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BIOMEDICAL AND ENVIRONMENTAL MASS SPECTROMETRY, VOL. 19, 248-252 (1990) Characterization of Glutathione Conjugates of Chlorambucil by Fast Atom Bombardment and Thermospray Liquid Chromatography/Mass Spectrometry Deanne M. Dulik,? 0. Michael Colvint and Catherine Fenselaug Department of Pharmacology and Molecular Sciences and Oncology Center, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA Chlorambucil @-(di-2chloroethyl)amino-y-phenylbutyric acid) is a bifunctional alkylating agent which exhibits acquired drug resistance upon repeated dosing in humans. This compound reacts with glutathione both nonenzymatically and enzymatically in the presence of immobilized microsomal glutathione-S-transferases to produce several glutathione conjugates. These conjugates result from displacement of one or both chlorines by the nucleophilic cysteine sul!l~ydrylmoiety of glutathione. The mono- and diglutathionyl conjugates of chlorambucil were purified by reversed-phase high-performance liquid chromatography and characterized by positive ion fast atom bombardment mass spectrometry. In addition, the mono- and dihydroxy hydrolysis products of chlorambucil were characterized by positive ion thermospray liquid chromatography/mass spectrometry (LC/MS). The glutathione conjugates of chlorambucil did not produce molecular ion species in thermospray LC/MS mode, but gave characteristic ions at m/z 147 corresponding to fragmentation of the glutathione moiety. The formation of glutathione conjugates of this class of alkylating agents may play a role in the development of acquired drug resistance. Glutathione (y-glutamylcysteinylglycine, GSH) is the most abundant intracellular nucleophile in mammalian cells, present at concentrations of approximately 5 mM. Chlorambucil (p-(di-2-chloroethyl)amino-y-phenylbuty- Several studies have suggested a relationship between GSH and the transferases involved in the enzymatic forric acid, I) is a bifunctional nitrogen mustard alkylating mation of glutathione conjugates (GST) and the develagent which has been used extensively in the treatment opment of ADR. Among these observations are: (a) in of ovarian and head/neck carcinomas. This compound, the presence of alkylating agents such as melphalan, along with several others in its therapeutic class, have intracellular levels of GSH are rapidly depleted;* (b) been associated with the development of acquired drug cells which are made resistant to alkylating agents by resistance (ADR), in which repeated dosing of the agent repeated exposure show higher initial levels of both results in a lack of target cell cytoxicity and therefore GSH and GST;3 and (c) depletion of intracellular GSH reduced clinical effectiveness. The development of ADR by GSH synthetase inhibitors, such as buthionine sulhas been associated with several possible factors, includfoximine, causes a concomitant increase in the cytoing (a) decreased drug uptake into target cells, (b) toxicity of administered alkylating agent^.^ These increased DNA repair mechanisms, and (c) increased results suggest a plausible relationship between the reaction of the alkylating agent with intracellular reaction of glutathione and alkylating agents and the nucleophiles with resulting loss of cytotoxic potential.' development of acquired drug resistance. CI--H C--H c \ N ~ C H 2 - C - H 2 - C H 2 - C c l o n The objective of this study was to synthesize and characterize the reaction products between chlorambuCI--H c-n c' 2 2 cil and glutathione. Conjugates of chlorambucil were prepared under non-enzymatic conditions and enzymatically using immobilized microsomal glutathione-SI pansferases from cynomolgus monkey liver. The use of t Present address: Department of Drug Metabolism, Smith Kline & 'immobilized GST for synthesis of glutathione conjuFrench Research Laboratories, L-711, P O Box 1539, King of Prussia, gates of a variety of electrophilic substrates has been Pennsylvania 19406-0939, USA. Author to whom correspondence should be addressed. described previously by our l a b ~ r a t o r yA . ~combination 3 Present address: Department of Pharmacology, Oncology Center, of fast atom bombardment (FAB) mass spectrometry Johns Hopkins School of Medicine, 600 N. Wolfe Street, Baltimore, and thermospray liquid chromatography/mass specMaryland 21205, USA. trometry (LC/MS) were employed for structural charac8 Department of Chemistry, University of Maryland Baltimore County, 5401 Wilkens Avenue, Baltimore, Maryland 21228, USA. terization of the resulting conjugates. INTRODUCTION 0887-6 134/90/04O248-O5 $05.00 0 1990 by John Wiley & Sons, Ltd. Received 24 July 1989 Accepted I November 1989 GLUTATHIONE CONJUGATES OF CHLORAMBUCIL MATERIALS AND METHODS Chemicals Chlorambucil was kindly provided by Dr John Hilton, Oncology Center, Johns Hopkins School of Medicine. Reduced glutathione was obtained from Sigma Chemical Company (St Louis, Missouri). All other chemicals were reagent grade or better and were used without further purification. Hydrolysis products of chlorambucil were prepared by reaction of chlorambucil in 0.1 M NaOH for 1 h at room temperature. Immobilized enzyme synthesis of glutathione conjugates Cynomolgus monkey liver microsomal g1utathione-Stransferases were immobilized onto cyanogen bromideactivated Sepharose 4B by a published method.' Incubation mixtures contained chlorambucil (1.0 mM), reduced glutathione (3.0 mM) and packed Sepharose beads carrying immobilized glutathione-S-transferases (15 ml packed beads) in aqueous phosphate buffer (0.1 M, pH 7.4). Total reaction volume was 20 ml. Reactions were run at 37°C for 1 h. A control non-enzymatic reaction was run in the absence of immobilized enzyme. After 1 h, the immobilized protein was removed by filtration through a coarse fritted funnel. The filtrate was concentrated using solid-phase extraction methods (Sep-Pak, C 8 cartridge, Waters, Milford, Massachusetts) and the organic eluent (methanol) was evaporated to dryness before thin-layer chromatography (TLC) and mass spectrometric analysis. Thin-layer chromatography TLC was done using aluminum-backed silica-gel plates (5 x 10 cm, E. Merck, Darmstadt) for crude reaction mixtures after solid-phase extraction. The mobile phase was ammonium hydroxide-absolute ethanol (70 : 30). After air drying, components of the incubation mixtures were visualized with ninhydrin spray reagent (0.2% in ethanol) and heated to 100°C on a hotplate for 5-10 min to produce a blue-purple color. High-performance liquid chromatography (HPLC) Incubation mixtures were purified using a Beckman Model 421 HPLC system with Model llOA pumps. Conditions were: c,, column (Brownlee, 100 x 4.6 mm, 5 pm); mobile phase, solvent A, 0.1 M ammonium acetate, pH 6.8, solvent B, methanol; linear gradient, 10-100% solvent B in 25 min; flow rate 1.0 ml min-'. Ultraviolet (UV) detection was done at 254 nm (Kratos Spectroflow Model 783 variable UV detector). Reaction products were collected into 50 ml round-bottom flasks and frozen on dry ice immediately. HPLC eluents were evaporated under vacuum and the residues stored at - 80 "C before analysis by mass spectrometry. Peak areas were integrated on an Isaac 41A Interface Module (Cyborg, Corp., Boston, Massachusetts) and Apple IIe computer. 249 Mass spectrometry Positive ion FAB mass spectra were obtained using a Kratos MS50 double-focusing magnetic sector mass spectrometer with a Kratos FAB source and DS-90 data system (accelerating voltage 8 kV, resolution 3000). Cesium iodide-glycerol was used to calibrate over the mass range 92-1005 u. Samples were dissolved in 25-50 pl methanol; thioglycerol was the liquid matrix. Samples were bombarded with a xenon atom beam at 7-8 kV kinetic energy. Spectra were plotted as averages of several scans at a scan rate of 10 s/decade. Positive ion thermospray LC mass spectra were acquired on a Finnigan TSQ-45 triple-quadrupole mass spectrometer in the Department of Drug Metabolism, Smith Kline & French Research Laboratories. A Finnigan thermospray ion source was used. No electron filament or discharge electrode was employed. Ion source conditions were : block temperature 240 "C; vaporizer temperature 120°C; repeller voltage +25 eV. Spectra were recorded in 1.95 s scans over the mass range 120-650 u. LC conditions were: RP-300 HPLC column (Brownlee, 100 x 4.6 mm, 5 pm); solvent A, 0.1 M ammonium acetate adjusted to pH 5.3 with glacial acetic acid, solvent B, methanol; linear gradient, 20% solvent B to 70% solvent B in 20 min; flow rate 1.1 ml min-'. A UV detector (Kratos Spectraflow Model 783) was placed in line before the mass spectrometer and the eluent was monitored at 254 nm. RESULTS AND DISCUSSION The HPLC chromatogram for the enzymatic reaction mixture between chlorambucil and reduced glutathione is shown in Fig. 1. Seven HPLC peaks were observed, designated as A-G. HPLC product G (retention time 19.3 min) coeluted with an authentic sample of chlorambucil and gave an identical FAB mass spectrum. HPLC products A and B coeluted with products obtained by reaction of chlorambucil with 0.1 M NaOH, suggesting that they are hydrolysis products formed by displacement of one or both of the chlorines in the chloroethyl side chains by water. FAB mass spectral analysis of purified peaks A and B gave no useful structural information when analyzed in several liquid matrices and in the presence of 0.1% HCl. The mono and dihydroxy hydrolysis products of a structural analog, melphalan, were also resistant to FAB analysk6 Thermospray LC/MS analysis of product A (retention time 4.0 min) produced a protonated molecular ion at m/z 268, corresponding to the dihydroxy analog of chlorambucil (Fig. 2). Thermospray LC/MS analysis of product B (retention time 5.3 min) produced a molecular ion M" at mlz 250, corresponding to the cyclic aziridinium ion form of the monohydroxy analog of chlorambucil (Fig. 3). Four ninhydrin-positive products were identified from enzymatic reaction mixtures containing chlorambucil and reduced glutathione (rf = 0.64, 0.58, 0.50 and 0.32). These products corresponded to HPLC fractions C, D, E and F, suggesting that four glutathione conjugates were present. The FAB mass spectrum of HPLC s- D. M. DULIK, 0. M. COLVIN AND C. FENSELAU 250 HO-H2C-H2C,N 0 \ / CH2-CH HO-H C-H C' 2 2 2 -CH-COOH 2 E mlz 0 5 15 10 25 20 Figure 2. Positive ion thermospray LC/MS spectrum of HPLC product A, dihydroxy chlorambucil. TIME (rnin) Figure 1. HPLC chromatogram of the reaction mixture of chlorambucil and reduced glutathione in the presence of immobilized microsomal glutathione-S-transferases (UV detection at 254 nm). Designated products are: A, dihydroxy chlorambucil; B, monohydroxy monochloro chlorambucil; C, diglutathionyl chlorambucil; D, monohydroxy monoglutathionyl chlorambucil; E, 4(glutathiony1)phenylbutyricacid; F, monochloro monoglutathionyl chlorambucil; G, chlorambucil. product C (retention time 8.0 min) shows a protonated molecular ion at mass 846, corresponding to the diglutathionyl conjugate of chlorambucil (Fig. 4). The FAB mass spectrum of HPLC product D (retention time 8.8 min) contains a protonated molecular ion peak at m/z 557 and a sodium adduct peak at m/z 579, corresponding to the monohydroxy monoglutathionyl conjugate (Fig. 5). This glutathione adduct of a hydrolysis product of chlorambucil is of special interest, since the analogous conjugate was not a major metabolite of the structurally similar alkylating agent, melphalan.6 The FAB mass spectrum of a minor HPLC product E (retention time 11.2 min) gave a weak signal at m/z 470 using 2nitrophenyl octyl ether as the liquid matrix. The mass H C M+ 2 I 100 %I 50 M+N~)+ 272 F,3TTT7 I m/z Figure 3. Positive ion thermospray LC/MS spectrum of HPLC product B, monohydroxy chlorambucil (cyclic aziridinium ion form). %I I x 20 50 200 300 400 800 900 Figure 4. Positive ion FAB mass spectrum of HPLC product C, diglutathionyl chlorambucil. SG = glutathionyl. GLUTATHIONE CONJUGATES OF CHLORAMBUCIL (M+H)+ 300 400 350 450 500 251 (M+Na)+ 550 600 m/z Figure 5. Positive ion FAB mass spectrum of HPLC product D, monohydroxy monoglutathionyl chlorambucil. SG spectrum is believed to correspond to 4-(glutathionyl) phenylbutyric acid. This unusual conjugate is analogous to that previously reported for melphalan, in which nucleophilic displacement of the mustard moiety occurs by glutathione, perhaps through the cyclic aziridinium ion inte~mediate.~ HPLC product F (retention time 13.0 min) was confirmed by FAB mass spectrometry as the monochloro monoglutathionyl conjugate of chlorambucil; a protonated molecular ion of mass 575 was observed as well as a potassium adduct of mass 613 (Fig. 6). Thermospray LC/MS analysis of products C-F gave no protonated molecular ions under the conditions employed; a common fragment ion of mass 147 was observed for each product, resulting from loss of glutamic acid from the intact glutathione conjugate. This characteristic loss of 147 u in the thermospray LC mass spectra of other glutathione conjugates has been and is most likely a result of the thermal lability of the tripeptide. Integration of the HPLC/UV peak areas for peaks A-G demonstrated that the enzyme-catalyzed reaction of chlorambucil with glutathione produced a modest two- to fivefold increase in the relative amounts of glu- C I - H2C-H2C C-H c' 2 2 GS-H 0 1 \ CH-CH 2 2 -CHZ-COOH 254 'O01 %I rt I I I I j 5 (M+H)+(M+K)+ 0 200 250 300 350 400 450 500 550 600 0 650 mlz Figure 6. Positive ion FAB mass spectrum of HPLC product F, monochloro monoglutathionyl chlorambucil. SG = glutathionyl. = glutathionyl tathione conjugates formed as compared with the nonenzymatic reaction. Formation of product E was negligible in the non-enzymatic reaction. A marked decrease in the amounts of peaks A and B were observed in the enzymatic reaction. In general, the presence of immobilized microsomal glutathione-s-transferases may serve to increase the relative nucleophilicity of the cysteine sulfhydryl moiety of glutathione and therefore provide increased competition for nonenzymatic hydrolysis of the active alkylating agent. These observations suggest that chlorambucil, as well as other alkylating agents in this class, are substrates for the microsomal glutathione-S-transferases. No studies have been done with the cytosolic forms of the enzyme in our laboratory to date. In summary, chlorambucil reacts with glutathione to produce several conjugates which are formed by nucleophilic displacement of one or both chlorines from the nitrogen mustard side-chain. The products formed are analogous to those produced by reaction of glutathione with the alkylating agent melphalan. The glutathione conjugates are amenable to characterization by positive ion FAB mass spectrometry and produce fragment ions in thermospray LC/MS which are characteristic of the glutathione moiety. The hydrolysis products of chlorambucil are resistant to analysis by FAB mass spectrometry, but produce molecular ions in thermospray LC/MS mode. This may in part be due to the relative polarity of the hydrolysis products in the liquid matrix in FAB which impedes desorption, or perhaps analysis is hindered due to the presence of interfering buffer salts. The formation of glutathione conjugates of chlorambucil may play a role in the development of acquired drug resistance in tumor cells. Further studies will address the presence of chlorambucil-glutathione conjugates in sensitive and resistant cells and the possible biochemical mechanisms responsible for the development of ADR. Acknowledgements The authors gratefully acknowledge the financial support of US Public Health Service Grants GM-21248 and CA-16783-11 from the 252 D. M. DULIK, 0. M. COLVIN AND C. FENSELAU National Institutes of Health and the US Department of Agriculture Specific Cooperative Agreement 58-579-4-14. We thank Connie Murphy, Mid Atlantic Mass Spectrometry Laboratory, a National Science Foundation Regional Instrumentation Facility, for providing FAB mass spectral data. We thank Dr. John Hilton, Johns Hopkins School of Medicine, for helpful discussions. REFERENCES 1. T. A. Connors, Handbook Exper. Pharmacol. 72,403 (1984). 2. K. Suzukake, B. P. Vistica and D. T. Vistica, Biochem. Pharmacol. 32,165 (1 983). 3. A. L. Wang and K. D. Tew, Cancer Treat. Rep. 69,677 (1985). 4. R. F. Ozols, K. G. Louie, J. Plowman, B. C. Behrens, R. L. Fine, D. Dykes and T. C. Hamilton, Biochem. Pharmacol. 36, 147 (1987). 5. S. L. Pallante, C. A. Lisek, D. M. Dulik and C. Fenselau, Drug Metab. 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