Evidence for the Bioactivation of 4-Nonylphenol to Quinone Methide

Chem. Res. Toxicol. 2010, 23, 1617–1628

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Evidence for the Bioactivation of 4-Nonylphenol to Quinone Methide and ortho-Benzoquinone Metabolites in Human Liver Microsomes Pan Deng,† Dafang Zhong,† Fajun Nan,† Sheng Liu,† Dan Li,† Tao Yuan,† Xiaoyan Chen,*,† and Jiang Zheng*,‡ Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China, and Center for DeVelopmental Therapeutics, Seattle Children’s Research Institute, DiVision of Gastroenterology, Department of Pediatrics, UniVersity of Washington, Seattle, Washington ReceiVed July 1, 2010

4-Nonylphenol (4-NP) is a well-known toxic environmental contaminant. The major objective of the present study was to identify reactive metabolites of 4-NP. Following incubations of 4-NP with NADPHand GSH-supplemented human liver microsomes, 6 GSH conjugates, along with 19 oxidized metabolites, were detected by UPLC/Q-TOF mass spectrometry utilizing the mass defect filter method. Several authentic key metabolite standards were chemically synthesized for structural identification. Three GSH conjugates were found to derive from quinone methide intermediates, and the other three resulted from ortho-benzoquinone intermediates. Conjugation of the quinone methides with GSH produced benzylicorientated GSH conjugates by 1,6-addition, while the reaction of the ortho-benzoquinone intermediates offered aromatic-orientated GSH conjugates. The conversion of 4-NP to the quinone methides and orthohydroquinones required cytochromes P450, specifically CYPs1A2, 2C19, 2D6, 2E1, and 3A4, while the oxidation of ortho-benzohydroquinones to the corresponding benzoquinones was apparently independent of microsomal enzymes. The ortho-benzoquinone derived from 4-NP was isomerized to the corresponding hydroxyquinone methide, and the former dominated the latter at a rate of approximately 20:1. The findings of the quinone methide and benzoquinone metabolites intensified the concern on the impact of 4-NP exposure on human health. Introduction Nonylphenol polyethoxylates (NPnEOs) are important nonionic surfactants that have been used for a variety of industrial purposes and the annual production volume is approximately 650,000 tons (1). One of their primary biodegradation products is 4-nonylphenol (4-NP1), which is one of the well-known persistent organic pollutants (POPs), principally interfering with endocrine function and inducing apoptotic tendencies (2). In the past decades, a wide variety of structurally diverse environmental compounds have been identified as POPs, which pose risks of causing adverse effects to human health. These compounds include pesticides, plasticizers, furans, polycyclic aromatic hydrocarbons, and polyhalogenated biphenyls (3, 4). It is known that the toxic effects of some of these compounds are potentiated after metabolism by P450 enzymes (5-9). In the case of polychlorinated biphenyl (7), benzo[a]pyrene (8), and furan (9), the toxicities were attributed to their metabolites, such as quinones and unsaturated dialdehyde, formed by P450 oxidation. Because of low solubility and high hydrophobicity, 4-NP easily accumulates in environmental compartments, enters the food chain, and has been found in aquatic environments, * To whom correspondence should be addressed. (X.C.) Tel/Fax: +8621-50800738. E-mail: [email protected]. (J.Z.) Tel: (206) 884-7651. Fax: (206) 987-7660. E-mail: [email protected]. † Shanghai Institute of Materia Medica. ‡ University of Washington. 1 Abbreviations: CYP, cytochrome P450; ESI, electrospray ionization; GSH, glutathione; HLM, human liver microsome; HMBC, heteronuclear multiple-bond correlation; MDF, mass defect filter; 4-NC, 4-nonylcatechol; 4-NP, 4-nonylphenol; POPs, persistent organic pollutants; UPLC/Q-TOF MS, ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

food-contact materials, foods, animals, and even humans (2, 4, 10). Recent studies indicated that 4-NP could induce apoptosis in sertoli cells of male Sprague-Dawley rats and that oxidative stress might play an important role in this process (11-13). The impacts of 4-NP in the environment include the feminization of living organisms and the decrease in male fertility at concentrations as low as 8.2 µg/L (2). The mean daily oral intake of nonylphenol by humans is estimated to be 0.16 mg/day (14). The potential risk of 4-NP to human populations is still unclear and is a topic of considerable debate (15-17). Aromatic compounds with oxygen-containing substituents belong to one class of the best known examples of POPs, such as phenols, hydroquinones, and catechols, all of which can be converted to chemically reactive quinones by various enzymes (7, 8). The cytotoxic and/or genotoxic effects elicited by these compounds were often attribute to intracellular reactions of electrophilic or free radical intermediates (18). Because of the para-methylene phenol moiety in the structure of 4-NP, we speculated that 4-NP might be metabolized to quinone methide and ortho-benzoquinone derivatives. In the present study, we report the successful characterization of quinone methide and ortho-benzoquinone metabolites of 4-NP, and the identification of the cytochrome P450 enzymes responsible for the bioactivation of 4-NP. In addition, this article describes the chemical synthesis of glutathione conjugates derived from the quinone methide and ortho-benzoquinone metabolites.

Materials and Methods Materials. The following chemicals were purchased from SigmaAldrich (St. Louis, MO, USA): 4-nonylphenol (PESTANAL, analytical standard), leucine enkephalin, GSH, and NADPH. Pooled

10.1021/tx100223h  2010 American Chemical Society Published on Web 09/15/2010

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Scheme 1. Synthesis of Hydroxylated Metabolites and GSH Conjugates of 4-Nonylphenola

a

Dashed arrows represent the possible carbons that may react with GSH.

human liver microsomes (HLMs) and recombinant P450 enzymes CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5 were purchased from BD Gentest (Woburn, MA, USA). The concentrations of the recombinant enzymes were all 50 nM in 100 mM potassium phosphate buffer (pH 7.4). All other reagents and solvents were of either analytical or HPLC grade. Synthesis. Three oxidative metabolites of 4-NP and three GSH conjugates were synthesized as shown in Scheme 1. A brief description of the synthesis procedure follows below. 4-(1-Hydroxynonyl)phenol (1), 4-(1-Hydroxynonyl)catechol (2), and 4-Nonylcatechol (3). Grignard reagent was prepared by adding 2 mL of 1-bromooctane (12.4 g, 64 mmol) to a suspension of magnesium (2.3 g, 96 mmol) in diethyl ether, and the reaction mixture was stirred for 1 h at 70-75 °C and allowed to cool down to room temperature with stirring. Next, the resulting Grignard reagent was added dropwise to a solution of 4-hydroxybenzaldehyde (2.0 g, 16 mmol) in tetrahydrofuran at 0 °C. After stirring overnight at room temperature, the reaction mixture was diluted with 200 mL of diethyl ether and washed with 200 mL of saturated NH4Cl. Finally, the organic layer was evaporated to dryness under reduced pressure to yield a white solid product 1 (3.6 g, 95%). lH NMR (CD3Cl): δ 7.21 (dd, J ) 8.7 Hz, 2 H, H-3, 5), 6.79 (d, J ) 8.7 Hz, 2 H, H-2, 6), 4.60 (t, J ) 6.7 Hz, 1 H, H-1′), 1.82 (m, 2 H, H2-2′), 1.29 (brs, 12H, (CH2)6), 0.88 (t, J ) 6.6 Hz, 3 H, H3-9′). Q-TOF MS: m/z 235.1700 [M H]- (calcd for C15H24O2: 235.1698). 4-(1-Hydroxynonyl)catechol

(2) was prepared using a procedure similar to that used for the synthesis of 1 except that 3,4-dihydroxybenzaldehyde replaced 4-hydroxybenzaldehyde. The resultant product was characterized by UPLC-Q/TOF high resolution MS: m/z 251.1669 [M - H](calcd for C15H24O4: 251.1647). The crude product was used in the next step without further purification. The crude 4-(1hydroxynonyl)catechol (1.4 g, 5.56 mmol) was hydrogenated with H2 over Pd/C in ethanol/HCl, and the mixture was stirred at room temperature for 2 h, then the Pd/C was filtered off, and the filtrate was purified by chromatography on a silica gel column eluted with hexane/EtOAc (4:1, v/v) to afford 4-nonylcatechol (4-NC, 3) as an oil, which was solidified at room temperature (1.17 g, 89%). lH NMR (CD3Cl): δ 6.80 (d, J ) 8.1 Hz, 1 H, H-6), 6.71 (brs, 1 H, H-3), 6.61 (d, J ) 8.1 Hz, 1 H, H-5); 2.50 (t, J ) 7.6 Hz, 2 H, H2-1′), 1.57 (m, 2 H, H2-2′), 1.28 (brs, 12 H, (CH2)6), 0.90 (t, J ) 6.4 Hz, 3 H, CH3-15). Q-TOF MS: m/z 235.1655 [M - H]- (calcd for C15H24O2, 235.1698). Exocyclic GSH Conjugate 4-(1-(Glutathione-S-yl)nonyl)phenol (4). Compound 1 (100 mg, 0.43 mmol) was added to a solution of GSH (130 mg, 0.43 mmol) in TFA. The reaction mixture was stirred for 2 h at room temperature. The solvent was removed under reduced pressure and purified by chromatography on a reversed-phase C18 column (gradient eluting with CH3CN/1%HCOOH 50:50 to 90:10) to afford target compound 4 (70 mg, 31%).

BioactiVation of 4-Nonylphenol Exocyclic GSH Conjugate 4-(1-(Glutathione-S-yl)nonyl)catechol (5). Compound 5 (65 mg, 30%) was prepared from 2 (120 mg, 0.48 mmol) by procedure similar to that used for the synthesis of 4. Ring GSH Conjugate 6-(Glutathione-S-yl)-4-nonylcatechol (7). 4-Nonylcatechol (3, 304 mg, 1.29 mmol) was dissolved in methanol and cooled to 0-5 °C. Upon the addition of silver oxide (900 mg, 3.87 mmol), the resulting suspension was stirred for 2 min and filtered to give compound 6 as a dark red liquid. This liquid was added dropwise into a GSH solution in methanol/water. After 10 min of stirring at room temperature, the solvent was removed under reduced pressure, and the residue was crystallized from ethyl acetate and water (1:1, v/v) to provide 7 (360 mg, 52%) as a gray solid. The structures of synthetic GSH conjugates were characterized by high resolution Q-TOF mass spectrometry and NMR. NMR spectra were recorded on a Bruker DRX 500 NMR spectrometer (Newark, DE, USA) operating at 400 and 100 MHz for proton and carbon, respectively. All compounds were dissolved in deuterated methanol. Chemical shifts are expressed as parts per million relative to tetramethylsilane. UPLC/Q-TOF MS Analyses. Chromatographic separation for metabolite profiling was achieved via an Acquity UPLC system (Waters Corp., Milford, MA, USA) on an Acquity UPLC HSS T3 column (1.8 µm, 2.1 mm × 100 mm, Waters Corp.). The mobile phase was a mixture of 5 mM ammonium acetate (A) and acetonitrile (B), and the gradient elution used was started from 5% B and maintained for 3 min, then increased to 100% B linearly in 15 min, maintained for 1 min, and finally decreased to 5% B to equilibrate the column. The column temperature was maintained at 40 °C, and the flow rate was 0.4 mL/min. The MS detection was conducted by a Synapt Q-TOF high-resolution mass spectrometer (Waters Corp., Milford, MA, USA) operated in negative ion electrospray (ES -ve) mode, using nitrogen and argon as API and collision gas, respectively. Data were acquired from 80-1000 Da, using a source temperature of 100 °C, a desolvation temperature of 350 °C, and a cone voltage of 40 V. Data were centroided during acquisition using an internal reference comprising a 400 ng/mL leucine enkephalin solution infused at 5 µL/min, generating a reference ion in ES -ve mode at m/z 554.2615. Data acquisition was achieved using MSE, which has two separate scan functions that are programmed with independent collision energies. In this way, a low collision energy scan can be immediately followed by a scan where the collision energy is ramped over a higher range to induce fragmentation of the ions transmitted through Q1. Acquiring data in this manner provided for the collection of information of intact precursor ions as well as fragment ions. The mass spectrometer and UPLC system were operated under MassLynx 4.1 software. The actual samples were compared with the control samples using a MetaboLynx subroutine of the MassLynx software. Mass defect filtering (MDF) was used for screening metabolites using a filter of 40 mDa between the filter template and the target metabolites. Fragmentations were proposed on the basis of plausible deprotonation sites, subsequent isomerization, and electron species, as well as bond saturation. Comparison between the parent and metabolite fragment ion spectra further aided in the identification of metabolite structures and sites of modifications in the parent molecule. In Vitro Incubations. A typical incubation mixture contained 1.0 mg/mL HLMs and 50 µM substrate [4-NP, 4-NC, or 4-(1hydroxynonyl)phenol], made up to 200 µL with 100 mM potassium phosphate (pH 7.4). The mixture was preincubated in a shaking water bath at 37 °C for 3 min, and then NADPH (2.0 mM) was added to initiate the reactions. After 60 min of incubation, the reactions were terminated by adding equal volumes of ice-cold acetonitrile. To trap reactive metabolites, separate incubations were performed in the presence of GSH at the final concentration of 10 mM. Control samples containing no NADPH or substrates were included. Each incubation was performed in duplicate. General incubation conditions of 4-NP with human recombinant P450 enzymes, including CYPs1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9,

Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1619 2C19, 2D6, 2E1, 3A4, or 3A5, were essentially identical to those for HLMs. In each P450 incubation system, the MS peak areas of detected metabolites were recorded to determine the contributions of the selected P450 enzymes to the formation of 4-NP metabolites. The highest peak area of each metabolite detected in the incubations with the corresponding P450 enzymes (10 pmol enzyme with a total volume of 200 µL in each incubation) was normalized to 100%, and the MS responses of the specific metabolite in the other P450 enzyme incubation samples were expressed as the percentage relative to this highest level. Moreover, the MS response of leucine enkephalin was used for monitoring the performance of the Q-TOF mass spectrometer, which was maintained at approximately 300 counts during the multiday analysis of in vitro samples, indicating an acceptable MS response reproducibility. Preparation of Samples. To a 100 µL aliquot of the above in vitro incubation samples was added 200 µL of methanol. After it was vortex-mixed and centrifuged at 11,000 g for 5 min, the supernatant was transferred into a glass tube, evaporated to dryness under a stream of nitrogen at 40 °C, and then reconstituted in 100 µL of acetonitrile and 5 mM ammonium acetate (20:80, v/v). A 10 µL aliquot of the reconstituted solution was injected onto UPLC/ Q-TOF MS for analysis.

Results Identification of Oxidative Metabolites in HLMs. To investigate potential metabolic pathways leading to the bioactivation of 4-NP, the metabolites obtained from HLM incubations of 4-NP were first analyzed by UPLC/Q-TOF MS. The LC/MS data was then processed by the MDF method, which takes advantage of high-resolution LC/MS data to filter out interferences and facilitate the detection of metabolites in complex biological matrixes (19). As shown in Figure 1, compared with the control sample, 19 oxidative metabolites of 4-NP were detected in HLM incubations. These metabolites could be mainly classified into six categories, i.e., dehydrogenated (M1, m/z 217.1592), monohydroxylated and dehydrogenated (M2, m/z 233.1542), monohydroxylated (M3, m/z 235.1698), dihydroxylated and dehydrogenated (M4, m/z 249.1491), dihydroxylated (M5, m/z 251.1647), and trihydroxylated (M6, m/z 267.1596) metabolites. The structures of these metabolites were characterized on the basis of high resolution MS and NMR spectral data. Table 1 summarizes the chemical formulas, accurate masses, and LC retention times (Rt) of these oxidative metabolites identified in HLMs. Q-TOF MS Analyses of Authentic Compounds and 4-NP Metabolites. The high energy mass spectral and chromatographic behaviors of metabolites were compared with those of the parent compound and synthetic authentic compounds to determine the sites of modification. Figure 2 shows the proposed fragmentation pathways of 4-NP and synthetic oxidative metabolites. Under negative ESI, 4-NP had a deprotonated molecule [M - H]- at m/z 219.1754. The characteristic product ion at m/z 106.0432 in the high collision energy mass spectrum was formed through the homolytic cleavage of the C1′-C2′ bond. Synthetic compound 4-(1-hydroxynonyl)phenol is an oxidative metabolite at the benzylic carbon of 4-NP with a molecular ion [M - H]- at m/z 235.1702. Product ions at m/z 217.1600 and 132.0573 were observed. The former ion was formed by the loss of a single molecule of water, indicating the existence of an aliphatic hydroxyl group. This fragment ion could further lose a hexane radical (-85.1027 Da) to give the ion at m/z 132.0573. Synthetic 4-NC had a molecular ion [M - H]- at m/z 235.1655. The observation of a major fragment ion at m/z 122.0342, which is 15.9910 Da higher than the fragment ion at m/z 106.0432 of the parent compound suggests that 4-NC had fragmentation patterns very similar to that of 4-NP. 4-(1-

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Figure 1. In vitro metabolism of 4-nonylphenol (50 µM) mediated by human liver microsomes (1.0 mg/mL). (A) Incubations performed in the presence of NADPH (the insert is the expanded chromatogram in the region of 7-12 min); (B) incubations performed in the absence of NADPH.

Table 1. Identification of 4-Nonylphenol Oxidative Metabolites in Human Liver Microsomes Using UPLC/Q-TOF Mass Spectrometry

M0 M1 M2-1 M2-2 M2-3 M3-1 M3-2 M3-3 M3-4 M4-1 M4-2 M4-3 M4-4 M5-1 M5-2 M5-3 M5-4 M5-5 M6-1 M6-2

description

Rt (min)

molecular formula

calculated mass (m/z)

measured mass (m/z)

error (in ppm)

parent dehydrogenation monohydroxylation and dehydrogenation monohydroxylation and dehydrogenation monohydroxylation and dehydrogenation hydroxylation hydroxylation hydroxylation hydroxylation dihydroxylation and dehydrogenation dihydroxylation and dehydrogenation dihydroxylation and dehydrogenation dihydroxylation and dehydrogenation dihydroxylation dihydroxylation dihydroxylation dihydroxylation dihydroxylation trihydroxylation trihydroxylation

13.81 12.96 7.84 7.95 10.58 10.22 10.33 11.18 12.82 8.66 8.78 9.69 9.85 8.15 8.24 9.38 9.49 10.44 7.35 7.46

C15H24O C15H22O C15H22O2 C15H22O2 C15H22O2 C15H24O2 C15H24O2 C15H24O2 C15H24O2 C15H22O3 C15H22O3 C15H22O3 C15H22O3 C15H24O3 C15H24O3 C15H24O3 C15H24O3 C15H24O3 C15H24O4 C15H24O4

219.1749 217.1592 233.1542 233.1542 233.1542 235.1698 235.1698 235.1698 235.1698 249.1491 249.1491 249.1491 249.1491 251.1647 251.1647 251.1647 251.1647 251.1647 267.1596 267.1596

219.1760 217.1605 233.1536 233.1539 233.1527 235.1698 235.1700 235.1709 235.1713 249.1490 249.1490 249.1514 249.1504 251.1640 251.1643 251.1650 251.1663 251.1652 267.1589 267.1610

5.0 6.0 -2.6 -1.3 -6.4 0.0 0.9 4.7 6.4 -0.4 -0.4 9.2 5.2 -2.8 -1.6 1.2 6.4 2.0 -2.6 5.2

Hydroxynonyl)catechol had a molecular ion [M - H]-at m/z 251.1669, which lost a water molecule easily to give a product ion at m/z 233.1541, and further fragmentation produced an ion at m/z 148.0521, which is an odd-electron ion, through the homolytic cleavage of the C3′-C4′ bond. Metabolite M1 at an Rt of 12.96 min exhibited a deprotonated molecule [M - H]- at m/z 217.1605, and accurate mass measurement revealed a two hydrogen atom loss in comparison with the [M - H]- of 4-NP, suggesting that the parent molecule

underwent dehydrogenation. A high collision energy mass spectrum showed an abundant fragment ion at m/z 132.0578, which was the same as that of synthetic 4-(1-hydroxynonyl)phenol. The dehydrogenation site was thus tentatively proposed at the side chain of 4-NP between C1′ and C2′. Metabolites M2-1 (Rt ) 7.84 min), M2-2 (Rt ) 7.95 min), and M2-3 (Rt ) 10.58) had deprotonated molecules [M - H]at m/z 233.1536. Accurate mass measurement showed the chemical formula of C15H22O2 (Table 1), suggesting the oxida-

BioactiVation of 4-Nonylphenol

Figure 2. Q-TOF mass spectra of synthetic standard compounds under high collision energy. (A) 4-Nonylphenol; (B) 4-(1-hydroxynonyl)phenol; (C) 4-nonylcatechol; (D) 4-(1-hydroxynonyl)catechol.

tion and dehydrogenation of molecule 4-NP. The high energy mass spectra of both M2-1 and M2-2 revealed a major product ion at m/z 132.0582, which is the same as that of M1. For metabolite M2-3, the major fragment ion was observed at m/z 106.0418, which is the same as that of 4-NP. The observed fragment ions indicated that the modification took place at the side chain of 4-NP, but the exact structures cannot be assigned on the basis of limited information. Metabolites M3-1 (Rt ) 10.22 min), M3-2 (Rt ) 10.33 min), M3-3 (Rt ) 11.18 min), and M3-4 min (Rt ) 12.82) gave the deprotonated molecule [M - H]- at m/z 235.1698. Accurate mass measurement demonstrated the chemical formula of C15H24O2, indicating an addition of an oxygen to molecule 4-NP. Metabolites M3-3 and M3-4 were identified to be 4-(1hydroxynonyl)phenol and 4-NC, respectively, by comparing their chromatographic behaviors and MS characteristics with the synthetic authentic standards. The high energy mass spectra for M3-1 and M3-2 revealed the major fragment ion at m/z 106.0425, which is the same as that of 4-NP. This indicates that hydroxylation occurred on the side chain but not at the C-1′ position. Metabolites M4-1, M4-2, M4-3, and M4-4 had the deprotonated molecule [M - H]- at m/z 249.1490, with an elemental composition of C15H22O3, suggesting the addition of two oxygens and the loss of two hydrogen atoms at molecule 4-NP. The chromatographic retention times were 8.66, 8.78, 9.69, and 9.85 min, respectively. In the high collision energy

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mass spectra of M4-1 and M4-2, two major fragment ions at m/z 120.0208 and 92.0277 were observed. The former ion revealed a 14-Da increase compared with the fragment ion at m/z 106.0432 for 4-NP, and further loss of CO (-27.9931 Da) resulted in the fragment ions at m/z 92.0277. These findings indicate that M4-1 and M4-2 presumably arose from dihydroxylation at the side chain of 4-NP, followed by further oxidation of the secondary alcohol at C-1′ to the corresponding ketone. The high energy mass spectrum of M4-3 showed major fragment ions at m/z 221.156, which were formed by the loss of CO (-27.9930 Da), and at m/z 122.038, the characteristic fragment ion of 4-NC. On the basis of the MS data, M4-3 was tentatively indentified as the ω oxidation of 4-NC to the corresponding aldehyde. The fragment ions for M4-4 included m/z 132.0580 and 106.0440, which were the characteristic product ions of 4-(1-hydroxynonyl)phenol (M3-3). Moreover, M4-4 lost a molecule of water easily to give a predominant fragment ion at m/z 231.1400, and further fragmentation generated an ion at m/z 203.146 (-CO). Metabolite M4-4 was thus tentatively assigned to be the ω oxidation of M3-3 to an aldehyde. Metabolites M5-1, M5-2, M5-3, M5-4, and M5-5 had the deprotonated molecule [M - H]- at m/z 251.1640, which is 31.9880 Da higher than that of the parent. Accurate mass measurement implicated the dihydroxylation of 4-NP. The chromatographic retention times were found to be 8.15, 8.24, 9.38, 9.49, and 10.44 min, respectively. Metabolites M5-1, M5-2, M5-3, and M5-4 had a characteristic fragment ion at m/z 122.037, which is the same as that of 4-NC, suggesting that these metabolites were derived from 4-NC via side chain oxidation, but the exact position of hydroxylation cannot be identified at this stage. Metabolite M5-5 was identified as 4-(1hydroxynonyl)catechol by comparing the chromatographic retention time and MS characteristics with those of the synthetic standard. Metabolites M6-1 (Rt ) 7.35 min) and M6-2 (Rt ) 7.46 min) displayed the deprotonated molecule [M - H]- at m/z 267.1589. Accurate mass measurement showed the chemical formula C15H24O4, suggesting the formation of trihydroxylated metabolites of 4-NP. Because of the low amount of these metabolites generated, no high energy mass spectra were available, and the sites of hydroxylation were not assigned. In summary, a total of 19 oxidative metabolites were identified in human liver microsomal incubations. The structures of these metabolites are confirmed after comparison with authentic standards or tentatively proposed on the basis of MS data (Scheme 2). Trapping Reactive Metabolites of 4-NP with GSH. 4-Nonylphenol was incubated with HLMs in the presence of GSH as trapping agent and analyzed by UPLC/Q-TOF MS. The GSH conjugates were screened by processing the LC/MS data with the GSH conjugate MDF template. A total of six GSH conjugates were observed in an NADPH-dependent manner. They had deprotonated molecules at m/z 524.2427 (M7, Rt ) 8.64 min), m/z 540.2376 (M8-1, Rt ) 6.80 min; M8-2, Rt ) 8.71 min; and M8-3, Rt ) 9.03 min), and m/z 556.2313 (M9-1, Rt ) 7.05 min; and M9-2, Rt ) 8.08 min) (Table 2). Among these six conjugates, M7 and M8-3 were the most abundant on the basis of the peak areas. Figure 3 illustrates UPLC/Q-TOF MS chromatograms of the GSH conjugates detected in HLM metabolism incubations of 4-NP. To investigate the formation mechanism of reactive intermediates, the oxidative metabolites 4-NC and 4-(1-hydroxynonyl)phenol were incubated separately with HLMs in the

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Scheme 2. Proposed Metabolic Pathways of 4-Nonylphenol Following Incubations with Human Liver Microsomes in the Presence of NADPH

Table 2. Identification of GSH Conjugates in Human Liver Microsomes Using UPLC/Q-TOF Mass Spectrometry description M7 M8-1 M8-2 M8-3 M9-1 M9-2

parent + GS parent + GS +O 4-nonylcatechol + 4-nonylcatechol + 4-nonylcatechol + 4-nonylcatechol +

GS GS GS + O GS + O

Rt (min)

molecular formula

calculated mass (m/z)

measured mass (m/z)

error (in ppm)

8.64 6.80 8.71 9.03 7.05 8.08

C25H39N3O7S C25H39N3O8S C25H39N3O8S C25H39N3O8S C25H39N3O9S C25H39N3O9S

524.2430 540.2380 540.2380 540.2380 556.2329 556.2329

524.2427 540.2376 540.2361 540.2353 556.2313 556.2297

-0.57 -0.74 -3.5 -5.0 -2.9 -5.8

presence of GSH. The metabolic profile of 4-NC in HLMs showed four GSH conjugates, corresponding to M8-2, M8-3, M9-1, and M9-2, respectively, and M8-3 was the most abundant conjugate. Moreover, M8-2 and M8-3 were formed by NADPH-independent processes. In the HLM incubation of 4-(1-hydroxynonyl)phenol, only M9-2 was detected. Figure 4 shows the extracted ion chromatograms of the GSH adducts from incubations of 4-NP, 4-nonylcatechol (M3-4), and 4-(1hydroxynonyl)phenol (M3-3) in HLMs, respectively, with or without NADPH.

Characterization of GSH Conjugates of 4-NP (M7 to M9). The MS spectrum of M7 showed a deprotonated molecular ion at m/z 524.2427, 305.0680 Da higher than that of 4-NP, indicating the incorporation of a GS moiety to molecule 4-NP (Table 2). In negative ESI analysis, the characteristic fragment ions of this GSH conjugate were mainly derived from the GSH moiety (Figure 5), which are the same as those reported for other GSH conjugates with different instruments (17, 18). The high energy mass spectrum of M7 revealed a diagnostic fragment ion of a GSH conjugate at m/z 306.0733 (deprotonated

Figure 3. Q-TOF MS analysis of 4-nonylphenol-derived GSH conjugates formed in the HLM incubations in the presence of GSH and NADPH.



Figure 4. Extracted ion chromatograms of GSH conjugates ([M - H]- 524.243, 540.238, 556.233) following incubations of 4-NP (A), 4-nonylcatechol (B), and 4-(1-hydroxynonyl) phenol (C) in GSH-supplemented HLMs. Left column: microsomes also supplemented with NADPH. Right column: microsomes supplemented without NADPH.

GSH moiety). Further elimination of the element of H2S generated the anion at m/z 272.0875, and other fragment ions originated from GSH were also observed at m/z 254.0769, 210.0871, 160.0060, 143.0444, and 128.0338 with low abundance. Unfortunately, the mass spectrometry data are unable to tell the regio-structure of the GSH conjugate. On the basis of our knowledge, 4-NP does not directly react with GSH and is likely metabolized to a quinone methide

intermediate which further reacts with GSH by 1,6-addition (Scheme 3). We chemically synthesized GSH conjugate 4 as outlined in Scheme 1. Briefly, a Grignard reaction gave intermediate 1, followed by an SN1 displacement reaction with GSH catalyzed by trifluoroacetic acid (TFA). As expected, the 1 H NMR spectrum showed p-substituted aromatic proton signals at δ 7.13 (d, J ) 8.3 Hz, H-3, 5) and 6.73 (d, J ) 8.3 Hz, H-2, 6) in an AB-A′B′ pattern (Table 3). The long-range coupling

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Figure 5. Q-TOF high collision energy mass spectra of GSH conjugates in human liver microsomes. (A) GSH conjugate of 4-nonylphenol, M7; (B-D) GSH conjugates of monohydroxylated 4-nonylphenol, M8-1, M8-2, and M8-3; (E-F) GSH conjugates of dihydroxylated 4-nonylphenol, M9-1, and M9-2.

between H2-1′′ (δ 2.77, dd, J ) 13.6, 6.1 Hz; 2.61, dd, J ) 13.6, 7.5 Hz) and C-1′ (δ 116.8) observed in the HMBC NMR spectrum (Figure 6) confirmed the structure of conjugate 4. The observed M7 was assigned as GSH conjugate 4 since they shared the same retention time, deprotonated molecule, and fragmentation pattern. At retention times of 6.80, 8.71, and 9.03 min, M8-1, M8-2, and M8-3 were detected with the deprotonated molecule at m/z 540.2380. Accurate mass measurement suggested the

incorporation of a GS molecule and an oxygen atom (15.9950 Da) to 4-NP. Conjugate 5, a 4-NC-derived GSH conjugate, was synthesized by the same procedure for the synthesis of conjugate 4 except for the use of 3,4-dihydroxybenzaldehyde as the starting material. The 1H NMR spectrum showed a typical ABX proton coupling system of an aromatic ring at δH 6.81 and 6.82 (d, J ) 1.7 Hz), 6.71 and 6.73 (d, J ) 8.5 Hz), and 6.66 and 6.64



Scheme 3. Proposed Metabolic Formation of Six GSH Conjugates from 4-Nonylphenol in Human Liver Microsomal Incubations Supplemented with NADPH and GSH

(dd, J ) 8.5, 1.7 Hz) (Table 3). This indicates the presence of a 1,2,4-trisubstituted phenyl ring. Conjugate 7, another 4-NC-derived GSH conjugate, was synthesized by three steps (Scheme 1), including (1) reduction of intermediate 2 prepared for the synthesis of GSH conjugate 5 to intermediate 3; (2) oxidation of compound 3 to 1,2-

benzoquinone derivative 6; and (3) adduction of intermediate 6 with GSH. We speculated that the conjugation of compound 6 with GSH would produce three regioisomers, i.e., GSH conjugates 7, 8, and 9, but only two GSH conjugates were detected by UPLC/Q-TOF MS. Unexpectedly, one of the peaks was characterized as conjugate 5 (Rt ) 8.71 min), on the basis

Figure 6. HMBC spectrum of synthetic GSH conjugate 4-(1-(glutathione-S-yl)nonyl)phenol. The long-range coupling between H2-1′′ and C-1′ was observed, which confirms the attachment of GSH to the benzylic carbon.

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Table 3. 1H- and 13C-NMR Data of Synthetic GSH Conjugates 4, 5, and 7 (δ in CD3OD) 5a

4 position 1 2 3 4 5 6 1′ 1′′ 2′′ 3′′

δH (mult; J, Hz) 6.73 (d, 8.3) 7.13 (d, 8.3) 7.13 6.73 3.79 2.77 2.61 4.39

(d, 8.3) (d, 8.3) (dd, 8.8, 6.1) (dd, 13.6, 6.1) (dd, 13.6, 7.5) (dd, 7.5, 6.1)

δC 158.1 116.8 130.6 134.9 130.6 116.8 51 34 54.9

7

δH (mult; J, Hz)

6.82 or 6.81 (d, 1.7) 6.66 or 6.64 (dd, 8.5, 1.7) 6.73 or 6.71 (d, 8.5) 3.73 (m) 2.61-2.83 (m) 4.47 (dd, 9.1, 4.7) or 4.41 (t, 6.6)

δH (mult; J, Hz)

δC 146 147 116.3 (116.2) 135.8 (135.6) 121.3 (121.2) 116.6 51.0 (51.3) 34.0 (34.1)

6.60 (d, 2.0) 6.71 (d, 2.0) 2.44 3.32 3.08 4.46

54.9

173.5

δC 145.3 146.9 117.4 136.4 126.2 120.8 36.7 37.7

(2H, t, 7.7) (dd, 13.9, 9.0) (dd, 13.9, 4.8) (dd, 9.0, 4.8)

55.2

173.8 (173.6)

173.6

a

NMR spectra of 5 exhibited two sets of signals. This was suggestive of the presence of two epimeric compounds resulting from the conjugation of GSH at the prochiral benzylic carbon of the reactant.

of its deprotonated molecule and fragmentation pattern. Conjugates 7-9 would be readily differentiated by their characteristic 1H NMR spectra. The 1H NMR spectra of both conjugates 7 and 9 would show two aromatic protons splitting into a doublet, but the coupling constant for 7 would be 1-3 Hz, while that for 9 would be 7-9 Hz. The 1H NMR spectrum of 8 would show two singlets. In fact, the 1H NMR spectrum of the second synthetic GSH conjugate revealed two aromatic protons as a doublet with a coupling constant of 2 Hz (Table 3). Clearly, the GSH conjugate obtained in the reaction was GSH conjugate 7. The structures of synthetic GSH conjugates 5 and 7 were confirmed by their 2D NMR HMBC spectra (refer to Supporting Information for the 2D NMR spectra). The observed M8-2 and M8-3 were assigned as GSH conjugates 5 and 7, respectively, since these metabolites showed the same retention time, deprotonated molecule, and fragmentation pattern as those of the corresponding authentic conjugates. After comparison with the authentic standards, M7 and M8-2 were confirmed as benzylic GSH conjugates, whereas M8-3 was identified as the aromatic-orientated one. The Q-TOF MS analyses of these two classes of GSH conjugates showed distinctive fragment characteristics. For M7 and M8-2, the most abundant product ion was observed at m/z 306.0733; however, M8-3 produced the major fragment ion at m/z 272.0869. Similar fragments have been reported for 4-methylphenol GSH conjugates (20). In the high energy mass spectrum of M8-1, the predominate fragment ion was at m/z 306.0771. Taken together, M8-1 was most likely a benzylic-orientated GSH conjugate. Accurate mass measurement of the deprotonated molecule of M9 indicated the chemical formula of C25H39N3O9S (Table 1), suggesting the introduction of a GS moiety (305.0682 Da) and two oxygen atoms (31.9898 Da) to molecule 4-NP. The high energy mass spectra of M9-1 and M9-2 showed fragment ions at m/z 272.0880 and 283.1372 via the characteristic cleavage of the C-S bond of the glutathionyl group. Metabolites M9-1 and M9-2 shared mass fragment patterns similar to those of the aromatic-orientated GSH conjugate M8-3. We thus anticipated that M9-1 and M9-2 held the glutathionyl group on the phenyl ring. However, the definitive structure of M9-1 and M9-2 could not be determined only on the basis of the MS data. Enzymes Involved in the Formation of 4-NC and GSH Conjugates. Metabolite 4-NC and the GSH conjugates detected in HLM incubations were monitored in incubations of 4-NP with individual recombinant human P450 enzymes, including CYPs1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1,

Table 4. Formation of 4-Nonylcatechol and GSH Conjugates Catalyzed by Individual CYP Enzymes 4-nonylcatechola P450

M3-4

1A1 1A2 1B1 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4 3A5

4.0 23 4.3 n.d.b 9.8 n.d. n.d. 25 100 27 58 n.d.

GSH conjugates M7 M8-1 M8-2 M8-3 M9-1 M9-2 42 45 1.9 1.7 4.8 2.3 1.2 32 100 46 66 1.1

37 38 n.d. n.d. n.d. n.d. n.d. 29 n.d. 100 n.d. n.d.

n.d. 100 13 n.d. 41 n.d. n.d. 37 65 54 38 n.d.

6.1 100 13 n.d. 43 n.d. n.d. 40 62 28 50 n.d.

n.d. 100 n.d. n.d. n.d. n.d. n.d. 57 19 61 44 n.d.

n.d. 100 n.d. n.d. n.d. n.d. n.d. n.d. 60 n.d. 63 n.d.

a The highest peak area of the metabolite detected in the incubations with the specified P450 enzymes was normalized to 100%, and the MS responses of the metabolite in the other P450 enzyme incubation samples were expressed as the percentage relative to this highest level. b n.d. denotes not detected.

3A4, and 3A5. As summarized in Table 4, 4-NP was metabolized by a wide arrange of human P450 enzymes. For the formation of 4-NC, CYP2D6 was the most active enzyme, followed by CYPs3A4, 2E1, 2C19, and 1A2. CYP2D6 was the most efficient enzyme catalyzing the formation of M7, followed by CYPs3A4, 2E1, 1A2, 1A1, and 2C19. CYPs2E1, 1A2, 1A1, and 2C19 were involved in the formation of M8-1. CYP1A2 was the major enzyme responsible for the generation of conjugates M8-2 and M8-3, followed by CYPs2D6, 2E1, 3A4, and 2C19. CYPs1A2, 2D6, and 3A4 participated in the production of M9-1 and M9-2. These data suggest that multiple P450 enzymes participate in the oxidative metabolism of 4-NP and that inhibition of a single enzyme will not have a significant impact on the formation of reactive metabolites. We did not examine the effects of inhibitors of individual P450 enzymes on the metabolism of 4-NP.

Discussion 14

The metabolism of C-labeled 4-NP has been studied in Wistar rats (21). The results indicated that most of the radioactivity was eliminated in urine and that the metabolites resulted from extensive side chain oxidation and from sulfate or glucuronic acid conjugation of the phenol group. Traces of ring-hydroxylated 4-NP were also characterized. Fecal excretion was mainly associated with unchanged 4-NP and side chain hydroxylated metabolites. In experiments in vitro, 4-hydroxy-


4-nonyl-2,5-cyclohexadien-1-one, 4′-hydroxynonanophenone, and hydroquinone were detected in rat liver microsomes, while 4-(1-hydroxynonyl) phenol was detected in HLMs (22). The present study was initiated with the intent of characterizing metabolic pathways of 4-NP in HLMs that might lead to bioactivation. A total of 18 new oxidative metabolites were identified in HLMs, which were mainly derived from hydroxylation of the phenol group and/or nonyl side chain, and further oxidized to the corresponding aldehydes or ketones. 4-NP and its catechol metabolites were expected to form both paraquinone methide and ortho-quinone intermediates, and orthoquinone could be isomerized to a hydroxyl-nonylphenol quinone methide (23). The scope of the present study mainly focused on the identification of reactive intermediates derived from 4-NP and its oxidative metabolites. Six GSH conjugates were detected in HLM incubations of 4-NP in the presence of NADPH and GSH, with 4-(1-(glutathione-S-yl)nonyl)phenol (M7) and 6-(glutathione-S-yl)-4-nonylcatechol (M8-3) as the major GSH conjugates. We succeeded in the synthesis of GSH conjugate 4 (M7) by reaction of GSH with 4-(1-hydroynonyl)phenol (1) in the presence of TFA (Scheme 1). Additionally, we detected 4-(1hydroynonyl)phenol (M3-3) in the HLM samples incubated with 4-NP. This made us speculate on the transformation of 4-(1-hydroynonyl)phenol to M7 as an alternative pathway. To address this question, we incubated 4-(1-hydroynonyl)phenol in NADPH-supplemented HLMs and failed to detect M7. This implies that GSH is incapable of displacing the benzylic hydroxyl group. However, we may not exclude the possible biotransformation of 4-(1-hydroynonyl)phenol to M7 in vivo. For instance, 4-(1-hydroynonyl)phenol is likely to be converted to the corresponding sulfate arising from the sulfation of the benzylic hydroxyl group. In turn, sulfate is a good leaving group, and the displacement of the sulfate group with GSH affords M7. A total of four GSH conjugates, including M8-2, M8-3, M9-1, and M9-2, were detected in HLM incubations with 4-NC. It appears that the generation of GSH conjugates M9-1 and M9-2 required NADPH, while the formation of conjugates M8-2 and M8-3 was independent of NADPH. This indicates that no CYP enzymes were involved in the oxidation of 4-NC to ortho-benzoquinone 6. Apparently, the oxidation of 4-NC to ortho-benzoquinone 6 occurred spontaneously. It has been reported that some catechol compounds such as nordihydroguaiaretic acid, 4-methylcatechol, and estrogens can be auto-oxidized to their ortho-quinone intermediates in HLMs (24-27). The chemical reaction of ortho-benzoquinone 6 with GSH produced two GSH conjugates identified as 5 and 7 at a ratio of 1:20. The observed formation of conjugate 5 indicates that an isomerization took place in the conjugation reaction. We propose that a hydroxyquinone methide intermediate (10) was formed via isomerization (Scheme 3). Similar isomerization has been reported by Bolton’s group (23, 28). Metabolites M8-2 and M8-3 characterized as conjugates 5 and 7 were detected in HLM incubations with either 4-NP or 4-NC, and the ratio of the two was also approximately 1:20. Three regioisomers of aromatic-orientated GSH conjugates, i.e., 7-9, were expected to form in the reaction of orthobenzoquinone 6 with GSH. Interestingly, we only detected conjugate 7, which is generated via 1,6-addition by which the resulting anion enjoys greater resonance stability (29). The failure to form conjugate 9 likely results from the steric hindrance at C-3, despite the availability of a 1,6-addition resonance system. The lack of conjugate 8 as a product of the


conjugation reaction may be explained by the less favored 1,4addition of GSH at C-5 relative to the 1,6-addition at C-6 and C-3. It appears that the transformation of 4-NP to ortho-benzoquinone 6 involves four-electron loss. We propose that two separated oxidation reactions (four electrons) occurred in the formation of M8-2 and M8-3 from 4-NP in microsomal incubations, including (1) CYP-dependent aromatic hydroxylation of 4-NP to 4-NC and (2) auto-oxidation of 4-NC to orthobenzoquinone 6, followed by conjugation with GSH as shown in Scheme 3. Multiple CYPs, including CYPs2D6, 3A4, 2E1,1A2, 1A1, and 2C19 in the order of their activities, were found to catalyze the formation of quinone methide 11 (Scheme 3). Particularly, CYP2D6 dominated the bioactivation of 4-NP to quinone methide 11. CYP2D6 was also reported to dictate the metabolic activation of 4-methylphenol, an analogue of 4-NP, to the corresponding quinone methide (30). Interestingly, the CYPs found to activate 4-NP were the same as those for the bioactivation of 4-methylphenol with a slight difference in the order of their activities. The array of CYPs responsible for the metabolic activation of different molecules to quinone methides can vary, such as trimethoprim (31), raloxifene (32-34), and dauricine (35), but it appeared that CYP3A4 was often found to participate in the bioactivation reactions. Phenols and catechols have been linked to numerous adverse events, including hepatotoxicity and carcinogenesis, believed in part to be associated with the in situ formation of quinone methides (36-38) and ortho-quinones (24, 32, 37). These reactive species are capable of alkylating key cellular proteins and/or DNA. Moreover, quinones are redox-active molecules that could produce reactive oxygen species, causing oxidative stress and damage to macromolecules (18, 39). Bolton et al. indicated that the toxicity of 4-allylphenols may be primarily dependent on the amount of quinone methide formed and the reactivity of their quinone methide metabolites (36). Particularly, studies on the bioactivation of selected SERMs (selective estrogen receptor modulators), such as tamoxifen, acolbifene, and raloxifene, suggested that electrophilic metabolites have the potential to cause genotoxicity (33). Moreover, a recent investigation demonstrated that there is a direct link between the metabolism of estrogens and the increased risk of breast cancer, and the factors that affect the formation, reactivity, and cellular targets of estrogen quinoids were thoroughly explored (40). The observed quinone methide and ortho-benzoquinone metabolites of 4-NP allow us to anticipate the involvement of the reactive metabolites in 4-NP induced toxicity. In conclusion, the present study provided clear evidence for the formation of quinone methides and ortho-benzoquinones from 4-NP after incubation with human liver microsomes. These electrophilic metabolites reacted with glutathione to produce the respective glutathione conjugates. Additionally, one orthobenzoquinone intermediate was isomerized to the corresponding hydroxyquinone methide. The dehydrogenation of 4-NP to quinone methide and hydroxylation of 4-NP to 4-NC were mediated by multiple CYP enzymes, including CYP1A2, 2C19, 2D6, 2E1, and 3A4. The observed bioactivation of 4-NP raises a serious concern about the impact of the environmental exposure of 4-NP on human health. Acknowledgment. This work was supported in part by the National Basic Research Program of China (No. 2009CB930300).

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Supporting Information Available: HMBC spectra of synthetic GSH conjugates. This material is available free of charge via the Internet at http://pubs.acs.org.

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Evidence for the Bioactivation of 4-Nonylphenol to Quinone Methide

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