Tandem Mass Spectrometric

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Chem. Res. Toxicol. 2004, 17, 1038-1046

Synthesis and Liquid Chromatography/Tandem Mass Spectrometric Characterization of the Adducts of Bisphenol A o-Quinone with Glutathione and Nucleotide Monophosphates Sheng-Xiang Qiu, Richard Z. Yang, and Michael L. Gross* Department of Chemistry, Box 1134, Washington University, One Brookings Drive, St. Louis, Missouri 63130 Received January 30, 2004

An environmental, estrogen-like substance, bisphenol A (BPA), is the monomer for the production of polycarbonate plastics used in baby bottles, dental sealants, and as a major component of epoxy resin for the lining of food cans. The oxidation of BPA leads to the reactive electrophilic BPA-o-3,4-quinone (BPA-Q), which can damage DNA and may be implicated in cancer initiation. BPA-Q reacts in vitro with 2′-deoxyguanosine 5′-phosphate (dGMP) and 2′deoxyadenosine 5′-phosphate (dAMP) but not with 2′-deoxycytidine-5′-phosphate and 2′deoxythymidine 5′-phosphate. In aqueous acetic acid, BPA-Q also reacts with 2′-deoxyguanosine (dG) and 2′-deoxyadenosine (dA) but not with 2′-deoxycytidine and 2′-deoxythymidine. The reactions are accompanied by loss of the modified base (depurination). We determined the structures of the modified bases by primarily tandem mass spectrometry. In mixtures of deoxynuclesides and deoxynucletides treated with BPA-Q, reactions occur more readily with dGMP/dG followed by dAMP/dA. With calf thymus DNA, significant apurinic sites must be produced because we detected the BPA-Q-guanosine adduct in the incubation mixture. We also found that BPA-Q reacts readily with glutathione (GSH) under acidic or neutral conditions, and we characterized the BPA-Q-GSH conjugate with tandem mass spectrometry (MS/MS). The results are consistent with a mechanism of carcinogenesis whereby BPA-Q, formed in vivo and not adequately detoxified by reactions with GSH, reacts with DNA, causing depurination. The adducts reported will also be appropriate references for identification of BPA-Q adducts in environmental and biological systems.

Introduction A growing body of evidence strongly suggests that excessive exposure to estrogens through early menarche, late menopause, estrogen replacement therapy, and environmental pollution by estrogenic chemicals (“environmental estrogens”) increases the risk of developing cancer in several tissues, especially the breast and endometrium (1-4). Environmental estrogens (xenoestrogens) are diverse groups of chemicals that mimic estrogenic actions (5). Many of these estrogen-like chemicals are substituted phenols (6). BPA,1 which is also estrogenic but occurs at levels that are 3-4 orders of magnitude lower than that of estradiol (7), is widely used as a monomer for the production of polycarbonate (PC) plastics that are used in baby bottles, in the lining of food cans, and in dental sealants. Although manufacturer and government claims assert that BPA is safe and its entry into the environment has no impact on human health (8), the safety/toxicology of BPA is of increasing public * To whom correspondence should be addressed. Tel: 314-935-4814. Fax: 314-935-7484. E-mail: [email protected]. 1 Abbreviations: dN, 2′-deoxynucleoside; dG, 2′-deoxyguanosine; dA, 2′-deoxyadenosine; dC, 2′-deoxycytosine; dT, 2′-deoxythymidine; dGMP, 2′-deoxyguanosine 5′-phosphate; dAMP, 2′-deoxyadenosine 5′-phosphate; dCMP, 2′-deoxycytidine 5′-phosphate; dTMP, 2′-deoxythymidine 5′-phosphate; BPA, bisphenol A; BPA-Q, BPA-o-3,4-quinone; 3-OHBPA, 3-OH-bisphenol A; CID, collision-induced dissociation; SPE, solid phase extraction cartridge.

concern. BPA can migrate from polycarbonate tableware and plastic packages into food (9-12) and water (13), from dental sealants into humans (14), from baby bottles into infants (15), and from mother to fetus (16). A recent publication (17) reported that exposure to BPA causes a chromosomal abnormality in the oocytes of female mice and implies that the abnormality can lead to untoward reproductive and developmental effects; this possibility is causing a new round of arguments on the safety of BPA. Although BPA-containing polymers are normally robust, the carbonate linkage can hydrolyze at a high temperature or be damaged by strong detergents to release BPA. BPA can also be liberated from incompletely polymerized resins. As a lipophilic substance, BPA, like other xenoestrogens, can access the human body by ingestion or absorption through the skin and mucosal membranes. Of particular public health concern are two recent reports that show significant amounts of BPA in foodstuffs and human saliva. The first reported BPA was found in canned vegetables (10-20 µg/can or 50-100 µM) (9). The second showed that 20-30 µg/mL BPA existed in saliva collected from subjects treated with composite dental sealants (14). There is also evidence demonstrating the absorption of large quantities of BPA through the skin, causing damage to the kidneys, liver, spleen, pancreas, and lungs (18). BPA exposure to male rats and mice may also be associated with increased

10.1021/tx049953r CCC: $27.50 © 2004 American Chemical Society Published on Web 07/02/2004

Characterization of the Adducts of Bisphenol A

cancers of the hematopoietic system (19, 20). High doses of BPA do cause reproductive toxicity and affect cellular development in rats and mice (21-23). Although there are concerns about the widespread exposure and side effects/toxicity of BPA, the mechanism whereby BPA causes these adverse effects is not clear. Moreover, little attention has been directed at the potential consequences of such exposure or to the chemical mechanisms that may underlie the genotoxicity of BPA. One report shows that BPA in vivo is converted to a hydroxylated BPA (24). Evidence from 32P-postlabeling shows that BPA, in the presence of the cofactor cumene hydroperoxide or NADPH, reacts with DNA to form adducts in a rat hepatic microsomal cytochrome P450 activation system (25). Although the chemistry was unknown at that time, Atkinson and Roy (26) later found that BPA-o-quinone-DNA adducts, which were chemically synthesized, and the adducts that were formed in the presence of the activation system have identical chromatographic mobility, suggesting that the compounds are identical although their structures were not characterized. Furthermore, BPA, as was shown by 32Ppostlabeling, reacts with DNA to give adducts in CD1 male rats; two major adducts from the liver matched those produced by the reaction of DNA or dGMP with bisphenol-o-quinone (27). These results suggest that BPA and endogenous estrogens, by modifying critical cellular macromolecules with electrophilic/redox active quinoids, share the same mechanism of imparting genotoxicity or carcinogenesis (28). If so, the genotoxicity of BPA should be viewed from a perspective that goes beyond simple estrogenic-related side effects. To understand BPA at the genome level and to shed light on the mechanisms of reaction with DNA and glutathione (GSH) in vivo, we need authentic reference compounds formed by reaction of BPA-Q with deoxyucleosides and deoxynucleotides and with GSH. Furthermore, we also need an analytical method to determine these species. We report herein the synthesis and characterization of BPA-Q adducts, which were formed by reaction with 2′-deoxynucleotides and dNs, and of a GSH conjugate. We utilize high resolving power quadrupole time-of-flight (Q-TOF) and ion trap mass spectrometry, both coupled with liquid chromatography. The data that result should facilitate future studies of DNA damage and the assessment of the importance of depurinating adducts in vivo.

Materials and Methods Caution: This work described involved the synthesis and handling of hazardous agents and was, therefore, conducted in accordance with NIH guidelines for the Laboratory Use of Chemical Carcinogens. Chemicals and Reagents. All chemicals were purchased from Sigma (St. Louis, MO) or Aldrich (Milwaukee, WI) unless stated otherwise. Preparation of BPA-3,4-Q (Scheme 1). BPA-3,4-Q was synthesized by following the procedures of Yoshida et al. (29) with minor modifications. Fremy’s salt (20. g, 0.090 mol) was dissolved in 1100 mL of 25 mM aqueous sodium phosphate. BPA (3.3 g, 1.4 mmol) was dissolved in 67 mL of ethyl ether. The two solutions were mixed and shaken for 20 min at room temperature. The resulting brown solution was extracted three times with 400 mL of chloroform. The extracts were combined and washed three times with 400 mL of deionized water. After the extracts were dried over anhydrous magnesium sulfate for 3 h, the solvent was removed under vacuum to afford a brown,

Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1039 Scheme 1. Structures of BPA and BPA-Q

amorphous powder. The crude BPA-Q was purified by using a silica gel column and isocratic elution (hexane:ethyl acetate, 3:1). The resulting dried reaction mixture was worked-up by using silica gel (60-90 mesh) column chromatography of the crude BPA-Q using hexane-EtOAc (3:1, v/v) as the eluting solvent. Each fraction (50 mL) was monitored by TLC, using the same solvent, and visualized by dipping the plate in the staining reagent p-anisaldehyde/10% H2SO4. The fractions containing pure BPA-Q were combined and concentrated under vacuum to afford a brown amorphous powder (yield of 17%). BPA was also recovered from the reaction mixture with a yield of 50%. The identity of the oxidative product was established by NMR spectrometry. 1H NMR (CDCl3): δ 1.48 (6H, s, β-H), 6.15 (1H, d, J ) 10.2 Hz, H-5), 6.44 (1H, d, J ) 2.1 Hz, H-2), 6.62 (1H, dd, J ) 10.2 and 2.1 Hz, H-6), 6.18 (2H, d, J ) 8.4 Hz, H-2′ and H-6′), 7.38 (2H, d, J ) 8.4 Hz, H-3′ and 5′). 13C NMR (CDCl3) (the signal simplicity from APT was shown in parentheses): δ 27.00 (q, β-CH3), 42.84 (s, R-C), 115.82 (d, C-3′ and 5′), 127.51 (d, C-2′ and 4′), 134.92 (s, C-1′), 155.51 (s, C-4′), 161.85 (s, C-1), 123.38 (d, C-2), 180.33 (s, C-3), 171.69 (s, C-4), 141.83 (d, C-2), 128.66 (s, C-5), 141.83 (s, C-6). Positive ion electrospray MS showed an [M + H]+ of m/z 243. Reaction of BPA-3,4-Q with Nucleosides. BPA-Q (24. mg, 0.10 mmol) was dissolved in 1 mL of acetone and then reacted in separate experiments with 4 mL of dA (270 mg, 1.0 mmol), dG (290 mg, 1.0 mmol), dC (1.0 mmol), and dT (1.0 mmol) solution in AcOH-H2O (1:1). The mixtures were kept at 37 °C for 6 h. An aliquot of 1 µL from each incubation solution was diluted with the spray solvent (50/50 V/V acetonitrile/H2O) to 1000 µL for ESI mass spectrometry analysis by direct infusion. Reaction of BPA-3,4-Q with Nucleotides. BPA-Q (1.7 mg, 7.0 µmol) was dissolved in 50 µL of acetone and then reacted in separate experiments with 400 µL of dAMP (2.6 mg, 7.8 µmol), dGMP (2.5 mg, 7.2 µmol), dCMP (2.9 mg, 9.4 µmol), and dTMP (2.8 mg, 8.7 µmol) solution in water. The mixtures were kept at 37 °C for 12 h, and the adducts were isolated from the aqueous phase by using C-18 cartridges (Waters Oasis TM HLB), which were eluted with methanol to remove impurities and then with one column volume of water. Each 100-µL adduct solution, after purification, was diluted with the spray solvent (50/50 V/V acetonitrile/H2O) to 1000 µL for ESI MS analysis. Reaction of BPA-3,4-Q with Reduced GSH. The reaction of BPA-3,4-Q with reduced GSH was conducted in two media. (i) Twenty microliters of GSH solution (0.089 mg, 0.014 M) in distilled water was reacted with 20 µL of BPA-Q solution (0.73 mg, 0.14 M) in acetone for 12 h at 37 °C. The crude product was diluted by 1000 with spray solvent (50/50 V/V acetonitrile/ H2O) and analyzed by ESI mass spectrometry. (ii) GSH (330 mg, 1.0 mmol) was dissolved in 4 mL of AcOH-H2O (1:1) and then reacted with a BPA-Q (24 mg, 1.0 mmol) solution in 1 mL of acetone for 6 h at 37 °C. The crude product was purified by passing it through a SPE, and the MeOH-eluted fraction was diluted by 1:10 with the spray solvent (50/50 V/V acetonitrile/ H2O) and infused directly into the ESI source for analysis by MS. Mass Spectral Data of Adducts. The proposed structures of the adducts, shown in Scheme 2, are based on the following mass spectral data. 3-OH-BPA-dG (BPA-Q-dG Adduct, Intermediate). Ion trap tandem MS [Mquinone +MdG + H]+ ) 510 (60), 394 (100), 376 (15), 260 (10). MS2 of the ion of m/z 394 (positive mode) [M + H]+: m/z 394 (30), 300 (10), 260 (100).

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Scheme 2. Reaction of BPA-Q with dNs and 2′-Deoxynucleotides

3-OH-BPA-Gua (BPA-Q-dGMP/dG Formed by Depurination). Q-TOF high-resolution mass spectrometry [M + H]+: m/z 394.1517 (calcd 394.1515 for C20H20N5O4), 300.1086 (calcd 300.1097 for C14H14N5O3), 285.0862 (calcd 285.0868 for C16H17N2O3), 260.0787 (calcd 260.0784 for C11H10N5O3), 243.0505 (calcd 243.0509 for C15H15O3), 152.0580 (calcd 152.0573 for C5H6O3), 135.0834 (calcd 135.0839 for C9H11O). Ion trap ESI MS/MS [M + H]+: m/z 394 (30), 300 (10), 260 (100). 3-OH-BPA-dA (BPA-Q-dA Intermediate Adduct). Ion trap tandem mass spectrometry [Mquinone + MdA + H]+ m/z 494 (30), 378 (100). MS2 of ion m/z 378 (positive ion mode) [M + H]+: m/z 378 (60), 363 (10), 284 (5), 244 (100), 135 (5). 3-OH-BPA-Ade (BPA-Q-dAMP/dA Formed by Depurination). Q-TOF [M + H]+: m/z 378.1576 (calcd 378.1566 for C20H20N5O3), 136.0627 (calcd 136.0623 for C5H6N5). Ion trap ESI tandem spectrometry [M + H]+: m/z 378 (60), 363 (10), 350 (12), 284 (10), 244 (100), 135 (5). 3-OHBPA-GSH. Q-TOF [M + H]: m/z 550.1840 (calcd 550.1859 for C25H32N3O9S, 25), 475.1528 (calcd 475.1539 for C23H27N2O7S, 25), 421.1425 (calcd 421.1433 for C20H25N2O6S, 100), 404.1164 (calcd 404.1168 for C20H22NO6S, 15), 327.0951 (calcd 327.0970 for C14H19N2O5S, 8), 318.1164 (calcd 318.1164 for C17H20NO3S, 52), 310.0741 (calcd 310.0749 for C14H16NO5S, 50), 287.0692 (calcd 287.0702 for C11H15N2O5S, 14), 275.0732 (calcd. 275.0742 for C15H15O3S, 80), 135.0811 (calcd. 135.0810 for C9H11O, 78). Ion trap ESI tandem spectrometry (positive ion mode) [M + H]+: m/z 550 (15), 475 (7), 421 (100). MS3 of ion of m/z 421 (68), 404 (64), 327 (6), 310 (100), 287 (20), 275 (66), 135 (28). Reaction of 3,4-BPA-Q with Calf Thymus DNA. BPA-Q (0.24 mg, 1.0 µmol) was dissolved in 100 µL of DMSO and reacted with 1.63 mg of calf thymus DNA in 900 µL of water. The mixture was kept at 37 °C for 18 h. The DNA was precipitated with ethanol and removed by filtration. A 100 µL aliquot of the adduct solution was diluted with the spray solvent (50/50 V/V acetonitrile/H2O) to 500 µL for HPLC/MS/MS analysis. Instrumentation. CapLC/ESI/product-ion spectra were obtained with a Finnigan Classic LCQ ion trap mass spectrometer (San Jose, CA) equipped with an ESI source. The sample (approximately 1 part in 10 000 of the original isolate) was introduced either by direct infusion at a rate of 5 µL/min or via an Agilent 1100C gradient HPLC system (Waldbronn, Germany) through a six pore Rheodyne 7725 manual sample injector. The

LC column was a Thermo Hypersil-Keystone Aquasil C18 (150 mm × 0.32 mm) column. The linear gradient HPLC started with 3% acetonitrile in 0.2% aqueous acetic acid, increased to acetonitrile/H2O/AcOH (97/3/0.2) in 35 min, isocratic for 10 min, and returned to initial conditions for 10 min before the next run. The spray voltage was 3.8 kV, and the N2 gas flow was set as 50 arbitrary units on the Thermo Finnigan LCQ. The capillary temperature was 200 °C to ensure efficient desolvation. The analyzer was operated at a background pressure of 3.6 × 10-5 Torr. All data were acquired in the positive ion mode with a unit resolving power between m/z 100 and 800. Normally, 10 scans were averaged and processed by using Finnigan Xcalibar 1.1 software. For accurate mass measurements, ESI mass spectra were also obtained with a Micromass Q-TOF Ultima mass spectrometer (Manchester, United Kingdom) equipped with a Z-spray ESI source. A solution of 80:19:1 (vol:vol:vol) of CH3CN:H2O:HCOOH was introduced into the mass spectrometer at a flow of 32 µL/ min. The capillary voltage was 3.4 kV, and the source and desolvation temperatures were 80 and 150 °C, respectively. The cone gas and the desolvation gas flows were 40 and 500 L/h, respectively. All data were acquired in the positive-ion mode at 10 000 resolving power over a mass range of m/z 100 to 1500. Normally, 10 scans were averaged and processed using MassLynx 3.5 and maximum entropy (MaxEnt1) from Micromass. The accurate masses of the fragments were determined in the MS/MS mode by adjusting the collision energy until an optimal ratio of parent ion-to-fragment ion abundances was obtained. Mass calibration used the parent ion as the internal standard by “locking” onto its signal and computing the m/z of the fragments.

Results Structure Identification of BPA-Q. We oxidized BPA by potassium nitrosodisulfonate [Fremy’s salt, (KSO3)2NO] according to the procedure of Yoshida (29) with minor modifications (Figure 1) and determined the structure of the purified product by using MS and NMR. The 1H NMR spectrum showed a signal pattern of a 1,2,4trisubstituted benzene in addition to a A2 × 2 pattern of a four-substituted phenol, which also pertains to the 1H NMR of BPA. Besides these typical 1H NMR signal patterns, two carbonyl signals in the 13C NMR confirmed


Figure 1. Positive ion CapLC/MS/MS analysis (full scan from m/z 100-800) of adducts from the incubation of BPA-Q with dG. (A) Total ion current; (B,C) the overlapping chromatographic peaks at 21.8 min, representing the elution of BPA-Q-dG of m/z 394 formed by depurination and the initially formed BPA-QdG adduct (intermediate) of m/z 510.

that this oxidative product contained not a catechol (3,4hydroquinone) moiety but a 3,4-quinone moiety. Characterization of BPA-Q-dGMP and BPA-Q-2′dG Adducts. The freshly synthesized BPA-Q was incubated in parallel with dGMP in water and dG under acidic conditions. Analysis by reverse phase CapLC indicated that the BPA-Q had been consumed and a small amount of guanine had appeared, presumably due to the hydrolysis of dGMP (dG) under the acidic reaction conditions or due to fragmentation in the source of the mass spectrometer. Analysis of the mixture by capLC/ESI/MS showed two protonated molecules (M + H+) at m/z 510 and 394; the substances coeluted at a time characteristic of the major reaction product (Figure 1). The molecular ion of m/z 394 is that of the BPA-Q-guanine adduct (i.e., BPA-Q-dG formed by depurination), and the product ion fragmentation pattern obtained upon CID of the [M + H]+ ion is consistent with the assigned structure (Scheme 2). The structure was further confirmed by using accurate mass measurements with the Q-TOF (Figure 2). The

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formulas of the fragment ions were assigned on the basis of the accurate mass measurements (to within 5 ppm); the ion structures in the figure are speculative but reasonable assignments. For example, a fragment of m/z 152.0580 (calcd 152.0573 for C5H6N5O, protonated guanine) confirms the presence of a guanine moiety and another of m/z 243.0505 (calcd 243.0509 for molecular formula of C15H15O3) confirms the presence of the quinone moiety. As depicted in Figure 2, other fragments of m/z 300.1086 (calcd 300.1097 for C14H14N5O3), 285.0862 (calcd 285.0868 for C16H17N2O3), 260.0787 (calcd 260.0784 for C11H10N5O3), and 135.0834 (calcd 135.0839 for C9H11O) are consistent with losses from either the guanine or the o-quinone portion of the product. The complementary product ion pair of m/z 260 and 135 suggests, considering steric effects, a linkage between the guanine and the 3,4di-OH-aromatic ring, mostly likely at the C-5 position, although we cannot rule out other possibilities (C-2 and C-6). The product ion of m/z 300, generated by loss of the hydroxylbenzene unit of BPA, further supports the attachment of the 3,4-di-OH-aromatic ring to the guanine moiety. The product ion of m/z 285 is evidence that the adduct involves coupling of the 3-OH-BPA to N7 of guanine rather than to the exocyclic amino group, as this product ion is generated by cleavage of the five-membered ring of guanine (Figure 2), and stable adducts will arise if the exocyclic amino group is the modification site (36). Similarly, the ESI mass spectra allowed the assignment of a molecular weight, [Mquinone + MdG + H]+ ) 510 to the other adduct, consistent with the reductive addition of dG to the quinone followed by isomerization and addition of H+ to give the substituted catechol. Upon collisional activation (ion trap MS/MS), the ion of m/z 510 [Mquinone + MdG + H]+ decomposed to a major fragment ion at m/z 394 [510 - C5H8O]+ (100%) and two minor fragment ions of m/z 376 [510 - C5H8O - H2O]+ (20%) and m/z 260, which was discussed previously to arise by cleavage of the BPA moiety (Figure 2). These fragments are consistent with the assigned structure of the intermediate in Scheme 3. CID of the ion at m/z 394 gave a nearly identical mass spectrum to that of the 3-OHBPAguanine, as shown in Figure 3, indicative of their identical structures. We note that the product-ion spectrum obtained with the Q-TOF is more informative than that obtained with the ion trap, consistent with the

Figure 2. Q-TOF product ion mass spectrum of the BPA-Q-Gua adduct of m/z 394.


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Scheme 3. Proposed Pathway for Formation of BPA-Q-dG via Depurination of an Intermediatea

a

Addition at C5 is not proven and is for illustrative purposes only.

Figure 3. Positive ion product ion analyses of the BPA-Q-dG depurinating adduct of m/z 394 eluting at 21.80 min (A); product ion spectra of BPA-Q-dG adduct (intermediate) of m/z 510 (B); and product ion spectrum (MS/MS/MS) of the BPA-Q-dG adduct (intermediate) of m/z 510 (C).

higher activation energy available with the Q-TOF. The results suggest that BPA-Q forms an adduct in vitro with dG at N7 rather than at the exocyclic amino group; the latter would lead to formation of an unstable intermediate that undergoes hydrolysis of the sugar moiety, leaving an arylated guanosine. The CapLC/MS/MS analysis of the in vitro incubation mixture of BPA-Q with dGMP revealed only the 3-OHBPA-guanine, formed by depurination, whose identity was confirmed to be the same as that generated by incubating BPA-Q with dG. That means the glycosidic bond undergoes rapid hydrolysis under the incubation conditions, presumably because the 5′-phosphate renders the 2′-deoxyribose a better leaving group than the sugar moiety. Characterization of BPA-Q-dAMP and BPA-Q-2′dA Adducts. We incubated the freshly synthesized BPA-Q in parallel with dAMP (in water) and dA (under

Figure 4. CapLC/MS/MS analysis (positive ion, ion trap) of the adducts from incubation of BPA-Q with dAMP: total ion current (A). The chromatographic peaks at 22.42 and 25.96 min represent the elution of BPA-Q-dA adducts (intermediates) of m/z 494, obtained by selected ion monitoring (B), and those at 22.66 and 25.99 min, selected ion monitoring, represent BPAQ-dA formed by depurination (m/z 378) (C).

acidic conditions), as previously described for dGMP and dG. The reactions occurred more slowly than those with dGMP and dG, as was seen by the slow losses of color of the starting material BPA-Q and by semiquantitative HPLC analysis. Analysis of the resulting incubation mixture by capLC/ESI/MS revealed two prominent [M + H]+ ions at m/z 494 and m/z 378 with retention times corresponding to the major reaction products. Each appeared as two peaks eluting at 22.4/25.9 and 22.7/26.0 min, respectively, which presumably represent two isomeric intermediates and two isomeric depurinating adducts (Figure 4). The [Mquinone + MdA + H]+ ions at m/z 494, appearing for eluents at 22.4 and 25.9 min, correspond to the BPA-Q-dA adduct (intermediate) whereas the [M + H]+ ions of m/z 378, appearing at 22.7 and 26.0 min, correspond to BPA-Q-adenine adducts (i.e., BPAQ-Ade formed by depurination). The product ion spectra of the [M + H]+ ions are consistent with the proposed structure (Scheme 2). As for the guanine adducts, the structures were further established by accurate mass measurements to within 5 ppm (Figure 5). Unexpectedly, MS/MS on the Q-TOF



Figure 5. Q-TOF product ion spectrum (MS/MS) of the 3-OHBPA-N7-Ade adduct of m/z 378, introduced by ESI.

Figure 7. LC/MS/MS analysis of 3-OHBPA-guanine formed in the incubation of BPA-Q with calf thymus DNA. The upper panel is the total ion current chromatogram, and the bottom panel is the product-ion spectrum of the material eluting at 19.87 min. The experiment was done on a different column than that used for the chromatogram in Figure 1, accounting for the small difference in retention time. Figure 6. Product ion spectra (positive ion, obtained with the ion trap) of the BPA-Q-dA of m/z 378, formed by depurination and eluting at 22.7 min (A); product ion spectrum of BPA-Q-dA adduct (intermediate) of m/z 494 eluting at 22.4 min (B); and product ion spectrum (MS/MS/MS) of the intermediate BPA-QdA adduct of m/z 494 eluting at 22.4 min (C).

gave an ion of m/z 136.0627 (calcd 136.0623 for C5H6N5) as the only significant fragment, which confirmed the presence of the adenine moiety. In contrast, ion trap mass spectrometry (Figure 6) gave more informative fragments. Upon CID, the [M + H]+ of m/z 378 decomposed to fragment ions of m/z 363 [M + H - CH3]+, 350 [M + H - CO]+, 284 [M + H - p-OHC6H6]+, 244 [M + H p-OH-isopropylbenzene]+, and 135 [p-OH-isopropylbenzene - H]+. These fragments are consistent with the structure of the BPA-Q-dA adduct depicted in Scheme 2; most likely, the C-5 position of 3-OHBPA and the N-7 position of adenine are involved in the linkage because the ion-trap, product-ion spectrum is nearly identical to that of the BPA-Q-N7-guanine adduct (from the Q-TOF), although the possibility of the diastereoisomers cannot be ruled out.

Similar to the results observed for the incubation with dG, both intermediates and depurinating adducts were found. A protonated molecule of m/z 494 comes from the component that elutes at 22.4 min, presumably the unstable intermediate of 3-OHBPA-N7-dA, which ultimately depurinates to give the 3-OHBPA-N7-adenine. We were able to show by ion trap tandem mass spectrometry that the 3-OHBPA-N7-dA intermediate is the precursor of 3-OHBPA-N7-adenine (Figure 6). Upon collisional activation, the [M + H]+ of m/z 494 decomposes to form the fragment ion of m/z 378 [M + H - deoxyribose]+, which further decomposes upon collisional activation to give a fragment pattern that is nearly identical to that of the 3-OHBPA-N7-adenine. Another potential binding site involved in adduct formation is N3, as was observed for dibenzo[a,l]pyreneDNA adduct formation in vitro (30). As shown in Figure 4, we saw additional intermediate adducts having [M + H]+ ions of m/z 494 and 378 and eluting at approximately 22 and 26 min, respectively. We tentatively assign them as 3-OHBPA-N3-dA and 3-OHBPA-N3-adenine on the basis of the similarity of their ion-trap, product-ion


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Figure 8. Product ion spectrum (LC/MS/MS, obtained with the ion trap) of the [M + H]+ ion of m/z 550 from 3-OHBPA-GSH.

Figure 9. Q-TOF product-ion spectrum of the [M + H]+ ion at m/z 550 from 3-OHBPA-GSH. Fragment ion structures given in the figure are suggested to account for the m/z values of the product ions.

spectral patterns (data not shown) to those of 3-OHBPAN7-dA and 3-OH-BPA-N7-adenine. Definite structures await isolation, purification, and characterization using NMR spectroscopy. When BPA-Q was incubated with calf thymus DNA, we found by capLC/MS/MS analysis only 3-OHBPA-N7guanine, and we saw no 3-OHBPA-adenine. We confirmed the identity of 3-OHBPA-N7-guanine by its retention time and product ion spectrum (Figure 7), which are nearly identical with those obtained of products in the incubation with dG/dGMP. This result underscores the relatively low reactivity of dA toward BPA-Q, as found in the incubation of BPA-Q with dA/dAMP. Characterization of the BPA-Q-GSH Adduct. BPA reacts readily with reduced GSH, as seen by the rapid loss of the brown color of BPA-Q, in both the water and the acidic AcOH-H2O media. The resulting mixture, as analyzed using capLC/MS/MS in a manner similar to that for the dG and dA adducts, has one major GSH conjugate. The structure of this conjugate was determined by ion trap MS/MS by using the protonated molecule of m/z 550 [M + H]+, corresponding to the 3-OHBPA-mono-GSH conjugate. The product ion spectrum shows production of diagnostic ions that are common to GSH adducts (31, 32), including an abundant y2 ion at m/z 421 and a moderately abundant b2 ion at m/z 475, as expected for the structure depicted in Figure 8.

We found that the Q-TOF and ion-trap, product-ion spectra (MS2 of m/z 550 and MS3 of m/z 421, data not shown) of 3-OHBPA-GSH are nearly identical. The superior mass resolving power of the Q-TOF and the capability to give accurate masses of the protonated molecules, [M + H]+, however, give more convincing evidence for the 3-OHBPA-GSH conjugate structure (see Figure 9). Although the most abundant product ions are due to fragmentation within the GSH moiety and afford little information regarding the linkage between 3-OHBPA and GSH, the product ion of m/z 275.0732, with an elemental composition of C15H15O3S, corresponds to [3-OHBPA-SH]+. It contains the sulfur of the GSH moiety and reveals some structural information about the BPA moiety. This product ion was selected as a precursor for collisional activation with a further stage of MS/MS by the ion trap to yield the product ion of m/z 135. This ion is likely to arise by the cleavage of the bond linking the two aromatic rings accompanied by a loss of HS-C6H6(OH)2. Another confirmation for the depurinating adducts comes from the Q-TOF experiment in which we showed that the elemental composition of the m/z 135 ion is C9H11O.

Discussion Because BPA is weakly estrogenic and routes exist for human exposure to this compound, it presents a possible



Figure 10. Optimized CapLC chromatogram and ion-trap, product-ion spectra of 3-OHBPA-N7-Gua and 3-OHBPA-S-GSH mixtures. The upper panel (from selected ion monitoring of the m/z 394.9 ion) indicates the elution of the Gua adduct, and the lower panel (from monitoring of the m/z 550.8 ion) indicates elution of the GSH adduct.

health hazard. Thus, following the pattern of other synthetic estrogens (e.g., hexestrol and diethylstilbestrol), BPA also has the potential to cause estrogenic endocrine disruption (33). Exposure of BPA to male rats and mice is associated with increased cancer of the hematopoietic system (19, 20), but the mechanism by which BPA elicits these adverse effects is not clear. Certainly by structural analogy to endogenous and synthetic estrogens, it is reasonable to envisage a “BPA-catechol-quinone-DNA conversion” to cause genotoxicity. Motivated by the need for understanding the pathways for chemical and biological conversion of BPA and the causes for its toxicity, we examined the reactivity of BPA-Q with model nucleotides and with GSH. The formation in vivo of covalent DNA adducts, both depurinating and stable, has been implicated in the initiation of some cancers (34-36). To exert its carcinogenic effects, BPA requires metabolic activation to an electrophilic species that can modify DNA. Unless the modified bases are promptly repaired, miscoding may result during DNA replication, leading to mutations (37). BPA can be converted to BPA-o-quinone chemically by a radical oxidant (29) or by enzymatic oxidation (29, 39). BPA can also be converted to a glucuronide and sulfate conjugate in rats (38). Using 32P-postlabeling techniques, Atkinson and Roy (26, 27) investigated the covalent modification of DNA caused by in vitro and in vivo BPA exposure and found that DNA adducts can be formed in both cases. They confirmed that the DNA adducts formed in vitro (rat hepatic microsomal cytochromes P450 with cofactor cumene hydroperoxide or NADPH) and in vivo (CD1 male rats) are identical; however, they did not characterize the structures of these adducts. The present investigation of the reactivity of BPA-oquinone with nucleotides, nucleosides, and calf thymus DNA uses capLC/tandem mass spectrometry to provide direct evidence that BPA, after metabolic activation to a reactive quinone, reacts with nucleosides, nucleotides, and DNA to form adducts. When BPA-Q reacts with dGMP and dG, the depurinating adduct 3-OHBPA-N7Gua arises, presumably via an intermediate 3-OHBPAN7-dG. This shows that modified DNA can undergo glycosidic hydrolysis (depurination) to release the modi-

fied nucleobase. The intermediate cannot be detected in the incubation with dGMP, indicating that the intermediate hydrolyzes readily. The incubation of BPA-Q with dA/dAMP gives a similar outcome to that of dG/dGMP, but the adducts are formed with lower yields except for two isomeric 3-OHBPA-Ade adducts that arise by depurination of both dA and dAMP. Two isomeric intermediates (3-OHBPA-dG) also come from the incubation of dA, indicating that adduct formation depends strongly on the structure of the nucleosides/nucleotides. Once we had characterized the BPA-Q-dN adducts, we were in the position to examine adduct formation in calf thymus DNA. Significant amounts of apurinic sites presumably arise through reaction with guanosine as we were able to detect 3-OHBPA-N7-Gua, which is formed by depurination. As apurinic sites are known to be mutagenic (40), in vivo formation of these sites in reaction with BPA-Q may elucidate a carcinogenic mechanism for BPA after metabolic activation. Conjugation of BPA-Q with GSH should represent a preventive pathway that competes with reaction of BPA-Q and DNA. GSH, the most abundant intramolecular nonprotein thiol, can react nonenzymatically or via the catalytic action of GSH-S-transferase with electrophilies and free radicals, leading to their ultimate removal (41). BPA-Q reacts readily, under both neutral and acidic aqueous conditions, with GSH, without GSH-S-transferase, to give the 3-OHBPA-GSH conjugate. Conjugation to GSH and adduction to DNA share similar chemistries. The ability to detect both classes of products is important for developing an understanding of carcinogenesis and for estimating the risk associated with exposure to BPA. HPLC/MS and HPLC/MS/MS (Q-TOF or ion trap) are useful tools for characterizing the underlying chemistry of BPA with biomolecules and for further studies of carcinogenesis (Figure 10). Our data suggest that several types of DNA lesions can be expected, including apurinic sites, in addition to BPA damage to DNA by telomeric association (42). The implications that these adducts have for the toxicity of BPA are not known. Given the exposure potential, however, the likelihood of genetic abnormality is sufficiently serious that it cannot be dismissed without further study.


Acknowledgment. This research was supported by the National Centers for Research Resource of NIH (Grant P41RR00954) and by the NIH (Grant P01CA49210). We thank Dr. Joy Du for her comments and suggestions on Q-TOF mass spectrometry.

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