Characterization of the Enzymatic and Nonenzymatic Reaction of 13

The enzymatic oxygenation of linoleic acid leads to the production of 13-hydroxyoctadecadienoic acid (13-HODE). Subsequent dehydrogenation of 13-HODE ...
0 downloads 0 Views 246KB Size
Download PDF
1364

Chem. Res. Toxicol. 1997, 10, 1364-1371

Characterization of the Enzymatic and Nonenzymatic Reaction of 13-Oxooctadecadienoic Acid with Glutathione Mary L. Blackburn,† Brian Ketterer,‡ David J. Meyer,‡ Amy M. Juett,† and Arthur W. Bull*,† Department of Chemistry, Oakland University, Rochester, Michigan 48309-4401, and Department of Biochemistry and Molecular Biology, University College London, Windeyer Building, Cleveland Street, London W1P 6DB, U.K. Received May 22, 1997X

The enzymatic oxygenation of linoleic acid leads to the production of 13-hydroxyoctadecadienoic acid (13-HODE). Subsequent dehydrogenation of 13-HODE by the NAD+-dependent 13-HODE dehydrogenase results in the formation of the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). These oxidized derivatives of linoleic acid have been shown to be involved in several cellular regulatory processes. In the present study, we have examined the enzymatic and nonenzymatic reaction of 13-OXO with glutathione (GSH) and N-acetylcysteine (NAcCySH). Nonenzymatic reaction rates were determined spectrophotometrically and exhibited a pH optimum of 9.0 which is consistent with attack of a thiolate anion. Product formation was evaluated by reverse-phase HPLC which showed formation of one major product upon reaction with either GSH or N-AcCySH. The HPLC-purified products were examined by FAB MS as well as one- and two-dimensional NMR. The products, with either GSH or N-AcCySH, were found to consist of an equal mixture of two diastereomers arising from addition of a thiolate to the 9 position of 13-OXO. Using GSH as the thiol, the reaction was also shown to be catalyzed by rat glutathione transferase 8-8. In the case of the enzymatic reaction there is stereoselective product formation. Furthermore, submicromolar concentrations of the 13-OXO-GSH conjugate were shown to significantly inhibit glutathione transferase activity in HT-29 homogenates. These investigations provide insight into the potential metabolic disposition of linoleate oxygenation products.

Introduction The oxygenation products of linoleic acid, 9-hydroxy(Z,E)-10,12-octadecadienoic acid and 13-hydroxy-(Z,E)9,11-octadecadienoic acid (9- and 13-HODE)1 are formed by both enzymatic and nonenzymatic pathways (1-8). The enzymatic oxygenation products are involved in the regulation of many cellular processes. Specifically, 9- and 13-HODE have been shown to be involved in cell-cell interactions, response to growth factors, and modulation of eicosanoid production, inter alia (9-14). Our laboratory has reported the enzymatic production of 13-oxo-(Z,E)-9,11-octadecadienoic acid (13-OXO) from 13-HODE. The product is formed in a variety of tissues by the action of an NAD+-dependent 13-HODE dehydrogenase (15). The activity of 13-HODE dehydrogenase is positively correlated with the degree of cellular differentiation, but the precise contribution of the enzyme and the mechanism by which differentiation is altered has not yet been elucidated (16, 17). The product of the dehydrogenase reaction, 13-OXO, is a 2,4-dienone and as such should be an excellent * Corresponding author. † Oakland University. ‡ University College London. X Abstract published in Advance ACS Abstracts, November 15, 1997. 1 Abbreviations: 9-HODE, 9-hydroxy-(E,Z)-10,12-octadecadienoic acid; 13-HODE, 13-hydroxy-(Z,E)-9,11-octadecadienoic acid; 13-OXO, 13-oxo-(Z,E)-9,11-octadecadienoic acid; GSH, glutathione; N-AcCySH, N-acetylcysteine; GSTase, glutathione transferase; CDNB, 1-chloro2,4-dinitrobenzene; 13-HPODE, 13-hydroperoxy-(Z,E)-9,11-octadecadienoic acid; TMSP, 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid; TMS, tetramethylsilane; FBS, fetal bovine serum.

S0893-228x(97)00087-8 CCC: $14.00

Michael acceptor. This raises the possibility that a mechanism of action of 13-OXO in regulating cellular processes could involve conjugation to cellular nucleophiles. Until now neither 13-OXO nor 13-OXO conjugates have been positively identified in vivo, although we have previously detected binding of radiolabel to cellular protein in colon organ cultures incubated with either [1-14C]-13-HODE or [1-14C]-13-OXO (18). In the current work we have investigated the nonenzymatic reactions between 13-OXO and both glutathione (GSH) and N-acetylcysteine (N-AcCySH). Additionally, we have identified the structure of the conjugates and evaluated the equilibrium between product and starting material. Finally we have examined the enzymatic reaction of 13-OXO with GSH catalyzed by rat liver 8-8 glutathione transferase (GSTase) and assayed the effect of the 13-OXO-GSH conjugate on GSTase activity in homogenates of HT-29 cells. The HT-29 cells are a human colon carcinoma cell line that is known to have GSTase activity and has been previously used in our laboratory for studies on the oxidative metabolism of linoleic acid (19, 20). These studies will aid in elucidating the role of 13-HODE dehydrogenase and the metabolic product, 13-OXO, in the regulation of cellular processes.

Materials and Methods Materials. Cell culture grade GSH and N-AcCySH were purchased from Sigma Chemical Co. Inc. McCoy’s medium and fetal bovine serum (FBS) were purchased from Gibco Life Technologies Co. The HT-29 cell line was obtained from ATCC. NMR solvents and 1-chloro-2,4-dinitrobenzene (CDNB) were

© 1997 American Chemical Society

Characterization of 13-OXO-Glutathione Conjugation obtained from Aldrich Inc. Fisher Optima HPLC solvents (ultrahigh purity) were used in all HPLC experiments. Linoleic acid was supplied by Nu-Check Prep. Inc., Elysian, MN; [1-14C]linoleic acid was obtained from DuPont NEN, Boston, MA. Solid-phase extraction columns, C18, were purchased from J. T. Baker Inc.; Phillipsburg, NJ. All other materials and supplies, of the highest quality available, were purchased through local suppliers. Preparation of 13-OXO and [1-14C]-13-OXO. Linoleic acid was oxygenated by soybean lipoxygenase to prepare 13-hydroperoxy-(Z,E)-9,11-octadecadienoic acid (13-HPODE) as described by Funk et al. (21). The resulting 13-HPODE was then directly dehydrated by acetyl chloride in pyridine to yield 13-OXO (22). The radiolabeled [1-14C]-13-OXO was prepared in the previously described manner starting with [1-14C]linoleic acid. Both 13OXO and [1-14C]-13-OXO were analyzed by normal-phase HPLC and found to be 95% pure with an isomeric composition of 88% (Z,E) and 12% (Z,Z). HPLC Conditions. Normal-phase HPLC conditions were as follows: A 3-mm × 30-mm Perkin-Elmer Pecosphere column was eluted isocratically with 0.7% isopropyl alcohol, 0.1% acetic acid in hexane. Analytes were detected at 255 nm. Reversephase HPLC was performed on a Beckman Ultrasphere C18, 5-µm, 80-µm pore, 4.6-mm × 250-mm column eluted with a 20min gradient from 20% acetonitrile in water to 100% acetonitrile. The gradient was followed by a 10-min flush of 100% acetonitrile and a 15-min equilibration with 20% acetonitrile. All solvents also contained 0.1% trifluoroacetic acid as a proton source. Analytes were detected at 235 and 280 nm. Chiral HPLC separations were performed using an ASTEC Chirobiotic V, 5-µm, 4.6- × 250-mm column, eluted isocratically with 10% acetonitrile in 54 mM ammonium acetate at pH 4.5. Samples were dissolved in mobile phase for injection. Analytes were detected at 235 nm. Analyte detections were performed on a Perkin-Elmer LC-235 diode array detector at the specified wavelengths. Determination of pH Optimum for Conjugation Reactions. UV absorbance experiments were performed on a MiltonRoy Spectronic 3000 Array spectrophotometer with a rate analysis and enzyme kinetics package. Reaction progress was monitored at 280 nm by measuring the decrease in absorbance due to the destruction of the 2,4-dienone chromophore ( ) 28 000 M-1 cm-1). Initial reactions were run at 50 µM 13-OXO and 5 mM glutathione in a total reaction volume of 1 mL. Reactions were run in 20 mM sodium borate from pH 7 to 11 against a reference of 5 mM GSH in 20 mM sodium borate at the appropriate pH. Large-Scale Synthesis of Conjugate. A large-scale preparation of conjugate was performed to provide sufficient material for structural identification and other experiments. The reaction was run in a 10-mL volume under the following conditions: 5 mM thiol, 500 µM 13-OXO in 10% ethanol, 20 mM sodium borate, pH 10.0. The reaction was run for 1 h under argon to minimize thiol oxidation. After 1 h the mixture was acidified to pH 4.0 with concentrated HCl. The acidified mixture was then loaded onto a 3-mL C18 solid-phase extraction column preconditioned with 20 mM sodium borate, pH 4.0. After sample application the column was washed with 10 mL of water and the product eluted with either 40% or 50% acetonitrile for the 13-OXO-GSH conjugate or the 13-OXO-N-AcCySH conjugate, respectively (see Figure 1). The product yield for the 13-OXO-GSH conjugate under the previous conditions was 34%; yield was not determined for the 13-OXO-N-AcCySH conjugate. The collected fractions were evaporated to dryness under vacuum and redissolved in a small volume of acetonitrile/ water for HPLC. For NMR analysis the dried product was dissolved in acetonitrile-d3/D2O (40:60) (see Figure 1). Enzyme-Catalyzed Conjugate Synthesis. An enzymecatalyzed preparation of conjugate was performed using rat liver GSTase 8-8 coupled to Pharmacia CNBr-activated Sepharose (23). To achieve maximum product formation, the immobilized enzyme was used in the synthesis. Use of an immobilized enzyme alleviates problems of product sequestration common

Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1365

Figure 1. Flow diagram of 13-OXO-GSH or 13-OXO-NAcCySH conjugate synthesis and analysis. to GSTases and potential inhibition of transferase activity by the product (24, 25). Briefly, argon-saturated 450 µM 13-OXO in 200 mL of 20 mM Tris‚HCl, pH 8.0, was mixed with GSH (10 mM final concentration), and the mixture was pumped through the immobilized enzyme column. Eluted fractions were acidified to pH 4.0 with acetic acid, and the product was applied to SPE columns for purification. The conjugate was eluted with 68% acetonitrile in water. The acetonitrile was removed in vacuo and the aqueous suspension lyophilized for storage at -80 °C until analysis. Stability of Conjugate. To assess stability of the 13-OXOGSH conjugate, a large-scale synthesis was performed and the resulting product collected off the SPE column and then injected directly onto reverse-phase HPLC. Other samples were collected off the SPE column, dried, redissolved, and then injected. A third set of samples was collected as they eluted from the HPLC and then were reinjected after various lengths of time. To assess the temperature dependence of the 13-OXOglutathione conjugate stability, a large-scale synthesis was performed and the sample purified by reverse-phase HPLC. Purified material was stored at -80 °C until the initiation of the experiment. For the experiment, samples were incubated at 4 or 37 °C for 0, 4, 8, 24, 48, and 96 h. At the end of the incubation period, samples were again stored at -80 °C until analysis by reverse-phase HPLC. Nuclear Magnetic Resonance/FAB MS. All experiments were run on a Bruker AMX\300 wide-bore spectrometer, operating at 300 MHz for 1H. Reaction products and N-AcCySH samples were dissolved in acetonitrile-d3/D2O; GSH was dissolved in D2O. All aqueous samples were referenced with 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TMSP). Experiments on unreacted 13-OXO were run in CDCl3 and referenced with tetramethylsilane (TMS). Both the GSH and N-AcCySH conjugates were purified by HPLC and analyzed by FAB MS (see Figure 1).

1366 Chem. Res. Toxicol., Vol. 10, No. 12, 1997

Blackburn et al.

HT-29 Culture Conditions. HT-29 cells were maintained in McCoy’s medium with 10% fetal bovine serum (FBS) and diluted 1:5 weekly; cells were refed 4 days after dilution. Homogenates of normally maintained HT-29 cells were prepared as follows: Confluent cells were harvested by scraping, and the resulting cell pellet was stored at -80 °C until assayed. On the day of the assay the cell pellet was thawed, 0.1 M potassium phosphate buffer, pH 6.5, was added, and cells were homogenized by sonication. Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard (26). GSTase Assay in Cell Homogenates. The GSTase activity of HT-29 homogenates was determined according to Habig et al., in 0.1 M potassium phosphate buffer, pH 6.5, with 1 mM GSH and 1 mM CDNB, in a total reaction volume of 1 mL, containing 1% ethanol (27). The reaction was initiated by the addition of CDNB. Data were collected at 340 nm on a Milton Roy Spectronic 3000 instrument with rate analysis and enzyme kinetics software. Reference cuvettes contained all components except CDNB. The formation of CDNB-GSH conjugate by HT29 homogenates in the absence of exogenous GSH was not detectable. Inhibition of GSTase by the 13-OXO-GSH conjugate was examined using 24 µg of HT-29 homogenate protein per assay. Various levels of 13-OXO-GSH conjugate were assayed against the following levels of CDNB: 200, 400, 800, and 1600 µM. The rate of nonenzymatic conjugation of GSH with CDNB was determined for each and subtracted from the enzymatic rate. The percent of control activity was calculated by comparing the rate of CDNB-GSH conjugate formation in the presence and absence of the 13-OXO-GSH conjugate. Lineweaver-Burk analysis for these data was performed to determine the mode of inhibition.

Results The 2,4-dienone moiety of 13-OXO reacts readily with thiol-containing compounds. The reaction can be monitored by measuring the decrease in absorbance at 285 nm. Preliminary investigations have suggested the reaction between 13-OXO and either cysteine or GSH is a Michael addition as opposed to formation of a Schiff base (18). The following experiments were performed to identify optimal reaction conditions and characterize the major products. The pH dependence of the reaction between 13-OXO and GSH was examined. The reaction was linear for the 15 min of the experiment and is highly dependent upon the pH of the solution. At pH 8 the rate of consumption of 13-OXO was minimally detectable at 0.71 µM/min. However at pH 9.0 the rate increased significantly to 5.71 µM/min, and the maximum rate of 9.29 µM/min was detected at pH 10. No additional increase in reaction rate was observed at pH 11. To determine the dependence of reaction rate on the buffer composition, reactions were also run in phosphate buffer at pH 8 and 11. The results were identical with those obtained in borate buffer. Therefore, subsequent preparative reactions were run in 20 mM sodium borate, pH 10.0. Major Product Formation and Stability. A series of experiments was performed to assess the distribution of products in the reaction between 13-OXO and GSH. The results in Figure 2A are from a reaction in which the eluant off the SPE column (40% acetonitrile fraction, see Figure 1) was injected directly onto the HPLC column. As shown, the conjugate fraction contains no starting material, and there appears to be a single major product. Removal of solvent in vacuo followed by dissolution of the residue in HPLC mobile phase leads to the formation of significant secondary products as well

Figure 2. Reverse-phase HPLC chromatogram of the reaction product between 13-OXO and GSH: A, direct injection of the 40% acetonitrile fraction from the solid-phase extraction column; B, injection of material in panel A after evaporation to dryness and dissolution of residue in HPLC mobile phase. Retention time for 13-OXO was 25.8 min, confirmed by injection of standard material (data not shown). The retention time for the 13-OXOGSH conjugate was 16.3 min. Chromatographic conditions are described in Materials and Methods.

as decomposition of some conjugate to 13-OXO (Figure 2B). Subsequent experiments determined that the unidentified products and the 13-OXO, observed in Figure 2B, were due to sample workup and were not present in the original reaction mixture. Product decomposition could be prevented by thorough washing to remove residual borate buffer and not taking the samples to dryness during solvent removal. Therefore, analysis of the decomposition products was not pursued. To determine if products were formed which did not absorb at the wavelengths being monitored, experiments were also performed using [1-14C]-13-OXO (data not shown). In these experiments, the only significant product in the 40% acetonitrile fraction was the one shown in Figure 2A. Measurement of radioactivity in other fractions from the SPE column did not indicate additional products. Therefore, under the conditions used, only one major product is produced in the reaction between 13-OXO and GSH. Michael additions to unsaturated carbonyl compounds are known to be reversible, and as seen above, the 13OXO-GSH conjugate undergoes partial reversion to

Characterization of 13-OXO-Glutathione Conjugation

Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1367

Figure 3. Formation of 13-OXO during decomposition of 13OXO-GSH conjugate. Conjugate purified by HPLC was incubated at either 4 or 37 °C for the indicated times. The formation of 13-OXO was monitored by HPLC analysis measuring the absorbance at 280 nm. Each point represents the mean ( range resulting from single injections of duplicate incubations.

starting material upon complete removal of solvent (2831). Therefore, to characterize the solution-phase stability of the conjugate, the 40% acetonitrile fraction from the SPE column was incubated at different temperatures and the appearance of 13-OXO measured by reversephase HPLC. The results are presented in Figure 3. As shown, the product is stable at 4 °C, whereas there is some decomposition at 37 °C. It is noteworthy that while there is detectable 13-OXO production at the higher temperature, it represents a relatively small amount of the total product since no decrease in the conjugate peak on HPLC was observed. The detection of 13-OXO formation in the apparent absence of a decrease in conjugate is most likely due to the different molar absorptivities of the two compounds (28 000 M-1 cm-1 for 13-OXO vs 6450 M-1 cm-1 for the conjugate). Structural Identification. To assist in assignment of the 13-OXO-GSH conjugate structure, reactions were also run using N-AcCySH as the nucleophile and the products characterized for comparison to the 13-OXOGSH product. Mass Spectrometry. Fast atom bombardment mass spectra of both the GSH and N-AcCySH conjugates were obtained. For the GSH conjugate, the FAB MS shows a M + H of m/z 602, while the corresponding peak for the N-AcCySH conjugate was at m/z 458. Attempts to obtain more informative fragmentation patterns were unsuccessful. However, these data confirm that in both cases the conjugates formed are from the addition of a single thiol to 13-OXO. NMR Analysis. As mentioned above, 13-OXO contains a 2,4-dienone moiety which should be an excellent Michael acceptor. In addition, previous experiments have shown that Schiff base formation, which is another likely mode of reaction, does not occur to any significant extent (18). The proposed addition of the thiol could occur at either carbon 9 of 13-OXO (1-6 addition) or at carbon 11 (1-4 addition). Both sites of addition result in destruction of the 2,4-dienone, and thus the chromophore at 280 nm, and leave a single carbon-carbon double bond. To assign the correct structure, HPLCpurified conjugates of 13-OXO with GSH and N-AcCySH were examined by 1H and COSY NMR. Figure 4 contains the proton spectra for 13-OXO (panel A), GSH (inset of panel B), and the 13-OXO-GSH conjugate (panel B). The proton assignments are indi-

Figure 4. 1H NMR spectra (300 MHz): A, 13-OXO in CDCl3, referenced against TMS; B, HPLC-purified 13-OXO-GSH conjugate, inset is the GSH spectrum from 3 to 4 ppm, both compounds referenced against TMSP; C, HPLC-purified 13OXO-N-AcCySH conjugate, inset is the N-acetylcysteine spectrum from 3 to 4 ppm, both compounds referenced against TMSP.

cated in the figure. Assignments for the reactants were made on the basis of expected chemical shift as well as decoupling experiments. The spectrum of the 13-OXOGSH conjugate was compared to the spectra of the reactants. The following changes are observed. In 13OXO, between 6 and 8 ppm, there are multiplets assigned to the protons at positions 9, 10, 11, and 12. In the conjugate these signals have disappeared, and there are new multiplets at 5.3 and 5.6 ppm. This corresponds to destruction of conjugation in the 2,4-dienone system with

1368 Chem. Res. Toxicol., Vol. 10, No. 12, 1997

the newly formed vinyl protons being shifted upfield. There is also the appearance, in the conjugate, of a multiplet at 3.25 ppm which corresponds to protons on carbons 9 and 10 of the 13-OXO moiety. The methylene region of the conjugate spectrum is also altered relative to that of 13-OXO. The multiplet at 2.3 ppm in 13-OXO is due to overlapping signals from protons at positions 2 and 8. In the conjugate, the signal at 2.3 ppm is a clean triplet due to the destruction of the double bond at C-9 with movement of the signal from the protons on carbon 8 upfield to 1.5 ppm. Comparison of the proton spectrum of GSH to that of the 13-OXO-GSH conjugate also indicates changes consistent with the proposed Michael addition product. In particular, the multiplet at 3 ppm (5g) in GSH (methylene adjacent to thiol) becomes a multiplet spread from 2.5 to 3.0 ppm due to addition of the thiol to 13OXO. There are no other significant changes in the proton signals from the GSH portion of the conjugate. To further verify the above assignments, a conjugate between N-AcCySH and 13-OXO was prepared. It was expected that the simpler spectrum of N-AcCySH would allow unequivocal assignment of the proton signals discussed above. In particular, the intent was to allow a differentiation between thiol addition to position 9 versus 11 of 13-OXO. In Figure 4C the proton spectra of the 13-OXO-N-AcCySH conjugate and N-AcCySH (inset of panel C) are shown. Comparison of the spectra of the starting materials to that of the product of the conjugation reaction reveals changes similar to those seen for the reactions using GSH as the thiol. COSY experiments were also run to identify coupled systems. The COSY spectra of the products from the conjugation reactions are shown in Figure 5. In both cases, the spectra are consistent with addition of the respective thiol to carbon 9 of 13-OXO. In the spectrum of the N-AcCySH conjugate, the multiplet at 5.4 ppm (peak 11 in Figure 5A) is coupled to the multiplets at 3.25 and 5.6 ppm (peaks 9 and 10 in Figure 5A). The multiplet at 5.6 ppm is only coupled to the multiplet at 5.4 ppm. This indicates the reaction is a 1-6 addition to carbon 9 of 13-OXO as only addition at carbon 9 results in an alkene proton coupled to a single proton. A similar analysis can be applied to the COSY spectrum of the 13OXO-GSH conjugate (Figure 5B). Stereochemical Analysis of Reaction Products. The spectra discussed above indicate the site of addition of the thiol to 13-OXO, but they do not explain the complex multiplicity associated with the protons in the vicinity of the addition site. For example, the proton on C-12 is a multiplet rather than the expected clean doublet (see Figure 4B). Since nonenzymatic addition of the nucleophiles to the 2,4-dienone moiety could occur from either side of the molecule, the possibility exists that mixtures of diastereomers are formed. To investigate this possibility, reverse-phase HPLC-purified 13-OXOGSH conjugate was analyzed by chiral-phase HPLC to resolve stereoisomers and the stereochemistry of nonenzymatic and enzymatic reactions was then compared. The results of these experiments are presented in Figure 6. Panel A contains the chromatogram of a nonenzymatic reaction between 13-OXO and GSH. As shown, roughly equal amounts of two diastereomeric products are formed. These peaks were collected and proton NMR spectra obtained; the signals associated with the protons on carbons 11 and 12 of the 13-OXO moiety were resolved into clean multiplets in each stereoisomer

Blackburn et al.

Figure 5. COSY NMR spectra: A, 13-OXO-N-AcCySH conjugate spectrum, proton assignments correspond to the numbering system shown in Figure 4C; B, 13-OXO-GSH conjugate spectrum, proton assignments correspond to the numbering system shown in Figure 4B.

(see Figure 4B). Mixing of the isolated compounds yielded a spectrum identical with that shown in Figure 4B. Figure 6B contains the results of chiral-phase analysis of a GSH transferase-catalyzed reaction between 13-OXO and GSH. As shown, the enzyme-catalyzed reaction produces an excess of one of the diastereomeric products. These data are consistent with addition of GSH to only one face of the 2,4-dienone moiety of 13-OXO. The precise stereoselectivity of the enzymatic reaction could not be determined as under the conditions of the experiment there was both enzymatic and nonenzymatic product formation. Inhibition of GSTase by the 13-OXO-GSH Conjugate. To explore potential biological activities of the 13-OXO-GSH conjugate, the effect of the compound on

Characterization of 13-OXO-Glutathione Conjugation

Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1369

Inhibition was detectable at 0.283 µM 13-OXO-GSH, the lowest level of conjugate assayed, and increased with increasing conjugate concentrations. The IC50 was determined to be 0.613 µM. The Lineweaver-Burk analysis of the data indicates a mixed mode of inhibition (Figure 7B).

Discussion

Figure 6. Chiral-phase HPLC elution profile of nonenzymatically and enzymatically formed 13-OXO-GSH conjugate: A, nonenzymatic products; B, product profile from enzymecatalyzed reaction.

Figure 7. Inhibition of GSTase activity in HT-29 homogenates by 13-OXO-GSH conjugate. Assays were performed as described in Materials and Methods. Panel A is percent of control GSTase activity for incubations containing the indicated concentrations of 13-OXO-GSH conjugate, each run in the presence of 200, 400, and 800 µM CDNB. Data are the mean ( SD of three incubations. Panel B is the Lineweaver-Burk analysis of the data in panel A.

GSTase activities in HT-29 cells was examined. Consistent with previous reports, significant levels of GSTase activity were detected in HT-29 homogenates (19, 20). As shown in Figure 7A, addition of the 13-OXO-GSH conjugate to HT-29 homogenates results in significant inhibition of GSTase activity compared to control levels.

The enzymatic metabolism of linoleic acid to oxygenated species has recently received attention as a participant in cellular signal transduction systems (10-12). Of special interest in our laboratory is the metabolism of linoleic acid to 13-HODE and 13-OXO. As a step toward elucidating the role of these products in biological systems, we have chemically characterized the reaction between 13-OXO and GSH, a major cellular thiol. The 13-OXO under investigation is a dehydrogenation product, produced enzymatically by 13-HODE dehydrogenase, from the biologically active species 13-HODE. The results of the current investigation suggest one mechanism by which oxygenated derivatives of linoleic acid can interact with biological systems. The reaction of 13-OXO with thiols is readily observed by monitoring a decrease in absorbance at 280 nm, which corresponds to destruction of the 2,4-dienone chromophore of 13-OXO. Not too surprisingly, the major product is the result of a Michael addition to carbon 9 of 13-OXO. Since the reaction rate becomes maximal at pH above 9, which is the pKa of the GSH thiol, it is reasonable to suggest the reaction occurs by attack of a thiolate anion. The fact the reaction requires deprotonation of the thiol indicates that in vivo the reaction could be mediated by glutathione transferases. Data presented in the current work demonstrate that 13-OXO, a product of linoleic acid metabolism, is in fact a substrate for GSH transferase 8-8. It is noteworthy that this represents one of the few examples of a nonxenobiotic, enzymatically derived compound that is a substrate for GSTase (32). The major product in the reaction between GSH and 13-OXO results from addition of GSH to carbon 9 of 13OXO. A similar conjugate is formed when the thiol is N-AcCySH. The addition to C-9 creates a chiral center with the potential for formation of diastereomers depending on the face of the 2,4-dienone to which the thiol adds. The complexity of the NMR spectra of the nonenzymatic products substantiates the formation of diastereomeric products. In addition, chiral-phase HPLC demonstrated the formation of approximately equal amounts of two diastereomers. In the current system, the product is a 1:1 adduct between 13-OXO and the thiol. There is no evidence for addition of a second molecule of thiol, or for rearrangement of the initial adduct. However, the unidentified peaks observed in preparations evaporated to dryness may represent cyclized products (Figure 2B). Given the conditions necessary to generate the unidentified products, it is unlikely such products are significant under physiological conditions. It is not yet clear the extent, and the condition under which, the conjugation reaction reported in the present work occurs in vivo. Moreover, it is not known whether the reaction represents a metabolic activation or inactivation event, although studies are currently underway to address this question. There are, however, biological implications for the fact the reaction is reversible at physiologic temperatures. In particular, the reversibility

1370 Chem. Res. Toxicol., Vol. 10, No. 12, 1997

of the reaction provides a mechanism for modulation of the signal due to formation of 13-OXO in biological systems. The possibilities include product sequestration of the conjugate on glutathione transferase or partitioning of 13-OXO between GSH and protein-derived thiols (24, 25). It is also conceivable that the reversibility of the reaction provides a means for transporting 13-OXO to distant parts of an organism, as occurs with several other GSH conjugates (28). Catalysis of the conjugation reaction by GSTase has significant implication for the potential biological role of these reactions. The data in the present report indicate that not only is 13-OXO a substrate for GSTase but also the resulting conjugate is an inhibitor of GSTase activity with activity in the submicromolar range. The mixed mode of inhibition observed is consistent with the fact the homogenate may contain different classes of GSTases which may have differing susceptibilities to 13-OXOGSH-mediated inhibition. It is also possible the different stereoisomers of the conjugate have varying inhibitory potencies. These issues are currently under investigation in our laboratory using purified GSTases. The fact that the 13-OXO-GSH conjugate is an inhibitor of GSTase activity is significant when one considers that 13-OXO is derived from an endogenous substrate as opposed to the xenobiotic substrates which constitute a majority of the known GSTase substrates. The results of the present investigation provide information for elucidating the role of 13-OXO and its precursors in biological systems. The nonenzymatic conjugation with cellular thiols and the in vitro catalysis by GSH transferase suggest that, in vivo, product distribution would ultimately be under metabolic control (33). In addition we have shown that the 13-OXO-GSH conjugate is a potent inhibitor of GSTase activity in HT-29 homogenates (34, 35). Subsequent investigations are aimed at further defining the biological function of these reactions.

Acknowledgment. We would like to thank Ms. Jennifer A. Bulgarella and Mr. Michael A. Faw for assistance with the GSTase assays. In addition we would like to acknowledge the MSU-NIH Mass Spectrometry Facility for FAB MS analysis of the 13-OXO-GSH and 13-OXO-N-AcCySH conjugates. This work was supported in part by the Oakland University Research Excellence Program in Biotechnology.

References (1) Funk, C. D., and Powell, W. S. (1985) Release of prostaglandins and monohydroxy and trihydroxy metabolites of linoleic and arachidonic acids by adult and fetal aortae and ductus arteriosus. J. Biol. Chem. 260, 7481-7488. (2) Engels, F., Drijver, A. A., and Nijkamp, F. P. (1990) Modulation of the release of 9-hydroxyoctadecadienoic acid and other fatty acid derived mediators from guinea-pig pulmonary macrophages. Int. J. Immunopharmacol. 12, 199-205. (3) Oosthuizen, M. J., Engels, F., Van Esch, B., Henricks, P. A. J., and Nijkamp, F. P. (1990) Production of arachidonic and linoleic acid metabolites by guinea pig tracheal epithelial cells. Inflammation 14, 401-408. (4) Reinaud, O., Delaforge, M., Boucher, J. L., Rocchiccioli, F., and Mansuy, D. (1989) Oxidative metabolism of linoleic acid by human leukocytes. Biochem. Biophys. Res. Commun. 161, 883-891. (5) Daret, D., Blin, P., and Larrue, J. (1989) Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets. Prostaglandins 38, 203-214.

Blackburn et al. (6) Kaduce, T. L., Figard, P. H., Leifur, R., and Spector, A. A. (1989) Formation of 9-hydroxyoctadecadienoic acid from linoleic acid in endothelial cells. J. Biol. Chem. 264, 6823-6830. (7) Dix, T. A., and Marnett, L. J. (1985) Conversion of linoleic acid hydroperoxide to hydroxy, keto, epoxyhydroxy, and trihydroxy fatty acids by hematin. J. Biol. Chem. 260, 5351-5357. (8) Hamberg, M. (1975) Decomposition of unsaturated fatty acid hydroperoxides by hemoglobin: structures of major products of 13L-hydroperoxy-9,11-octadecadienoic acid. Lipids 10, 87-92. (9) Buchanan, M. R., Bertomeu, M.-C., and Bastida, E. (1990) Fatty acid metabolism and cell/cell interactions. Agents Actions 29, 1620. (10) Glasgow, W. C., and Eling, T. E. (1994) Structure-activity relationship for potentiation of EGF-dependent mitogenesis by oxygenated metabolites of linoleic acid. Arch. Biochem. Biophys. 311, 286-292. (11) Glasgow, W. C., Afshari, C. A., Barrett, J. C., and Eling, T. E. (1992) Modulation of the epidermal growth factor mitogenic response by metabolites of linoleic and arachidonic acid in Syrian hamster embryo fibroblasts: differential effects in tumor suppressor gene (+) and (-) phenotypes. J. Biol. Chem. 267, 1077110779. (12) Glasgow, W. C., and Eling, T. E. (1990) Epidermal growth factor stimulates linoleic acid metabolism in BALB/c 3T3 fibroblasts. Mol. Pharmacol. 38, 503-510. (13) Setty, Y. B. N., Berger, M., and Stuart, M. J. (1987) 13Hydroxyoctadecadienoic acid (13-HODE) stimulates prostacyclin production by endothelial cells. Biochem. Biophys. Res. Commun. 146, 502-509. (14) Setty, Y. B. N., Berger, M., and Stuart, M. J. (1987) 13Hydroxyoctadeca-9,11-dienoic acid (13-HODE) inhibits thromboxane A2 synthesis, and stimulates 12-HETE production in human platelets. Biochem. Biophys. Res. Commun. 148, 528-533. (15) Earles, S. M., Bronstein, J. C., Winner, D. L., and Bull, A. W. (1991) Metabolism of oxidized linoleic acid: characterization of 13-hydroxyoctadecadienoic acid dehydrogenase activity from rat colonic tissue. Biochim. Biophys. Acta 1081, 174-180. (16) Bull, A. W., Branting, C., Bronstein, J. C., Blackburn, M. L., and Rafter, J. J. (1993) Increases in 3-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase activity during differentiation of cultured cells. Carcinogenesis 14, 2239-2243. (17) Bronstein, J. C., and Bull, A. W. (1993) The correlation between 13-hydroxyoctadecadienoate dehydrogenase (13-HODE dehydrogenase) and intestinal cell differentiation. Prostaglandins 46, 387-395. (18) Blackburn, M. L., Earles, S. M., Juett, A. M., Bronstein, J. C., and Bull, A. W. (1995) Reaction of 13-hydroxyoctadecadienoic acid and 13-oxooctadecadienoic acid with cellular thiols including glutathione. Proc. Am. Assoc. Cancer Res. 36, 505. (19) Ranganathan, S., Ciaccio, P. J., and Tew, K. D. (1993) Principles of drug modulation applied to glutathione S-transferases. In Structure and Function of Glutathione Transferases (Tew, K. D., Pickett, C. B., Mantle, T. J., Mannervik, B., and Hayes, J. D., Eds.) pp 249-256, CRC Press, Boca Raton, FL. (20) Kuzmich, S., Vanderveer, L. A., Walsh, E. S., LaCreta, F. P., and Tew, K. D. (1992) Increased levels of glutathione S-transferase π transcript as a mechanism of resistance to ethacrynic acid. Biochem. J. 281, 219-224. (21) Funk, M. O., Isaac, R., and Porter, N. A. (1976) Preparation and purification of lipid hydroperoxides from arachidonic and γ-linolenic acids. Lipids 11, 113-117. (22) Porter, N. A., and Wujek, J. S. (1987) Allylic hydroperoxide rearrangement: β-scission or concerted pathway. J. Org. Chem. 52, 5085-5089. (23) Meyer, D. J., Lalor, E., Coles, B., Kispert, A., A ° lin, P., Mannervik, B., and Ketterer, B. (1989) Single-step purification and HPLC analysis of glutathione transferase 8-8 in rat tissues. Biochem. J. 260, 785-788. (24) Meyer, D. J. (1993) Significance of an unusally low Km for glutathione in glutathione transferases of the, R, µ, and π classes. Xenobiotica 23, 823-834. (25) Meyer, D. J., Crease, D. J., and Ketterer, B. (1995) Forward and reverse catalysis and product sequestration by human glutathione S-transferases in reaction of GSH with dietary aralkyl isothiocyanates. Biochem. J. 306, 565-569. (26) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. (27) Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974) Glutathione S-transferases. J. Biol. Chem. 249, 7130-7139.

Characterization of 13-OXO-Glutathione Conjugation (28) Baillie, T. A., and Slatter, J. G. (1991) Glutathione: A vehicle for the transport of chemically reactive metabolites in vivo. Acc. Chem. Res. 24, 264-270. (29) Ploemen, J. H. T. M., Van Schanke, A., and Van Bladeren, P. J. (1994) Reversible conjugation of ethacrynic acid with glutathione and human glutathione S-transferase P1-1. Cancer Res. 54, 915919. (30) Ploemen, J. H. T. M., Van Ommen, B., Bogaards, J. J. P., and Van Bladeren, P. J. (1993) Ethacrynic acid and its glutathione conjugate as inhibitors of glutathione S-transferases. Xenobiotica 23, 913-923. (31) Witz, G. (1989) Biological interactions of R,β-unsaturated aldehydes. Free Radical Biol. Med. 7, 333-349. (32) Jakschik, B. A., Kuo, C. G., and Wei, Y. F. (1985) Enzymatic formation of leukotrienes. In Biochemistry of Arachidonic Acid

Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1371 Metabolism (Lands, W. E. M., Ed.) Martinus Nijhoff Publishing, Boston. (33) Ketterer, B. (1982) The role of nonenzymatic reactions of glutathione in xenobiotic metabolism. Drug Metab. Rev. 13, 161187. (34) Chien, C., Kirollos, K. S., Linderman, R. J., and Dauterman, W. C. (1994) R,β-Unsaturated carbonyl compounds: inhibition of rat liver glutathione S-transferase isozymes and chemical reaction with reduced glutathione. Biochem. Biophys. Acta 1204, 175180. (35) Mannervik, B., and Danielson, U. H. Glutathione transferasesstructure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283-337.

TX970087A