ARTICLE pubs.acs.org/est
New Evidence for Toxicity of Polybrominated Diphenyl Ethers: DNA Adduct Formation from Quinone Metabolites Yongquan Lai, Minghua Lu, Xiang Gao, Hanzhi Wu, and Zongwei Cai* Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR, China
bS Supporting Information ABSTRACT: This study investigated the formation of DNA adducts of polybrominated diphenyl ethers (PBDEs) and the possible mechanisms. DNA adduction was conducted by in vitro reaction of deoxyguanosine (dG) and DNA with PBDEquinone (PBDE-Q) metabolites, and DNA adducts were characterized by using electrospray ionization tandem mass spectrometry. The results suggested DNA adduction involved Michael Addition between the exocyclic NH2 group at the N-2 position of dG and the electron-deficient carbon of quinone, followed by reductive cyclization with loss of (bromo-)1-hydroperoxy-benzene or water to form a type I or type II adduct. PBDE-Q with substituted bromine on the quinone ring was proven to be a favorable structure to form a type I adduct, while the absence of bromine on the quinone ring resulted in a type II adduct. Lower reactivity of adduction was also observed with increasing the number of bromine atoms on the phenoxyl ring. Our data clearly demonstrated PBDEs could covalently bind to DNA mediated by quinone metabolites, depending on the degree of bromine substitution. This study opened a new view on the mechanism of toxicity of PBDEs and reported the structure of PBDEDNA adducts, which might be valuable for the evaluation on potential in vivo formation of PBDEDNA adducts.
’ INTRODUCTION Polybrominated diphenyl ethers (PBDEs) have been used as flame retardants in many commercial and household products including polyurethane foam, textiles, furniture, and electronics.13 The noncovalent binding of PBDEs in polymers allows the chemicals to enter the environment easily from the product surface during use.4 PBDEs have become contaminants of worldwide concern because of their widespread use, ubiquitous environmental distribution, great bioaccumulation potential, and toxicity.5 It is of particular worth to note that the concentrations of PBDEs in human blood, breast milk, and other body tissues have been increasing with a doubling time of approximately 46 years over the last 30 years.6,7 PBDEs could induce phase I enzyme activities, such as cytochrome P450 isozymes,810 and could also be the substrates for the isozymes with the formation of several metabolites in vivo.3,1113 It has been reported that PBDE phase I metabolism occurred via three possible metabolic pathways: oxidation, debromination,14 and oxidative debromination.15 For the oxidation metabolism, PBDEs were initially metabolized to arene oxides, followed by cytochrome P450 enzyme-catalyzed hydroxylation to form monohydroxylated PBDEs (OH-PBDEs) or dihydroxylated PBDEs (diOH-PBDEs).1618 For example, OH-PBDEs have been determined in pooled serum from humans living or working at municipal waste disposal site.19 OH-PBDEs have received great attention due to their contribution to the recognized toxic effects of PBDEs such as endocrine disruption and development neurotoxicity. r 2011 American Chemical Society
It is of particular concern to note that metabolic activation of PBDEs could result in hydroquinone and catechol metabolites (diOH-PBDEs with two hydroxyl group at para and ortho positions) that might possess more toxic effects than PBDEs and OH-PBDEs. However, little attention has been paid to the toxicity of hydroquinone and catechol metabolites. Hydroquinone metabolites might undergo peroxidase-catalyzed oxidation to quinones that could create a variety of the hazardous effects in biological systems, including acute cytotoxicity, immunotoxicity, and carcinogenesis.20 The structure change of DNA as a result of covalent binding to carcinogens or their active metabolites was considered as an early critical step in chemical carcinogenesis. If not being repaired before DNA replication, DNA adducts can cause misrepairing, resulting in mutations and chromosomal alternations.21 To take as an example of polycyclic aromatic hydrocarbon (PAH) carcinogenesis, it has been shown that metabolic activation of PAH occurred via a two-step process involving cytochrome P450-mediated formation of hydroquinone metabolites and their further peroxidase-dependent oxidation conversion to PAH-derived quinones. The formed PAHquinones played a critical role in the initiation of cancer by covalent binding to DNA.20 Based on the mechanism for PAH carcinogenesis, it Received: September 2, 2011 Accepted: November 3, 2011 Revised: October 26, 2011 Published: November 03, 2011 10720
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Environmental Science & Technology was postulated that PBDE-derived quinones (PBDEQs) might covalently bind to DNA, and the DNA adducts might become biomarkers for the potential of PBDEs to initiate cancer. Although 32P-postlabeling is one of the most sensitive methods commonly used for the analysis of DNA adducts, this method provides no structural information. Mass spectrometry (MS) has become a powerful and alternative technology to characterize and in most cases determine DNA adducts with the sensitivity comparable to that of the other analytical approaches. In addition, the LC-MS method is more specific compared to 32 P-postlabeling, because adducts can be identified by their molecular weights and LC retention times. To date, there has been no report on the analysis of DNA adducts formed by PBDEQ metabolites. This study investigated the covalent binding of PBDEQs to DNA by using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Identification and characterization of the chemical structures of the DNA adducts may facilitate efforts to establish the occurrence of DNA adduction and its biological significance in complex biological system.22
’ EXPERIMENTAL SECTION Chemicals and Reagents. 20 -Deoxyguanosine (dG), nuclease
P1, phosphodiesterase I, alkaline phosphatase, calf thymus DNA, and 2,4-dibromophenol were obtained from Sigma (St. Louis, MO, USA). 2-Bromobenzoquinone (2BrBQ) was obtained from Tokyo Chemical Industrial Co., Ltd. (Tokyo, Japan). 2,6-Bromobenzoquinone (26BrBQ) was purchased from APIN Chemicals Ltd. (Abingdon, UK). CDCl3, phenol, 4-bromophenol, and 2,4,6-tribromophenol were purchased from Acros Organics (Geel, Belgium). Horseradish peroxidase (HRP) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). β-Nicotinamideadenine dinucleotide phosphate, reduced (NADPH) was obtained from Oriental Yeast Co., Ltd. (Tokyo, Japan). 60 -OHBDE-17, 30 -OH-BDE-7, and 6-OH-BDE-47 were received from Dr. Michael H.W. Lam (Department of Biology and Chemistry, City University of Hong Kong, Hong Kong). Precoated thinlayer chromatographic (TLC) plates (DC-Fertigplatten SIL G-25 UV254) were purchased from Macherey-Nagel (D€uren, Germany). Dimethyl sulfoxide (DMSO) in analytical grade was purchased from AJAX Chemicals (Sydney, Australia). Water was purified by employing a Milli-Q reagent water system (Millipore, Billerica, MA, USA). Synthesis of PBDEQs. PBDEQs were synthesized from the reaction of phenol or bromophenol with bromobenzoquinones. A solution of phenol or bromophenol (0.4 mmol) in DMF (0.5 mL) was added to the solution of bromobenzoquinone (0.25 mmol) in DMF (1 mL). The reaction was initiated by adding Na2HPO4 (0.25 mmol) and K2CO3 (0.08 mmol). The mixture was stirred at room temperature for 3 h. The reaction mixture was pour into 40 mL of ice cold H2O and extracted with ethyl acetate. The organic layer was washed with H2O and brine, dried (Na2SO4), and removed in vacuo. The crude product was purified on a TLC plate (CH2Cl2hexane, v/v, 1:1). The purified sample was submitted to NMR and GC-MS analysis. The 1H and 13C NMR spectra were recorded on a Bruker Avance-III spectrometer (at 400 and 100 MHz, respectively) in CDCl3. GCMS spectra were recorded on an Agilent 6890N GC coupled to an Agilent 5973 mass spectrometer (70 eV). More information on the GC-MS conditions and data is available in the Supporting Information.
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2-(20 ,40 -Bromophenoxyl)-benzoquinone (20 40 BrPhO-BQ). H NMR (400 MHz, CDCl3): δ = 5.62 (d, J = 2.4 Hz, 1 H), δ = 6.75 (dd, J = 2.4 and 10.4 Hz, 1 H), δ = 6.84 (d, J = 10.0 Hz, 1 H), δ = 7.05 (d, J = 8.4 Hz, 1 H), δ = 7.53 (dd, J = 2.4 and 8.8 Hz, 1 H), δ = 7.83 (d, J = 2.4 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 111.47, 116.55, 120.48, 124.03, 132.50, 134.66, 136.72, 137.02, 148.70, 156.51, 180.58, 187.04 ppm. MS (EI) m/z (relative intensity) = 358 (8) [M], 277 (100) [M Br], 249 (48) [M Br CO]. 2-Phenoxyl-6-bromo-benzoquinone (PhO-6-BrBQ). 1H NMR (400 MHz, CDCl3): δ = 5.74 (d, J = 2.4 Hz, 1 H), δ = 7.08 (d, J = 8.8 Hz, 2 H), δ = 7.21 (d, J = 2.0 Hz, 1 H), δ = 7.297.47 (m, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 110.99, 120.82, 126.91, 130.50, 134.49, 138.26, 152.41, 158.28, 174.40, 184.72 ppm. MS (EI) m/z (relative intensity) = 280 (97) [M], 199 (25) [M Br], 171 (100) [M Br CO]. 2-(20 -Bromophenoxyl)-6-bromo-benzoquinone (20 BrPhO-6BrBQ). 1H NMR (400 MHz, CDCl3): δ = 5.76 (d, J = 2.4 Hz, 1 H), δ = 6.98 (d, J = 8.8 Hz, 2 H), δ = 7.22 (d, J = 2.0 Hz, 1 H), δ = 7.57 (d, J = 8.8 Hz, 2 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 111.29, 120.09, 122.60, 133.64, 134.54, 138.26, 151.44, 157.75, 174.14, 184.41 ppm. MS (EI) m/z (relative intensity) = 358 (90) [M], 277 (90) [M Br], 249 (100) [M Br CO]. 2-(20 ,40 -Bromophenoxyl)-6-bromo-benzoquinone (20 40 BrPhO6-BrBQ). 1H NMR (400 MHz, CDCl3): δ = 5.63 (d, J = 2.4 Hz, 1 H), δ = 7.04 (d, J = 7.2 Hz, 1 H), δ = 7.23(d, J = 2.4 Hz, 1 H), δ = 7.52 (dd, J = 2.4 and 8.4 Hz, 1 H,), δ = 7.82 (dd, J = 2.4 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 111.53, 116.47, 120.73, 123.92, 132.60, 134.65, 136.81, 138.28, 148.72, 156.11, 173.56, 184.19 ppm. MS (EI) m/z (relative intensity) = 438 (17) [M], 357 (100) [M Br], 329 (48) [M Br CO]. 2-(20 ,40 ,60 -Bromophenoxyl)-6-bromo-benzoquinone (20 40 60 BrPhO-6-BrBQ). 1H NMR (400 MHz, CDCl3): δ = 5.62 (d, J = 2.0 Hz, 1 H), δ = 7.25 (d, J = 2.0 Hz, 1 H), δ = 7.78 (s, 2 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 111.33, 117.56, 121.07, 134.77, 135.76, 138.32, 146.28, 154.19, 173.07, 184.06 ppm. MS (EI) m/z (relative intensity) = 516 (10) [M], 435 (100) [M Br], 407 (35) [M Br CO]. Reaction of dG with PBDEQs. PBDEQs (0.3 μmol in DMSO, 5 μL) and dG (0.6 μmol in DMSO, 5 μL) were added into 300 μL of potassium phosphate buffer (0.1 M, pH = 7.4). The resulted mixtures were incubated in a shaking water bath at 37 °C for 2 h. The reaction mixture was extracted with ethyl acetate (2 0.5 mL), and the collected organic fractions were washed with DI water. The solvent was removed under a stream of nitrogen at room temperature. The residues were reconstituted with 0.5 mL of 0.1% formic acid in ACN before being submitted to ESI-MS/MS analysis. For control samples, PBDEQ or dG was absent in the incubation reactions. Microsomal-Mediated Metabolism of OH-PBDEs. In addition to chemically synthetic PBDEQs, the biotransformed PBDEquinone metabolites via microsomal-mediated metabolism of OH-PBDEs were also investigated. Liver microsomes from phenobarbital exposed male SpragueDawley rats were prepared as described previously.23 OH-PBDE congeners (0.3 μmol) dissolved in DMSO were added to 1.2 mL of potassium phosphate buffer (pH 7.4, 100 mM) containing 5 mM MgCl2 and microsomal protein (2.0 mg/mL). Incubations were prewarmed for 5 min at 37 °C in a shaking water bath before adding NADPH (2 mg) to initiate the reactions. After incubation for 1 h, the reaction mixtures were freeze-dried in a vacuum chamber, and the residues were extracted with ethyl acetate (2 0.5 mL). 1
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Environmental Science & Technology The combined extracts were dried under a stream of nitrogen at room temperature. Control reactions were carried out in the same manner, except that the NADPH or OH-PBDE congener was excluded. Adduction of dG with PBDE Metabolites. The residues of the microsomal-generated PBDE metabolites were dissolved in potassium phosphate buffer (pH 7.4, 100 mM) containing dG (0.3 μmol), HRP (0.2 U), and H2O2 (1 mM) to a final volume of 200 μL. Reactions were carried out for 2 h at 37 °C in a shaking water bath. The reaction mixtures were extracted with ethyl acetate (2 0.5 mL). The solvent was removed under a stream of nitrogen at room temperature, and the residues were reconstituted with 0.2 mL of 0.1% formic acid in ACN. Reaction of Calf Thymus DNA with 2BrBQ, 26BrBQ, and PBDEQs. DNA adduct formation in calf thymus DNA treated with bromobenzoquinone or PBDEQ was measured as follows. Briefly, quinone (0.15 μmol in DMSO, 5 μL) and calf thymus DNA (50 μg in H2O, 50 μL) were added into 250 μL of potassium phosphate buffer (0.1 M, pH = 7.4). The mixture was incubated in a shaking water bath at 37 °C for 12 h. Control incubation of quinone or DNA alone was carried out under the same conditions. The DNA was precipitated by adding 30 μL of 3 M sodium acetate solution (1/10 of the incubation buffer volume) and 0.9 mL of ice-cold ethanol. The precipitated DNA was recovered by centrifugation at 14 000 g for 30 min and rinsed with 0.9 mL of 70% ethanol to remove excess salts. After centrifugation at 14 000 g for 30 min, the wash liquid was removed, and the pellets were further dried under a nitrogen flow. The resulting DNA pellets were sequentially digested by nuclease P1, alkaline phosphatase, and phosphodiesterase using a previously published method.2426 More information regarding DNA digestion is available in the Supporting Information. The digestion solution was freeze-dried in a vacuum chamber, and the residue was reconstituted with 0.1% formic acid in the ACN before submitted to ESI-MS/MS analysis. ESI-MS/MS Analysis. Accurate mass values of DNA adducts were acquired in positive ion mode using a QTOF mass spectrometer (API QStar Pulsar i, Applied Biosystems, Foster City, USA) equipped with a TurboIonspray source. Experiments were performed at an ion spray voltage of 5000 V, declustering potential I (DP I) of 30 V, declustering potential II (DP II) of 15 V, and focusing potential (FP) of 80 V. The mass detection range was set to m/z 100900. The ion source gas I (GS I), gas II (GS II), curtain gas (CUR), and collision gas (CAD) were set at 30, 15, 30, and 3 psi, respectively. The temperature of GS II was set at 300 °C. Samples were directly infused to into the ESI source at a flow rate of 5 μL/min. Data acquisition and processing were performed by using Analyst QS software. The DNA adducts were analyzed by using triple-quadrupole mass spectrometry equipped with an ESI source (Waters ACQUITY TQ Detector, Waters Corporation, Milford, MA, USA). Separations of PBDEDNA adducts were performed on an UPLC (Waters ACQUITY UPLC system, Waters Corporation, Milford, MA, USA) with a reversed-phase ethylene bridged hybrid phenyl column (2.1 mm 150 mm, 1.7 μm). The mobile phase consisted of two components: A (H2O) and B (ACN). The mobile phase gradient was from 95% A to 5% A over 2.0 min, at which point it was held for 1.0 min. The column was then allowed to re-equilibrate back to the starting mobile phase of 95% A for 2.0 min before the next injection. An injection volume of 10 μL was selected with a flow rate of 0.3 mL/min. PBDEDNA adducts were detected by monitoring their precursor-product
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transition ions in selected reaction monitoring (SRM) mode according to the results obtained from the ESI-QTOFMS. The optimized MS parameters were described as follows: the capillary voltage was 3000 V; the dwell time was 0.05 s; the extractor voltage was 2.5 V; the temperatures of the negative ESI source and desolvation gas were 118 and 500 °C, respectively; the cone gas and the desolvation gas flows were 40 and 650 L/h, respectively. Instrument operation and data acquisition were processed by using the Waters MassLynx V4.1 SCN562 software package.
’ RESULTS AND DISCUSSION Previous studies suggested polychlorinated biphenyls (PCBs) could bind to DNA with an apparent preference for the guanine residue, which was mediated by the corresponding quinone metabolites.21,27,28 It was also found that PCB-derived paraquinones rather than ortho-quinones were involved in the major DNA adduction. Due to the structural analogy to PCBs, it was reasonable to envisage a PBDEDNA adduction mediated by PBDEquinone metabolites. Motivated by the need for understanding biological conversion of PBDEs and the causes for their potential genotoxicity, we examined the reactivity of PBDEQs with dG and calf thymus DNA. Understanding their interactions with DNA will provide useful information to guide future toxicological studies of PBDEs in vivo. In an effort to better understand the reactivity of PBDEQs, a series of PBDE-derived para-quinones were synthesized (Figure 1). The availability of these compounds has allowed us to examine whether the chemical structures of PBDEQs affect their ability to bind to DNA at physiological pH. The structures of purified products were confirmed from GC-MS and NMR analysis (Figures S1S10 in the Supporting Information). For structural characterization of DNA adducts by ESI-MS/ MS, the reaction of PBDEQ with dG was carried out to produce respectable quantities synthetically. The analysis of the reaction mixture by ESI-QTOFMS clearly indicated that a mixture of dG adducts was produced. The observed adducts could be divided into two major types based on the reaction mechanism (Table 1 and Figure 2). Benzoquinone that is a di-α,β-unsaturated carbonyl compound has been found to undergo Michael Addition resulting in the formation of nucleoside adduction.29 PBDEQs have the same core structure as that of benzoquinone. Therefore, it was proposed that PBDEQs might also undergo DNA adduction at the quinone moiety, preferentially with dG followed by reductive cyclization with loss of a small molecule. According to the mechanism of Michael Addition, PBDEQs have four electron-deficient carbons (C-2, C-3, C-5, and C-6) available for nucleophilic attack by the exocyclic NH2 group at position N-2 of dG, which in turn can result in the formation of multiple adducts (Figure 2). As for the formation of a type I adduct, Michael Addition was initiated between the electrondeficient C-2 from the quinone and the N-2 from the dG, and led to the formation of unstable intermediate. Another nucleophilic attack on the carbonyl group of the intermediate generated a fivemember ring, which was followed by the loss of a molecule of (bromo-)1-hydroperoxy-benzene before forming an aromatic system that stabilized the molecule. Similarly, a type II adduct was formed by nucleophilic attack of N-2 from the dG at C-5 position of quinone followed by reductive cyclization with loss a molecule of water. Table 1 summarized the accurate mass values of the identified dG adducts by ESI-QTOFMS, and the 10722
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Figure 1. Chemical structures of OH-PBDEs (30 -OH-BDE-7, 60 -OH-BDE-17, and 6-OH-BDE-47), bromobenzoquinones (2BrBQ and 26BrBQ), and synthetic PBDEQs (20 40 BrPhO-BQ, PhO-6-BrBQ, 20 BrPhO-6-BrBQ, 20 40 BrPhO-6-BrBQ, and 20 40 60 BrPhO-6-BrBQ).
Table 1. Protonated Molecular Ions of the Identified PBDEdG Adducts by ESI-QTOFMS PBDEdG adduct ([M + H]+) type I adduct PBDEQ
expt
calcd
2 4 BrPhO-BQ
358.1142
358.1151
PhO-6-BrBQ
436.0237
436.0257
20 BrPhO-6-BrBQ
438.0235 436.0261
0 0
20 40 BrPhO-6-BrBQ 20 40 60 BrPhO-6-BrBQ
type II adduct error (ppm)
expt
calcd
error (ppm)
2.51
607.9652
607.9603
8.06
4.59
528.0461
528.0519
10.98
438.0236 436.0257
0.23 0.92
530.0454 607.9588
530.0498 607.9603
8.30 2.47
438.0257
438.0236
4.80
436.0252
436.0257
1.15
685.8634
685.8708
10.79
438.0226
438.0236
2.28
687.8667
687.8668
0.14
not detected
436.0224
436.0257
7.57
438.0237
438.0236
0.23
mass errors were within 11 ppm. The molecular weights of type I and II adducts were consistent with M (dG + PBDEQ (bromo-)1-hydroperoxy-benzene) and M (dG + PBDEQ H2O), respectively, which supported the proposed mechanism for the formation of dG adducts. It was clear to note that a type I adduct with two isotopic protonated ions at m/z 436 and 438 was observed from PhO-6-BrBQ, 20 BrPhO-6-BrBQ, 20 40 BrPhO-6BrBQ, and 20 40 60 BrPhO-6-BrBQ due to the same substitution pattern of bromine at the quinone ring (Table 1 and Figure 1). For comparison, the above identified DNA adducts were confirmed from UPLC-ESI-MS/MS analysis. A fragmentation pattern of loss of the deoxyribose moiety from the molecular ion was observed for each adduct. The unique product ions generated by loss of deoxyribose moiety were chosen for the SRM analysis with increased detection sensitivity. The results from UPLC-MS/MS analysis were similar with those from the Q-TOF-MS/MS analysis, except that the UPLC-MS/MS in SRM mode provides much better sensitivity. A type I adduct with retention time at 1.80 min was detected from PhO-6-BrBQ,
20 BrPhO-6-BrBQ, 20 40 BrPhO-6-BrBQ, and 20 40 60 BrPhO-6-BrBQ by monitoring the same transition ions at m/z 436 > 320 (Figure 3). In addition, no type II adduct was observed from 20 40 60 BrPhO-6-BrBQ, which indicated that higher PBDEQs exhibited less reactivity compared to lower congeners. It was also demonstrated that all PBDEQs could form a type I adduct, which might be due to the more facile cleavage group of bromophenol compared to hydrogen. Theoretically, nucleophilic attack of N-2 from the dG at the C-3 or C-5 positions of quinone will generate two isomers of type II adducts. For example, two isomers of a type II adduct were observed from PhO-6-BrBQ, and they shared the same molecular ion and product ion with loss of a deoxyribose moiety from the molecular ion (Figure 3B). However, only one peak was identified as a type II adduct for 20 BrPhO-6-BrBQ and 20 40 BrPhO-6BrBQ. One possibility was that the bromophenoxyl group, unlike a nonsubstituted phenoxyl group, exerted a regioselective effect on the Michael addition of quinone. It was proposed that nucleophilic attack at C-5 (para to the bromophenoxyl group) formed a 10723
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Figure 2. Proposed mechanism for the formation of PBDEdG adducts. Michael addition was first initiated between the electron-deficient carbon of quinone and the exocyclic nitrogen, NH2, at the N-2 position of dG. Another nucleophilic attack formed a five-member ring, which was followed by loss of water or (bromo-)1-hydroperoxy-benzene before forming an aromatic system that stabilized the molecule.
Figure 3. UPLC-ESI-SRM chromatograms of PBDEdG adducts. For type I adducts of PhO-6-BrBQ, 20 BrPhO-6-BrBQ, 20 40 BrPhO-6-BrBQ, and 20 40 60 BrPhO-6-BrBQ, the chromatograms were obtained in negative ion mode with a collision energy value of 30 V. The type I adduct of 20 40 BrPhO-BQ and type II adducts of 20 40 BrPhO-BQ, PhO-6-BrBQ, 20 BrPhO-6-BrBQ, and 20 40 BrPhO-6-BrBQ were detected in positive ion mode with a collision energy value of 15 V.
sterically favorable adduct, whereas nucleophilic attack at C-3 (ortho to the bromophenoxyl group) generated a sterically unfavorable adduct due to the steric hindrance from the bulky bromophenoxyl group (Figure 2).22 Similar results were also obtained from the analysis of the type II adduct of 20 40 BrPhO-BQ (Figure 3A). To further confirm the mechanism for the DNA adduction and the structures of the identified adducts, multiple-stage tandem mass spectra were obtained by using ESI-QTOFMS. By taking the type I adduct of PhO-6-BrBQ as an example, the mass values and isotopic distribution of its protonated molecular ions agreed well with the theoretical ones (Figure 4A). Two major
isotopic protonated ions were observed at m/z 436.0237 and 438.0235 (calcd. m/z 436.0257 and 438.0236) with mass errors of 4.59 and 0.23 ppm, respectively. In addition, the MS2 spectrum exhibited one characteristic fragment ion at m/z 322 after the release of 116 mass units from the molecular ion at m/z 438 (Figure 4B). This was corresponding to the loss of deoxyribose with proton transferring from the sugar to the guanine moiety. This also revealed that the DNA adduct was modified on the base moiety.22 As depicted in Figure 4C, other fragments of m/z 293.9869, 266.9779, 241.0670, 186.0638, and 134.0511 were consistent with the losses from either the guanine or the quinone portion of the product. Structural information on other adducts 10724
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Figure 4. ESI-QTOFMS (A), MS2 (B and C) spectra of a type I adduct generated by incubating PhO-6-BrBQ with dG. For the MS2 spectrum (B), a MS/MS experiment was carried out on a selected molecular ion at m/z 438.0236 by collision-induced dissociation (CID) with a collision energy value of 10 V. For the MS2 spectrum (C), a MS/MS experiment with a higher collision energy (45 V) was performed on the ion at m/z 321.9847 that was produced from the molecular ion at m/z 438.0236 through “source fragmentation”.
was also obtained from the corresponding tandem mass spectra (Figures S11S14 in the Supporting Information). Confirmation of the PBDEDNA structures by NMR should be carried out if sufficient amount of adducts could be collected. In addition to the DNA adducts formed from chemically synthetic PBDEQs, the possibility of DNA adduct formation from microsomal-mediated quinone metabolites of PBDEs was also investigated. Metabolic activation of PBDEs has been reported to result in mono- and dihydroxylated metabolites.1618 It has also been reported that OH-PBDEs are more potent than PBDEs regarding the disruption of thyroid function and alteration of steroidogenesis.23,30 Compared to PBDEs and OHPBDEs, little attention has been paid to the toxicity of hydroquinone and catechol metabolites. PBDEs are first metabolized to OH-PBDEs that can be further oxidized to dihydroxylated metabolites by microsomal cytochrome P450s (Figure 5A).
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Figure 5. Reaction pathway and UPLC-ESI-MS/MS analysis of dG adducts formed by PBDEQ metabolites of 60 -OH-BDE-17. Reaction pathway for the biotransformation of 60 -OH-BDE-17 and adduction of dG with PBDEQ metabolites (A); UPLC-ESI-SRM chromatograms of 60 -OH-BDE-17 and diOH-BDE metabolites (B), type I and type II DNA adducts (C). The collision energy values for type I and II adducts were 30 and 15 V, respectively. The UPLC and MS conditions used for the analysis of 60 -OH-BDE-17 and diOH-BDE metabolites were the same as that described in Figure S15 in the Supporting Information.
For example, four diOH-PBDEs were detected as metabolites of 60 -OH-BDE-17 as shown in Figure 5B. It is of particular concern to note that hydroquinone and catechol metabolites might be oxidized by peroxidases to quinones that are electrophilic and capable of reacting with DNA and sulfur nucleophiles such as glutathione and N-acetylcysteine.28 UPLC-ESI-MS/MS analysis indicated that one type I adduct with deprotonated molecular at m/z 436 and two type II adducts with protonated molecular ions at m/z 686 were detected from the reaction of dG and diOHPBDE metabolites of 60 -OH-BDE-17 in the presence of HRP and H2O2 (Figure 5C). Similarly, 30 -OH-BDE-7 produced a type II adduct with a protonated molecular ion at m/z 608 after sequential oxidation by microsomal cytochrome P450s and peroxidase (Figure S15 in the Supporting Information). Further more, a type I adduct was also observed from the reaction of dG with diOH-PBDE metabolites of 6-OH-BDE-47 (Figure S16 in the Supporting Information). It should be noted that 6-OH-BDE-47 was detected with the highest level among the reported OHPBDE congeners.1,31 This evidence suggested PBDE metabolites could be oxidized to species capable of covalently binding to DNA. 10725
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Figure 6. Effects of chemical structures of PBDEQs on the formation of DNA adducts. After treatment of DNA with 2BrBQ, 26BrBQ, PhO-6BrBQ, 20 BrPhO-6-BrBQ, and 20 40 BrPhO-6-BrBQ, the same DNA adduct with transition ions at m/z 436 and 320 was detected from the digestion mixture of DNA by using UPLC-ESI-MS/MS. N.D. = not detected. The experiment was repeated twice.
After the analysis of dG adducts of PBDEQs was completed, the adduct formation in calf thymus DNA was examined. The generated DNA adducts were not detected in Q-TOF-MS analysis, probably due to the problem of detection limit. UPLC-ESIMS/MS analysis, however, provided good detection on the DNA adducts because much better sensitivity was obtained compared to Q-TOF-MS/MS. The UPLC-ESI-MS/MS analysis of the digestion mixture of DNA treated with PhO-6-BrBQ, 40 BrPhO6-BrBQ, and 20 40 BrPhO-6-BrBQ revealed that only one type I adduct was detected and confirmed (Figure S17 in the Supporting Information), probably because the substituted bromine exists at the C-6 position of the quinone ring in these PBDEQs. On the other hand, one type II adduct was observed for 20 40 BrPhOBQ that does not have bromine on the quinone ring (Figure S17(F) in the Supporting Information). Thus, PBDEQs with a substituted bromine on the quinone ring is favored for forming a type I adduct, while the absence of bromine on the quinone ring resulted in a type II adduct. 2BrBQ and 26BrBQ were found to form the same adduct with the protonated isotopic ions at m/z 436 and 438 as the adduct generated from the incubation of dG with PhO-6-BrBQ, 40 BrPhO-6-BrBQ, or 20 40 BrPhO-6-BrBQ.26 In order to further examine the effects of the bromophenoxyl group on the DNA adduction from PBDEQs, 2BrBQ and 26BrBQ were also incubated with calf thymus DNA under the same conditions. The obtained results shown in Figure 6 clearly demonstrated that chemical structure exerted effects on the formation of the DNA adducts. 2BrBQ without the bromine substitution at the C-6 position was not as reactive as 26BrBQ. Compared to 2BrBQ and PhO-6-BrBQ, much more DNA adducts were detected from 40 BrPhO-6-BrBQ, suggesting that the occurrence of a bromophenoxyl group increased the reactivity of PBDEQs. Furthermore, 40 BrPhO-6-BrBQ was found to have higher reaction yield for the adduct formation than 20 40 BrPhO-6-BrBQ. No adduct was detected from the reaction with 20 40 60 BrPhO-6-BrBQ. Thus, the increase in the number of bromine substitutions in the phenoxyl group greatly lowered the reactivity of adduct formation due to the steric hindrance, which was consistent with results obtained from the DNA adduct formation from PCB-derived quinones.21,27,28 This data has important implications for understanding the mechanism of metabolic activation of PBDEs to genotoxins.
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In summary, the results obtained from this investigation clearly demonstrated that PBDEs could covalently bind to DNA in vitro to form DNA adducts mediated by quinone metabolites. The mechanism for the DNA adduct formation involved the Michael addition followed by reductive cyclization with loss of a molecule of water or (bromo-)1-hydroperoxy-benzene. Our data also suggested that ESI-MS/MS was a powerful analytical tool for the detection of suspected DNA adducts. The identified adducts might provide validated biomarkers for future in vivo PBDE risk assessment study. In addition, the implications that these adducts have for the cytotoxicity and genotoxicity of PBDEs are not known. Given the exposure potential to PBDEs and PBDEQ metabolites, however, the likelihood of genetic abnormality is sufficiently serious that it cannot be dismissed without further study.
’ ASSOCIATED CONTENT
bS
Supporting Information. Details of the identification of synthetic PBDEQs by GC-MS and NMR, DNA digestion procedures, ESI-QTOF-MS and UPLC-ESI-MS conditions, and analysis of PBDEDNA adducts, OH-PBDEs, and diOHPBDEs by ESI-MS/MS. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: 852-34117070; fax: 852-34117348; e-mail: zwcai@ hkbu.edu.hk.
’ ACKNOWLEDGMENT The authors thank the special postgraduate studentship program of “Persistent Toxic Substances” from Research Grant Council, University Grants Committee of Hong Kong SAR. We also thank Dr. Michael H.W. Lam (Department of Biology and Chemistry, City University of Hong Kong, Hong Kong) for providing the 30 -OH-BDE-7, 60 -OH-BDE-17, and 6-OH-BDE-47. ’ REFERENCES (1) Valters, K.; Li, H. X.; Alaee, M.; D’Sa, I.; Marsh, G.; Bergman, A.; Letcher, R. J. Polybrominated diphenyl ethers and hydroxylated and methoxylated brominated and chlorinated analogues in the plasma of fish from the Detroit River. Environ. Sci. Technol. 2005, 39 (15), 5612–5619. (2) Alaee, M.; Arias, P.; Sjodin, A.; Bergman, A. An overview of commercially used brominated flame retardants, their applications, their use patterns in different countries/regions and possible modes of release. Environ. Int. 2003, 29 (6), 683–689. (3) Qiu, X. H.; Bigsby, R. M.; Hites, R. A. Hydroxylated metabolites of polybrominated diphenyl ethers in human blood samples from the United States. Environ. Health Perspect. 2009, 117 (1), 93–98. (4) de Wit, C. A. An overview of brominated flame retardants in the environment. Chemosphere 2002, 46 (5), 583–624. (5) Wan, Y.; Wiseman, S.; Chang, H.; Zhang, X. W.; Jones, P. D.; Hecker, M.; Kannan, K.; Tanabe, S.; Hu, J. Y.; Lam, M. H. W.; Giesy, J. P. Origin of hydroxylated brominated diphenyl ethers: natural compounds or man-made flame retardants. Environ. Sci. Technol. 2009, 43 (19), 7536–7542. (6) Hites, R. A. Polybrominated diphenyl ethers in the environment and in people: A meta-analysis of concentrations. Environ. Sci. Technol. 2004, 38 (4), 945–956. (7) Vonderheide, A. P. A review of the challenges in the chemical analysis of the polybrominated diphenyl ethers. Microchem. J. 2009, 92 (1), 49–57. 10726
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