Document vVeY2vOq4QV2G70orNZqZ29q6

Chemico-Biological Interactions 153154 (2005) 261266 Investigation of cysteinyl protein adducts of benzene diolepoxide Suramya Waidyanatha , Stephen M. Rappaport Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7400, USA Available online 8 April 2005 Abstract Metabolism of benzene is fairly complex yielding several reactive metabolites that could form adducts with macromolecules such as DNA and proteins. Systemic availability of these reactive species can be estimated by measuring the corresponding adducts of these species with serum albumin or hemoglobin. We have shown previously that serum albumin adducts of benzene oxide and 1,4-benzoquinone increased in a dose-dependent manner in rodents and humans exposed to benzene. However, formation of macromolecular adducts of other reactive metabolites, such as benzene diolepoxide and E,E-muconaldehyde, has not received much attention despite the demonstrated mutagenicity and/or carcinogenicity of these metabolites. We studied the reaction of isomers of benzene diolepoxide, namely syn-(()BDE1) and anti-BDE (()BDE2), with sulfhydryl groups in vitro. The half-life of BDE2 in 0.1 M ammonium acetate buffer (pH 7.6) was (5.46 h) longer that that in serum albumin in the same buffer (3.83 h) indicating the reaction of BDE with nucleophilic sites on albumin. Reaction products between these and l-cysteine, and GSH were identified and characterized by mass spectrometry in an attempt to develop an assay to quantify these adducts in serum albumin. 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Benzene; Reactive metabolites; Benzene diolepoxide; Protein adducts; Sulfhydryl groups 1. Introduction Metabolism of benzene is complex, yielding several reactive metabolites that could form adducts with macromolecules such as DNA and proteins within the body (Fig. 1). Systemic availability of these species can be estimated by measuring the corresponding adducts with serum albumin or hemoglobin. We have Corresponding author. E-mail address: suramya@unc.edu (S. Waidyanatha). shown previously that serum albumin adducts of benzene oxide and 1,4-benzoquinone increased in a dosedependent manner in rodents and humans exposed to benzene [13]. However, formation of macromolecular adducts of other metabolites, such as benzene diolepoxide (BDE) and muconaldehyde, has not received much attention despite the demonstrated mutagenicity and/or carcinogenicity of these metabolites [49]. BDEs arise from benzene oxide, the initial metabolite of benzene, via the dihydrodiol pathway and 0009-2797/$ see front matter 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2005.03.033 262 S. Waidyanatha, S.M. Rappaport / Chemico-Biological Interactions 153154 (2005) 261266 Fig. 1. Metabolism of benzene showing reactive metabolites. a second P450 oxidation (Fig. 1). The isomers of BDE are designated as syn-(()BDE1) and anti-BDE (()BDE2) (Fig. 2). Although no direct evidence is available for the formation of these diolepoxides from benzene, ()BDE2 has been shown to induce lung tumors in newborn mice [4]. The objective of this was to study the reaction between BDE isomers and sulfhydryl groups in an attempt to develop an assay to quantify these adducts in blood proteins. We synthesized racemic BDE1 and BDE2 as shown in Fig. 2 and studied the reactions between these isomers and sulfhydryl groups on cys- S. Waidyanatha, S.M. Rappaport / Chemico-Biological Interactions 153154 (2005) 261266 263 Fig. 2. Scheme for the synthesis of ()BDE1 and ()BDE2 [10,11]. teine (BDE-Cys), glutathione (BDE-Glu) and albumin (BDE-Alb). 2. Methods 2.1. Stability of ()BDE2 in aqueous media To 1 ml aliquots of either 0.1 M ammonium acetete (pH 7.6) or 25 mg/ml albumin in 0.1 M ammonium acetate (pH 7.6) was added ()BDE2 in ethyl acetate to give a final concentration of 200 M BDE2. Samples were reacted for desired times at room temperature and extracted immediately with 2 ml ethyl acetate. Ethyl acetate layers were dried with anhydrous Na2SO4 and were brought to dryness under nitrogen. The residue was resuspended in 100 l hexane, reacted with Trisil reagent (Pierce, IL) and the corresponding trimethylsilyl derivative was analyzed by GCEI-MS in selective ion monitoring mode. 2.2. Reaction of ()BDE2 with l-cysteine and GSH and characterization of adducts ()BDE2 (7.8 mol) was reacted overnight at room temperature, with a 1.2 molar excess of either lcysteine or GSH in 3 ml of 0.1 M ammonium acetete (pH 7.6). Reaction mixtures were analyzed on an LCQDECA quadrupole ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an API2 electrospray ion source operating in the negative mode. Direct injections were performed by introducing 5 l of each sample into the mass spectrometer to obtain full scans as well as product ions. The MS/MS 264 S. Waidyanatha, S.M. Rappaport / Chemico-Biological Interactions 153154 (2005) 261266 Fig. 3. Reaction of BDE-Cys and BDE-Alb with methanesulfonic acid and trifluoroacetic anhydride releases the trifluoroacetylated derivatives for GCMS. Fig. 4. ESIMS and MS/MS spectrum of BDE-Cys adduct. S. Waidyanatha, S.M. Rappaport / Chemico-Biological Interactions 153154 (2005) 261266 265 Fig. 5. GCEI-MS of cys derivative of ()BDE2. collision energy was 35 V and the argon pressure was 2.5 mTorr. 2.3. Reaction of BDE2-Cys, BDE2-Glu and BDE-Alb with methanesulfonic acid and trifluoroacetic anhydride (MT assay) (BDE2-Cys, BDE2-Glu and BDE2-Alb were reacted using the MT assay as described elsewhere for measuring benzoquinone and benzene oxide adducts [12]. Existing conditions were modified to enhance the cleavage of BDE adducts as shown in Fig. 3. Samples were analyzed by GCNICI- and EI-MS using HP 5890 GC coupled to a 5989 B MS engine. 3. Results and discussion Reaction of BDE-Cys with methanesulfonic acid and trifluoroacetic anhydride under modified MT assay conditions released a derivative, in moderate yields, suitable for GCMS. However, BDE-Glu and BDEAlb gave poor yields of this derivative and hence is not a suitable method to quantify these adducts (Fig. 3). We looked into an alternate approach involving enzyme hydrolysis to release the adducted cysteine followed by derivatization and GCMS to detect the final derivatives. Initial investigation of digestion of BDE2-Glu with pronase E and subsequent derivatization and GCMS of the adducted cysteine shows the expected product (Fig. 5). Chromatogram shows two major and two minor products due to nucleophilic attack by sulfhydrl group on both carbons of the epoxide ring of ()BDE2. ()BDE2 is fairly stable in 0.1 M ammonium acetete (pH 7.6) (half-life 5.46 h) giving a suitable medium to study the reaction between this diolepoxide and nucleophiles. Half-life of ()BDE2 in in serum albumin in the same buffer was 3.83 h indicating the reaction of ()BDE2 with nucleophilic sites on albumin. ESI/MS and MS/MS confirms the formation of adducts of ()BDE2 with l-cysteine and GSH (Fig. 4). Acknowledgements The authors thank Drs. Ramiah Sangaiah and Yutai Li in the chemistry core for synthesizing BDE isomers and for helping with ESI/MS experiments, respectively. This work was supported by the National Institute of Environmental Health Sciences through grant nos. 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