Evidence for the bioactivation of 4-nonylphenol to quinone methide and ortho -benzoquinone metabolites in human liver microsomes

Article abstract of DOI:10.1021/tx100223h

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 NADPH- and GSH-supplemented human liver microsomes, 6 GSH conju

Full text of DOI:10.1021/tx100223h

Chem. Res. Toxicol. 2010, 23, 1617–1628
1617
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 NADPH-
and 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 benzylic-
orientated 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 ortho-
hydroquinones 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.
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 bioacti-
vation of 4-NP. In addition, this article describes the chemical
synthesis of glutathione conjugates derived from the quinone
methide and ortho-benzoquinone metabolites.
Introduction
Nonylphenol polyethoxylates (NPnEOs) are important non-
ionic 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: +86-
21-50800738. E-mail: xychen@mail.shcnc.ac.cn. (J.Z.) Tel: (206) 884-7651.
Fax: (206) 987-7660. E-mail: jiang.zheng@seattlechildrens.org.
† 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.
Materials and Methods
Materials. The following chemicals were purchased from Sigma-
Aldrich (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

1618 Chem. Res. Toxicol., Vol. 23, No. 10, 2010
Scheme 1. Synthesis of Hydroxylated Metabolites and GSH Conjugates of 4-Nonylphenola
Deng et al.
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.
(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-(1-
hydroxynonyl)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
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-hy-
droxybenzaldehyde (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
l
(1.17 g, 89%). H 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)phe-
nol (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 chroma-
tography 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%).
l
1 (3.6 g, 95%). H 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

BioactiVation of 4-Nonylphenol
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1619
Exocyclic GSH Conjugate 4-(1-(Glutathione-S-yl)nonyl)cat-
echol (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.
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.
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 spec-
trometer (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.
Results
Identification of Oxidative Metabolites in HLMs. To
investigate potential metabolic pathways leading to the bioac-
tivation of 4-NP, the metabolites obtained from HLM incuba-
tions 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., dehydroge-
nated (M1, m/z 217.1592), monohydroxylated and dehydroge-
nated (M2, m/z 233.1542), monohydroxylated (M3, m/z 235.1698),
dihydroxylated and dehydrogenated (M4, m/z 249.1491), dihy-
droxylated (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 chro-
matographic 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 me-
tabolites. 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-
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 Mass-
Lynx 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-(1-
hydroxynonyl)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,

1620 Chem. Res. Toxicol., Vol. 23, No. 10, 2010
Deng et al.
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
molecular
formula
calculated
mass (m/z)
measured
mass (m/z)
error
(in ppm)
description
Rt (min)
M0
M1
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
13.81
12.96
7.84
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
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
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
-0.4
-0.4
9.2
5.2
-2.8
-1.6
1.2
6.4
2.0
trihydroxylation
trihydroxylation
-2.6
5.2
7.46
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)phe-
nol. 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
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1621
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 dihy-
droxylation 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-(1-
hydroxynonyl)catechol by comparing the chromatographic
retention time and MS characteristics with those of the synthetic
standard.
Figure 2. Q-TOF mass spectra of synthetic standard compounds under
high collision energy. (A) 4-Nonylphenol; (B) 4-(1-hydroxynonyl)phe-
nol; (C) 4-nonylcatechol; (D) 4-(1-hydroxynonyl)catechol.
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.
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-(1-
hydroxynonyl)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.
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-Non-
ylphenol 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.
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
To investigate the formation mechanism of reactive inter-
mediates, the oxidative metabolites 4-NC and 4-(1-hydroxy-
nonyl)phenol were incubated separately with HLMs in the

1622 Chem. Res. Toxicol., Vol. 23, No. 10, 2010
Deng et al.
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
Rt
(min)
molecular
formula
calculated
mass (m/z)
measured
mass (m/z)
error
(in ppm)
description
M7
parent + GS
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
M8-1
M8-2
M8-3
M9-1
M9-2
parent + GS +O
4-nonylcatechol + GS
4-nonylcatechol + GS
4-nonylcatechol + GS + O
4-nonylcatechol + GS + O
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-(1-
hydroxynonyl)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.

BioactiVation of 4-Nonylphenol
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1623
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 abun-
dance. Unfortunately, the mass spectrometry data are unable to
tell the regio-structure of the GSH conjugate.
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
1H 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
On the basis of our knowledge, 4-NP does not directly react
with GSH and is likely metabolized to a quinone methide

1624 Chem. Res. Toxicol., Vol. 23, No. 10, 2010
Deng et al.
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 fragmenta-
tion 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

BioactiVation of 4-Nonylphenol
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1625
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.

1626 Chem. Res. Toxicol., Vol. 23, No. 10, 2010
Table 3. 1H- and 13C-NMR Data of Synthetic GSH Conjugates 4, 5, and 7 (δ in CD3OD)
Deng et al.
4
5a
7
position
δH (mult; J, Hz)
δC
δH (mult; J, Hz)
δC
δH (mult; J, Hz)
δC
1
2
3
4
5
6
1′
1′′
158.1
116.8
130.6
134.9
130.6
116.8
51
146
147
116.3 (116.2)
135.8 (135.6)
121.3 (121.2)
116.6
145.3
146.9
117.4
136.4
126.2
120.8
36.7
6.73 (d, 8.3)
7.13 (d, 8.3)
6.82 or 6.81 (d, 1.7)
6.60 (d, 2.0)
6.71 (d, 2.0)
7.13 (d, 8.3)
6.73 (d, 8.3)
3.79 (dd, 8.8, 6.1)
2.77 (dd, 13.6, 6.1)
2.61 (dd, 13.6, 7.5)
4.39 (dd, 7.5, 6.1)
6.66 or 6.64 (dd, 8.5, 1.7)
6.73 or 6.71 (d, 8.5)
3.73 (m)
51.0 (51.3)
34.0 (34.1)
2.44 (2H, t, 7.7)
34
2.61-2.83 (m)
3.32 (dd, 13.9, 9.0)
3.08 (dd, 13.9, 4.8)
4.46 (dd, 9.0, 4.8)
37.7
2′′
3′′
54.9
4.47 (dd, 9.1, 4.7)
or 4.41 (t, 6.6)
54.9
55.2
173.5
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.
Table 4. Formation of 4-Nonylcatechol and GSH Conjugates
of its deprotonated molecule and fragmentation pattern. Con-
jugates 7-9 would be readily differentiated by their character-
Catalyzed by Individual CYP Enzymes
4-nonylcatechola
GSH conjugates
istic 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.
P450
M3-4
M7 M8-1 M8-2 M8-3 M9-1 M9-2
1A1
1A2
1B1
2A6
2B6
2C8
2C9
2C19
2D6
2E1
3A4
3A5
4.0
23
42
45
37
38
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
4.3
n.d.b
9.8
n.d.
n.d.
25
100
27
58
1.9
1.7
4.8
2.3
1.2
32
n.d.
n.d.
n.d.
n.d.
n.d.
29
100 n.d.
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.
46
66
1.1
100
n.d.
n.d.
n.d.
63
n.d.
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 metabo-
lized 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 meta-
bolism of 4-NP.
Discussion
The metabolism of 14C-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-
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,

BioactiVation of 4-Nonylphenol
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1627
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).
conjugation reaction may be explained by the less favored 1,4-
addition of GSH at C-5 relative to the 1,6-addition at C-6 and
C-3.
The present study was initiated with the intent of character-
izing 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 hydroxy-
lation 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 para-
quinone methide and ortho-quinone intermediates, and ortho-
quinone 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-(glu-
tathione-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-(1-
hydroynonyl)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 nordihydroguai-
aretic 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.
It appears that the transformation of 4-NP to ortho-benzo-
quinone 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 hydroxy-
lation of 4-NP to 4-NC and (2) auto-oxidation of 4-NC to ortho-
benzoquinone 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 ortho-
benzoquinone 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.
Three regioisomers of aromatic-orientated GSH conjugates,
i.e., 7-9, were expected to form in the reaction of ortho-
benzoquinone 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
Acknowledgment. This work was supported in part by the
National Basic Research Program of China (No. 2009-
CB930300).

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