Characterization and Quantification of Cysteinyl Adducts of Benzene

Microsomal epoxide hydrolase (EPHX1) polymorphisms are associated with aberrant promoter methylation of ERCC3 and hematotoxicity in benzene-exposed ...
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Characterization and Quantification of Cysteinyl Adducts of Benzene Diol Epoxide Suramya Waidyanatha, Ramiah Sangaiah, and Stephen M. Rappaport* Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7431 Received April 9, 2005

The production of macromolecular adducts of benzene diol epoxide (BDE), a toxic metabolite of benzene, has received little attention despite the demonstrated mutagenicity and carcinogenicity of BDE in rodents. Syn and anti enantiomers of BDE were relatively stable in 0.1 M ammonium acetate buffer, pH 7.6 (half times were greater than 5 h), and showed evidence of pseudo-first-order reactions with albumin (half times were about 4 h) and glutathione (GSH) (half times were about 0.3-0.4 h). Reaction products of BDE isomers with L-cysteine, N-acetylL-cysteine, N-acetyl-L-cysteine methyl ester, and GSH were characterized by a combination of electrospray ionization mass spectrometry and/or gas chromatography-mass spectrometry with electron impact ionization of trimethylsilyl derivatives of the adducts. Products corresponded to 1:1 addition of BDE isomers with each nucleophilic species, suggesting that adduction occurred primarily at the free sulfhydryl group. To investigate the disposition of the BDEs in vivo, we developed an assay for cysteinyl BDE-protein adducts. The assay involves enzymatic hydrolysis of the protein followed by derivatization of the released adducts and gas chromatography-negative ion chemical ionization-mass spectrometry. Preliminary applications of the assay showed linear increases in the formation of BDE-GSH adducts in samples of GSH incubated with increasing concentrations of BDE (10-300 µM) and showed the presence of BDE-albumin following incubation of albumin with 10 µM BDE.

Introduction Benzene is an important industrial chemical that is ubiquitous in the environment due to its presence in petroleum-based fuels, engine exhausts, and cigarette smoke (1, 2). Although benzene causes human hematotoxic effects and leukemia (3-5), the exact mechanism is not known (3, 5, 6). However, there is evidence that metabolism of benzene plays a major role in the production of toxic effects (3, 7). As shown in Figure 1, benzene is metabolically transformed to a host of reactive electrophiles, namely, benzene oxide, 1,2- and 1,4-benzoquinone, the muconaldehydes, and benzene diol epoxide (BDE)1 (reviewed in ref 6), all of which can react with DNA and proteins to form adducts. Because these reactive electrophiles are shortlived, protein adducts in serum albumin and hemoglobin have been used to study the in vivo disposition of benzene oxide and 1,2- and 1,4-benzoquinone (8-17). Of all of the reactive intermediates derived from benzene, BDE has received the least attention, despite its demonstrated mutagenicity in bacterial and mammalian cells and carcinogenicity in rodents (18-20) and despite evidence that diol epoxide metabolites of some * To whom correspondence should be addressed. Tel: 919-966-5017. Fax: 919-966-0521. E-mail: [email protected]. 1 Abbreviations: BDE, benzene diol epoxide; BDE-Alb, albumin adduct of BDE; BDE1, syn-benzene diol epoxide; BDE2, anti-benzene diol epoxide; BDE-CYS, L-cysteine adduct of BDE; BDE-GSH, glutathione adduct of BDE; EI, electron ionization; GC-MS, gas chromatography-mass spectrometry; GSH, glutathione; BDE-NAC, N-acetylL-cysteine adduct of BDE; BDE-NACME, N-acetyl-L-cysteine methyl ester adduct of BDE; NCI, negative ion chemical ionization; TFAA, trifluoroacetic anhydride; TMS, trimethylsilyl derivative.

polycyclic aromatic hydrocarbons are potent carcinogens (21). Various isomeric forms of BDE are postulated to arise from benzene oxide via epoxide hydrolase and a second CYP oxidation (Figure 1) (22), a pathway analogous to the formation of diol epoxides of polycyclic aromatic hydrocarbons (21, 23-25). Because we seek an assay for blood protein adducts of BDEs to complement our existing assays of other reactive benzene metabolites (14, 26), we undertook the current investigation to characterize products of reactions between syn and anti diastereomers of BDE, BDE1 and BDE2, respectively, and various nucleophiles containing sulfhydryl moieties and to quantify these adducts in modified proteins. Toward this goal, we synthesized racemic BDE1 and BDE2 and reacted them with Lcysteine, N-acetyl-L-cysteine, N-acetyl-L-cysteine methyl ester, glutathione (GSH), and human serum albumin. Then, we developed an assay to quantify levels of cysteinyl adducts of the BDEs, based upon enzymatic digestion of the adducted protein, derivatization, and gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI-MS).

Materials and Methods Caution: Trifluoroacetic anhydride (TFAA) reacts violently with water and should only be used to derivatize samples that are completely dry. Chemicals. BDE isomers were synthesized employing the methods of Platt and Oesch and Aleksejczyk et al. (22, 27) as outlined in Figure 2. Human serum albumin (fraction V, lyophilized powder), L-cysteine, N-acetyl-L-cysteine, N-acetyl-L-cysteine methyl ester, GSH, pronase E (EC 3.4.24.4), methanesulfonic

10.1021/tx0500981 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/24/2005

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Figure 1. Metabolism of benzene showing reactive metabolites.

Figure 2. Scheme for the synthesis of BDE isomers based upon refs 22 and 27.

acid, and triethylamine were obtained from Sigma-Aldrich Inc. (St. Louis, MO). Hydrochloric acid (concentrated), hexane (nanograde), methanol, and ethyl acetate were from Fisher Scientific (Pittsburgh, PA). Aminopeptidase M (EC 3.4.11.2), Trisil reagent, and TFAA were purchased from Pierce (Rockford, IL). [2H6]Hydroquinone was from Cambridge Isotope Laboratories

(Woburn, MA). TFAA was distilled once before use. The TFAIPA amino acid derivatization kit was from Alltech (Deerfield, IL). Stability of (()BDE1 and (()BDE2 in Buffered Media. The stability of BDE isomers in 0.1 M ammonium acetate (pH 7.6) was studied in the presence and absence of GSH and

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albumin. To 1 mL aliquots of either 0.1 M ammonium acetate (pH 7.6), GSH (10 mg/mL in 0.1 M ammonium acetate, pH 7.6), or albumin (10 mg/mL in 0.1 M ammonium acetate, pH 7.6) was added 25 µL of 0.1 mg/mL (()BDE2 or (()BDE1 in ethyl acetate to give a final BDE concentration of 20 µM. Samples were incubated at room temperature for varying times and extracted immediately with 1.5 mL of ethyl acetate containing 5 ng of [2H6]hydroquinone as internal standard. After drying with anhydrous Na2SO4, ethyl acetate was removed under nitrogen. The residue was resuspended in 100 µL of hexane and reacted with 100 µL of Trisil reagent at 70 °C for 30 min, and the resulting trimethylsilyl derivative (TMS) of BDEs was analyzed by GC-electron impact ionization (EI)-MS in selective ion monitoring mode. Reaction of BDE with N-Acetyl-L-cysteine Methyl Ester, L-Cysteine, N-Acetyl-L-cysteine, and GSH and Characterization of Adducts. The products of reaction between BDE and N-acetyl-L-cysteine-methyl ester, L-cysteine, N-acetyl-L-cysteine, and GSH are designated as N-acetyl-L-cysteine methyl ester adduct of BDE (BDE-NACME), L-cysteine adduct of BDE (BDECYS), N-acetyl-L-cysteine adduct of BDE (BDE-NAC), and the GSH adduct of BDE (BDE-GSH), respectively. Samples containing 117 µmol of either (()BDE1 or (()BDE2 in ethyl acetate were reacted with a 1.2 molar excess of N-acetylL-cysteine methyl ester in 1 mL of ethyl acetate for 17 h at room temperature in the presence of 40 µL of triethylamine to produce BDE-NACME. Similarly, 7.8 µmol of (()BDE2 or (()BDE1 was reacted with a 1.2 molar excess of either L-cysteine, N-acetylL-cysteine, or GSH in 3 mL of 0.1 M ammonium acetate buffer, pH 7.6, to produce BDE-CYS, BDE-NAC, or BDE-GSH, respectively. Five to ten microliters of the above reaction mixtures, except that of BDE-NACME, were directly analyzed by ESIMS as described below. One-half milliliter portions of the reaction mixtures of BDE-NACME and BDE-NAC were dried under vacuum, dissolved in 100 µL of hexane, and derivatized with 100 µL of Trisil reagent at 70 °C for 30 min and analyzed by GC-EI-MS in scan mode as described below. Reaction of Cysteinyl Adducts of BDE with Methanesulfonic Acid and TFAA. One-half milliliter portions of the above reaction mixtures containing BDE-NACME, BDE-NAC, BDE-CYS, and BDE-GSH were brought to dryness under vacuum, and the residues were reacted with either 750 µL of TFAA and 20 µL of methanesulfonic acid at 100 °C for 40 min as described previously for adducts of benzene oxide and the benzoquinones (14, 26) or with 750 µL of TFAA and 40 µL of methanesulfonic acid at 100 °C for 17 h (modified assay conditions) to generate derivatives suitable for quantitation by GC-MS. Enzymatic Digestion of BDE-Adducted Proteins or Peptides and Subsequent Derivatization of Adducts. Ten milligrams of either albumin or GSH in 1 mL of 0.1 M ammonium acetate, pH 7.6, was reacted overnight with (()BDE2 or (()BDE1 (dissolved in ethyl acetate) to give final BDE concentrations of 10 µM and 1 mM. The reaction products were dried briefly in a Speed Vac to remove traces of ethyl acetate. To 100-300 µL portions of these solutions containing the protein adducts [i.e., albumin adduct of BDE (BDE-Alb) and BDE-GSH] was added 700-900 µL of 0.1 M ammonium acetate, pH 7.6. Samples were digested with pronase E (EC 3.4.24.4) (8% w/w) for 24 h at 50 °C with the addition of the second portion of the enzyme after 4 h. The digestion was continued at 50 °C for an additional 24 h with the addition of 1 µL of 1 µg/µL aminopeptidase M (EC3.4.11.2). Samples were brought to dryness in a Speed Vac and reacted with 200 µL of 0.2 M HCl at 110 °C for 5 min. Each sample was dried in a Speed Vac, and the resulting residue was reacted with 400 µL of a mixture of isopropyl alcohol:acetyl chloride: (5:1) at 100 °C for 15 min. After the mixture was cooled to room temperature, the excess reagents were removed in a Speed Vac and the residue was reacted with 400 µL of TFAA at 100 °C for 15 min. Volatile byproducts were removed under a gentle stream of nitrogen, and the residue was reconstituted in 1 mL of hexane. The hexane solution was

Waidyanatha et al. washed once with 1 mL of deionized water and concentrated to 200 µL. The samples were analyzed by GC-MS in either scan or selected ion monitoring mode. Concentration-Dependent Formation of (()BDE1 and (()BDE2 Adducts with GSH in Vitro. To 3 mg portions of GSH in 1 mL of 0.1 M ammonium acetate buffer (pH 7.6) was added 20 µL of an ethyl acetate solution containing sufficient (()BDE1 or (()BDE2 to give final concentrations of BDE of 0, 10, 30, 100, and 300 µM. Samples were reacted at room temperature for 17 h on an orbital shaker, briefly dried in a Speed Vac to remove the ethyl acetate, and then carried through the enzymatic digestion and derivatization steps as described above. Final samples were analyzed by GC-NICI-MS in selected ion monitoring mode as described below, and adducts were quantified by external calibration against BDE-CYS. Standard curves were prepared by spiking 3 mg portions of GSH with 0-100 nmol of BDE-CYS and carrying out the assay as described for the samples. Mass Spectrometry. Reactions between BDEs and sulfhydryl groups of L-cysteine, N-acetyl-L-cysteine, and GSH were characterized by ESI-MS. These reaction mixtures were analyzed on an LCQ DECA 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 5-10 µL of each sample into the mass spectrometer to obtain full scans and product ions. The MS/MS collision energy was 35 V, and the argon pressure was 333 Pa. All other samples were analyzed on either a HP 5980 plus series II gas chromatograph coupled to a HP 5989-B MS engine or an Agilent 6890N gas chromatograph coupled to a Agilent 5973N mass selective detector. A DB-5 fused silica capillary column (60 m, 0.25 mm i.d., 0.25 µm film thickness) was used with He as the carrier gas at a flow rate of 1.5 mL/min. The injector and MS transfer line temperatures were 250 and 280 °C, respectively. Source and quadrupole temperatures were, respectively, 130 and 100 °C for NICIMS and 200 and 100 °C for GC-EI-MS. For the quantitation of BDEs from the stability experiment, the GC oven was held at 75 °C for 3 min and was then ramped at 8 °C/min to 165 °C where it was held for 10 min. Because the TMS derivatives of racemic BDEs were not separable under the GC-MS conditions used, they were quantified as a single peak. Ions monitored in GC-EI-MS, for TMS derivatives of BDE and [2H4]HQ, the internal standard, were m/z 272 and m/z 258, respectively. For the characterization of BDE-NAC and BDE-NACME by GC-MS, the adducts were first converted to their corresponding TMS derivatives as described previously. These derivatives were subsequently analyzed by GC-EI-MS by scanning the mass spectrometer from m/z 50-700. The GC oven was held at 75 °C for 3 min and was then ramped at 10 °C/min to 270 °C where it was held for 20 min. The adducted BDE-CYS, either in standard form or following release from BDE-GSH or BDE-Alb, was derivatized using isopropyl alcohol:acetyl chloride (5:1) and TFAA to convert it to S[O2,O3,O4-(trifluoroacetyl)-cyclohex-5-nyl]-N-trifluoroacetylcysteine isopropyl ester and was characterized in both GC-EIMS and GC-NICI-MS by scanning the mass spectrometer from m/z 50 to 700. The GC oven was held at 75 °C for 3 min and was then ramped at 10 °C/min to 250 °C where it was held for 15 min. For the quantitation of these adducts by GC-NICI-MS, in selected ion monitoring mode, the GC oven was held at 75 °C for 3 min and was ramped at 5 °C/min to 195 °C where it was held for 12 min. Late eluting compounds were removed by raising the oven temperature to 250 °C and holding there for 15 min. Retention times of the two adducts were 35.48 and 36.84 min, and ions monitored were m/z 447, 371, and 225.

Results and Discussion Stability of (()BDE1 and (()BDE2. Because the initial concentration of BDE was low relative to that of

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Table 1. Pseudo-First-Order Reaction Rate Constants (ke) and Half Lives (t1/2) of 20 µM (()BDE1 and (()BDE2 in 0.1 M Ammonium Acetate Buffer (pH 7.6) with and without 10 mg/mL GSH or Albumin electrophile

medium

ke (h-1)

t1/2 (h)

(()BDE1 (()BDE1 (()BDE1 (()BDE2 (()BDE2 (()BDE2

buffer buffer + GSH buffer + albumin buffer buffer + GSH buffer + albumin

0.118 1.61 0.173 0.127 2.38 0.182

5.85 0.429 4.08 5.46 0.291 3.83

the available nucleophiles, the overall rate of disappearance of (()BDE in the medium should have been pseudofirst-order, with a rate constant (ke) representing the sum of all first-order rate constants of individual reactions. Because the concentration of BDE at time t, [BDE]t ) [BDE]0e-ket, then ke and the corresponding half-time t1/2 ) ln(2)/ke can be estimated from the slope of the linear regression of ln[BDE]t on t. Pseudo-first-order reaction rate constants and corresponding half-times of (()BDE1 and (()BDE2 at 25 °C, in the presence and absence of GSH and albumin, are presented in Table 1. The BDEs were fairly stable in 0.1 M ammonium acetate buffer, pH 7.6, with values of t1/2 greater than 5 h for both syn and anti diastereomers. Following the addition of albumin (10 mg/mL) to the buffer, the BDE half-times were marginally reduced to about 4 h; the addition of GSH (10 mg/ mL) reduced the BDE half-times more than 10-fold to about 0.3-0.4 h. This is in contrast to what we have previously observed for benzene oxide where the t1/2 at pH 7 and 25 °C was 34 min indicating that BDE is more stable than benzene oxide. Furthermore, unlike with BDE, the t1/2 of benzene oxide did not change significantly in the presence of 2-15 mM GSH at 37 °C and at varying pH values (pH 7, 8, 8.5, and 10). This indicates less efficient reaction of benzene oxide with GSH as compared to BDE (28). Reactions of BDEs and Sulfhydryl-Containing Nucleophiles. Reaction products of the BDEs and

various sulfhydryl-containing nucleophiles were studied by mass spectrometry. Racemic mixtures of BDE1 and BDE2 were reacted with L-cysteine, N-acetyl-L-cysteine, N-acetyl-L-cysteine methyl ester, and GSH, and the resulting adducts were characterized by GC-MS and/or ESI-MS/MS. In each case, the addition of one molecule of BDE to the nucleophile was observed. This indicates that the major site of adduction was a free sulfhydryl group in the above molecules. We previously observed similar behavior for the reaction of benzene oxide with the same nucleophiles (28). The negative ion mass spectrum from ESI-MS is shown in Figure 3A for the reaction product of (()BDE2 and GSH. The base peak at m/z 434 corresponds to (M - H)-. The daughter ion spectrum (MS/MS) of m/z 434, shown in Figure 3B, has a base peak at m/z 416 that corresponds to a loss of water from (M - H)-. The adducts formed by reactions between (()BDEs and N-acetyl-L-cysteine and N-acetyl-L-cysteine methyl ester were converted to the corresponding TMS derivatives, which were characterized by GC-EI-MS. The total ion chromatogram, shown in Figure 4A for the TMS derivative of BDE2-NACME, has two peaks of equal intensity, suggesting the preferential attack by the sulfhydryl moiety on one carbon of the epoxide ring of (+)- and (-)BDE2. Because we do not have pure (+) and (-) enantiomers of BDE2, we were unable to distinguish between the diastereomeric adducts arising from the different enantiomers. Spectra of both peaks show similar fragmentation, with the largest fragment ion being m/z 431, corresponding to a loss of (SiCH3OH) from the molecular ion of m/z 521 (Figure 4B). Assay for Detection of Cysteinyl BDE Adducts. Portions of BDE-CYS, BDE-NAC, BDE-GSH, and BDEAlb were reacted with methanesulfonic acid and TFAA according to a procedure originally developed for cysteinyl adducts of benzene oxide and 1,4-benzoquinone (14). That procedure involves the reaction of the intact adducted

Figure 3. Mass spectra of cysteinyl adducts produced by reaction of racemic BDEs with GSH. (A) ESI-MS mass spectrum; (B) MS/MS mass spectrum of parent ion at m/z 434.

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Figure 4. (A) GC-EI-MS total ion chromatogram of products of reaction between racemic BDE2 and N-acetyl-L-cysteine methyl ester after trimethylsilylation; peaks 1 and 2 correspond to products between racemic BDE and sulfhydryl groups. (B) Mass spectrum of peak 1.

Figure 5. Scheme for analysis of cysteinyl protein adducts of BDE (designated BDE-Y) based upon enzyme hydrolysis and reaction with TFAA and isopropyl alcohol:acetyl chloride (5:1) to produce volatile derivatives suitable for GC-NICI-MS.

protein with methanesulfonic acid:TFAA (1:40) at 100 °C for 40 min. Under these conditions, the expected fluorinated derivative was not observed. However, by changing the ratio of methanesulfonic acid:TFAA to 1:20 and carrying out the reaction with BDE-CYS at 100 °C for 17 h, the expected derivative, S3,O4,O5,O6-tetra(trifluoroacetyl)cyclohex-1-ene, was observed at less than 50% yield. This low yield, coupled with rather extreme reaction conditions, suggested that the original assay was unsuitable for quantitation of cysteinyl BDE adducts. As an alternative approach to the quantitation of cysteinyl BDE-protein adducts, we exhaustively hydrolyzed an adducted protein (BDE-Alb) and an adducted peptide (BDE-GSH) with enzymes to release BDE-CYS and reacted the hydrolysates successively with isopropyl alcohol:acetyl chloride (5:1) and TFAA to produce the

derivative, suitable for analysis by GC-MS (Figure 5). Figure 6A shows a GC-EI-MS total ion chromatogram of this derivative released from BDE2-GSH by this reaction scheme. The chromatogram shows two isomers of equal intensity, from the reaction between (-)BDE2 and (+)BDE2 and the sulfhydryl group of GSH. The corresponding mass spectra of both isomers gave very similar fragmentation patterns, illustrated in Figure 6B for peak 1. The spectrum contains a fragment ion at m/z 616, which corresponds to a loss of 57 amu (C3H7O) from the molecular ion of m/z 675. Other characteristic fragment ions in the spectrum of this derivative are shown in Figure 6B. The corresponding GC-NICI-MS spectrum is shown in Figure 7. The largest fragment ion in the spectrum, at m/z 447, results from loss of two molecules of CF3COOH from the molecular ion of m/z 675 (Figure

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Figure 6. (A) GC-EI-MS total ion chromatogram of cysteinyl GSH adducts of racemic BDE2 after enzymatic hydrolysis and derivatization according to the scheme in Figure 5; peaks 1 and 2 refer to isomeric adducts from racemic BDE2. (B) Mass spectrum of peak 1.

Figure 7. NICI mass spectrum of cysteinyl GSH adducts of racemic BDE2 after enzymatic hydrolysis and derivatization according to the scheme in Figure 5. The base peak at m/z 371 corresponds to the circled fragment ion.

7). The base peak in the spectrum is m/z 371, which is characteristic of the derivative as shown in Figure 7. This characteristic ion should be suitable for quantitation of BDE-CYS by GC-NICI-MS in selected ion monitoring mode. As shown in Figure 8, applications of the assay to portions of albumin that had been incubated with 10 µM (()BDE2 also released BDE-CYS that was detected as

two peaks via GC-NICI-MS in selected ion monitoring mode using m/z 371. Dose-Dependent Formation of BDE-GSH. To study the feasibility of quantifying cysteinyl adducts of BDEs, the production of adducts was investigated by reacting GSH with (()BDE2 in vitro at concentrations of 10, 30, 100, and 300 µM. Samples were quantified by GC-NICI-

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Figure 8. GC-NICI-MS selected ion monitoring chromatogram of cysteinyl albumin adducts following incubation of albumin with racemic BDE2 (10 µM) after enzymatic hydrolysis and derivatization according to the scheme in Figure 6. Peaks 1 and 2 refers to isomeric adducts from racemic BDE2.

Conclusions

Figure 9. Formation of GSH adducts of BDE2 following incubation of GSH with 0-300 µM (()BDE2 in 0.1 M ammonium acetate buffer. The two curves correspond to diastereomeric adducts from racemic BDE2.

MS in selected ion monitoring mode using m/z 371, corresponding to the sulfhydryl-bound adduct, based on external calibration against BDE-CYS. Levels of adducts are presented in Figure 9 as nmol adduct/mg of GSH vs the initial concentration of (()BDE2. Linear production of BDE2-GSH was observed with increasing concentrations of (()BDE2. The two curves represent adducts corresponding to the reaction of (+)- and (-)BDE2 with GSH. Because we do not have pure (+) and (-) enantiomers of BDE2, we cannot identify each diastereomeric adduct at this time. The slopes of the dose-response curves for the two enantiomeric adducts were 0.086 and 0.100 (nmol BDE2GSH/mg GSH) (µM BDE2). As indicated previously, without standards, we cannot determine which curves represent the diastereomeric adduct formed from the (+) and (-) enantiomers. However, assuming 50% each of (+)- and (-)BDE2 in racemic BDE2, we estimate that 0.52 and 0.60 nmol of (+)- and (-)BDE2-GSH were produced per nmol of (+)- or (-)BDE2 used in the reaction. For example, [0.1 nmol (+ or -)BDE2 - GSH/mg GSH](3 mg GSH) ) [nmol (()BDE2/mL](1 mL)[0.5 (+ or -)BDE2/(()BDE2] 0.60 nmol (+ or -)BDE2 - GSH nmol (+ or -)BDE2

We observed that racemic BDEs are fairly stable in aqueous media in the absence of nucleophiles and found evidence of pseudo-first-order reactions of BDEs in the presence of albumin and GSH. Reactions of racemic BDEs with moieties containing free cysteine residues indicate that BDEs react predominantly with sulfhydryl groups to produce adducts. We developed a GC-NICI-MS assay to quantify these cysteinyl adducts and demonstrated that adducts were formed in a dose-dependent fashion by reacting GSH with increasing concentrations of racemic BDEs. Future work will synthesize isotopically labeled internal standards to quantify these adducts in serum albumin of humans exposed to benzene.

Acknowledgment. We thank Dr. Yutai Li for helping with the ESI-MS experiment and Dr. A. P. Henderson for helpful discussions. This work was supported by the National Institute of Environmental Health Sciences through Grants P42ES05948 and P30ES10126.

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