by resuspending it in fresh buffer and repeating the centrifugation.

Immunohistochemistry-Kidney tissue was fixed in a formalin solution (4% formaldehyde in phosphate-buffered saline, pH 7.5), embedded in paraffin, and cut into 5-pm sections using standard techniques. After blocking with 0.1% BSA in phosphate-buffered saline (pH 7.5) (PBS) for 2 h, and then 1.5% normal goat serum in PBS for 30 min at room temperature, anti-trifluoroacetyl serum was applied at a dilution of 1:1,000 in PBS containing 1% BSA. After 30 min at room temperature, the sections were washed with PBS for 10 min. Next, biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA; 1:200 dilution in PBS containing 1% BSA) was

applied following the supplier's instructions. After a 30-min incuba- tion at room temperature, the sections were washed with PBS for 10 min. Finally, biotin-avidin-horseradish peroxidase complex (Vector Laboratories) was applied and incubation continued for 1 h at room temperature. After a final 10-min wash with PBS, immunoreactive protein was visualized by addition of horseradish peroxidase sub- strates (100 pl of diaminobenzidine solution (60 mg/ml) and 12.5 pl of a solution containing 3% H202, 3% NiCl,, and 3% CoC12 diluted to a total volume of 5 ml with 50 mM phosphate buffer (pH 7.6)) (Nibbering and van Furth, 1987). Sections were counterstained with 0.5% methyl green in water.

Immunoblotting-SDS-PAGE was performed according to the pro- cedure of Laemmli (1970). Samples were diluted into sample buffer containing 2-mercaptoethanol and heated at 95 "C for 5 min before loading onto gels. Protein (50 pg/lane) was separated on 5 X 10-cm gels and transferred onto nitrocelluose paper using a mini-blot ap- paratus (Bio-Rad). After electroblotting, the nitrocellulose paper was washed three times with Tris-buffered saline (pH 7.5) (TBS), and either stained with Amido Black or blocked for 30 min with a 5% solution of powdered milk in TBS. After washing three times with TBS, blocked nitrocellulose paper was incubated with anti-trifluo- roacetyl serum at a dilution of 1:10,000 in TBS containing 1% bovine serum albumin (BSA) for 1 h at room temperature. After washing three times with TBS, the nitrocellulose paper was next incubated with goat anti-rabbit IgG alkaline phosphatase conjugate (Promega, Madison, WI) for 1 h at room temperature. Finally, after washing three times with TBS, immunoreactive protein was visualized by addition of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as alkaline phosphatase substrate (Promega).

Enzyme-linked Immunosorbent Assay (ELISA)-ELISA experi- ments were performed by coating the wells of a 96 well microtiter plate with 100 pl of DFTA-BSA (2.5 pg/ml; -3.5 X lo-" mol of adduct) in 50 mM NaHC03 (pH 9.6). After blocking with 150 pl of 1% BSA in TBS, each well was washed three times with TBS containing 0.05% Tween 20. Next, 25 p1 of TBS were added to each well, followed by various amounts of the indicated hapten. Finally, 100 pl of rabbit anti-trifluoroacetyl serum diluted 1:10,000 in TBS containing 1% BSA were added to each well and incubated for 1 h at room temperature. After washing three times with TBS containing 0.05% Tween 20, goat anti-rabbit IgG horseradish peroxidase conju- gate (Bio-Rad) in TBS containing 1% BSA were applied to each well and incubated for 30 min at room temperature. Finally, after washing three times with TBS containing 0.05% Tween 20, 100 pl of horse- radish peroxidase substrate (4 mg of 2,2'-azino-bis-(3-ethylbenzthia- zoline-6-sulfonic acid) in 10 ml of citrate buffer (pH 4.0) containing 5 pl of H202 was added. After color development for 15 min, absorb- ance was measured at 490 nm using a microtiter plate reader. Com- petition between various haptens and DFTA-BSA was determined by adding the test hapten to the well in the presence of antibody.

RESULTS AND DISCUSSION

Rat kidney cytosol (which contains ,&lyase activity) was employed to generate cysteine conjugate metabolite adducts of 35S-PCBC, 35S-DCVC, 35S-CTFC, and %-TFEC with cy- tosolic proteins. As determined by incorporation of 35S, 56 f 15.9, 72 -+ 14.8, 86 _+ 8.3, and 14 -+ 1 nmol of adduct/mg of protein were obtained from TFEC, CTFC, DCVC, and PCBC, respectively. The labeled proteins were then applied to nitro- cellulose paper and assayed for cross-reactivity with anti- trifluoroacetyl serum. Results shown in Fig. 1 reveal cross- reactivity of the antibody with protein adducts formed from TFEC and CTFC, but not PCBC or DCVC.

The cross-reactivity of the anti-trifluoroacetyl serum with several structurally related haptens was further characterized by competitive inhibition ELISA experiments. N'-Trifluo- roacetyl-L-lysine and N-difluorothionoacetyl-N-acetyl-L-ly- sine (DFTAL), the analogous products obtained from reaction of protein lysyl residues with halothane and TFEC metabo- lites, respectively, were comparable in their ability to compete with DFTA-BSA for antibody binding (Fig. 2). N-Acetyl-L- lysine and S-(1,1,2,2-tetrafluoroethyl)-N-difluorothionoace- tyl-L-cysteine (DFTA-TFEC) were approximately 500-fold less reactive. N"-Acetyl-L-lysine, TFEC, and CTFC did not

Cysteine Conjugate Metabolite Adduct Formation 18417

FIG. 1. Immunoreactivity of various cysteine conjugate me- tabolite adducts with anti-trifluoroacetyl serum. Adducts were formed by reacting rat kidney cytosol with various cysteine conju- gates. Cytosolic proteins were then applied to nitrocellulose paper and assayed for immunoreactivity with anti-trifluoroacetyl serum as described under “Experimental Procedures.” Control cytosol had no conjugate treatment.

0.41 1

0.3

E 8 0.2 d

P u)

m 0.1

0.0 - 1.0 0.0 1.0 2.0 3.0 4.0

log conc (pM)

FIG. 2. Enzyme-inked immunosorbent assay for immuno- reactivity of various haptens with anti-trifluoroacetyl serum. Difluorothionoacetyl-BSA (DFTA-BSA) was coated onto the wells of a 96-well microtiter plate. Various haptens were then assayed for ability to prevent the binding of anti-trifluoroacetyl serum to the DFTA-BSA as described under “Experimental Procedures.” Results are the mean of 3 experiments. DFTAL (O), N’-trifluoroacetyl-L- lysine (0); N’-acetyl-L-lysine (0); CTFC, (A; TFEC (A); N”-acetyl- L-lysine (X); DFTA-TFEC, (M).

affect antibody binding at concentrations up to -3 mM (Fig. 2). Adducts formed from metabolites of several inhalation anesthetics which have structural similarities to halothane arc also known to cross-react with this antibody (Pohl et aZ., 1989). Thus, the anti-trifluoroacetyl antibody displays a high specificity for halogen-substituted epsilon amides of lysine.

The cross-reactivity between halothane metabolite adduct antibodies and TFEC and CTFC metabolite adducts has allowed us to apply this antibody as a tool to address impor- tant questions regarding the mechanism of cysteine conjugate nephrotoxicity. Since nephrotoxic cysteine conjugates exert a toxicity which is specific for the S3 segment of the proximal tubule (Terracini and Parker, 1965; Nash et al., 1984; Jaffe et al., 1984; Wolfgang et al., 1987; and Jones et al., 1988), both the localization of metabolite binding to specific cell types within the complicated architecture of the kidney and the specificity of metabolite binding to individual molecules within the cell were of particular interest.

With these goals in mind, experiments investigating the binding of TFEC metabolites in vivo to rat kidney tissue were performed. SDS-PAGE and immunoblotting time course ex- periments of whole kidney tissue revealed a specific pattern of metabolite binding within 6 h. This pattern persisted for 24 h, after which it gradually diminished (Fig. 3). After 3 days, immunoreactive protein could no longer be detected. Three

kDa 200

97

68

43

29

FIG. :

r

- 120 96 72 48 24 18 12 6 0

Time (hours)

i. Immunoblot time course of in vivo binding of im- munoreactive TFEC metabolites to rat kidney homogenate proteins. TFEC-treated rats were sacrificed at the indicated times, and samples from total kidney homogenates were analyzed for im- munoreactive protein by Western blot analysis. Experimental con- ditions are described under “Experimental Procedures.” The major binding proteins have molecular masses of 87, 79, and 61 kDa, respectively.

Homogenate Mitochondria I cytosol r 1 * * Microsomes

FIG. 4. Immunoblot analysis of in vivo formation of immu- noreactive TFEC metabolite adducts with subcellular frac- tions of rat kidney protein. Total rat kidney homogenate (6-h time point) was fractionated by differential centrifugation. Proteins were then assayed for immunoreactivity with anti-trifluoroacetyl serum by Western blot analysis (8.5% polyacrylamide gel) as de- scribedunder “Experimental Procedures.” Lane l , Amido Black stain- ing; lane 2, antibody-specific staining for TFEC metabolite adducts.

proteins with apparent molecular masses of 87, 79, and 61 kDa were predominately labeled. Subsequent immunoblot analysis of subcellular fractions prepared from kidney tissue of rats treated with TFEC demonstrated that the modified proteins were almost exclusively confined to the mitochon- drial fraction (Fig. 4). The formation of protein adducts specifically in the mitochondrion is somewhat surprising be- cause both cytosolic and mitochondrial P-lyase isozymes that can use TFEC as a substrate exist (Stevens et al., 1986,1988). The role of the mitochondrion as a major site of amino acid metabolism may explain the targeting of cysteine conjugates to this organelle.

Immunohistochemical staining of kidney slices revealed binding of metabolites in a pattern consistent with localiza- tion to the medullary ray, which contains the S3C and S3M segments of the proximal tubule, within 6 h (data not shown). Some cell death was evident by this time. However, binding was also observed in cells that appeared morphologically healthy. By 12 h, the binding had increased and significant tissue damage was apparent, with dead cells and debris de-

18418 Cysteine Conjugate Metabolite Adduct Formation

taching from the basement membrane and obstructing the lumen of the tubules (Fig. 5). Thus, the difluorothioamide adducts formed by TFEC metabolites in uitro (Hayden et al., 1991) were also formed in significant amounts in uiuo, and death occurred only in cells containing these adducts. Tissue slices of control animals (no TFEC treatment) did not show anti-trifluoroacetyl serum-specific staining. Additionally, control experiments using anti-trifluoroacetyl serum in the presence of N’-trifluoroacetyllysine showed dramatically re- duced staining (data not shown), further confirming the spec- ificity of the staining for TFEC-labeled proteins.

As was clearly evident from immunohistochemical experi- ments described above, lysine adduct formation with mito- chondrial proteins may be sufficient to cause cell death. The identity of these proteins is presently unknown. Although cysteine conjugate metabolites are known to inhibit the p- lyase enzyme in uitro, in these in uiuo experiments, relatively little immunoreactive protein was detected in the molecular mass range (43-55 kDa) previously reported for this protein (Cooper, 1978; Jones et al., 1988; Stevens et al., 1986; Mac- Farlane et al., 1989). Cysteine conjugates have also been reported to inhibit pyruvate and 2-oxoglutarate dehydrogen- ase complexes (Stonard and Parker, 1971a), lipoyl dehydro- genase, which is a key enzyme of these complexes (Lock and Schnellmann, 1990), and succinate:cytochrome c and isocit- rate dehydrogenase activities (Lash and Anders, 1987). Sev- eral of the enzyme subunits present in the above dehydrogen- ase complexes have the appropriate molecular masses to be considered as possible candidates for adduct formation. How- ever, TFEC metabolites may also react with cysteinyl residues to form adducts which are unstable and are not detected in these experiments (Hayden et al., 1991), and with lipids, suggesting that other adducts may also play a role in toxicity?

In summary, a cross-reactivity between halothane metabo- lite adduct antibodies and TFEC and CTFC metabolite ad- ducts has been discovered. This antibody was used as a tool to investigate the binding of TFEC metabolites to cellular proteins in uiuo. A number of results of general significance were produced by these investigations. 1) There is a correla- tion between the formation of difluorothionoacetyl adducts and the death of proximal tubule epithelial cells after in uiuo treatment with TFEC. 2) Metabolite binding to specific pro- teins of the mitochondrial fraction provides compelling evi- dence for in vivo targeting of cysteine conjugates to the mitochondrion. Identification of these modified proteins may provide insights into mechanisms of cell death. 3) The for- mation of TFEC and CTFC metabolite adducts raises the

possibility of hypersensitivity reactions as possible alternate mechanisms of toxicity for cysteine conjugates, and suggests the possibility of cross-sensitization after exposure to halo- thane, TFE, or CTFE. It remains to be determined if TFE or CTFE are immunogenic in animal models or humans.

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