Chemical Interaction of Protein Cysteine Residues with Reactive Metabolites of Methyleugenol

Article abstract of DOI:10.1021/acs.chemrestox.6b00290

Methyleugenol (ME), an alkenylbenzene compound, is a natural ingredient of several herbs and is used as flavoring agent in foodstuffs and fragrance in cosmetics. The hepatotoxicity, cytotoxicity, and carcinogenesis of ME have been well documented, and met

Full text of DOI:10.1021/acs.chemrestox.6b00290

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES
Article
Chemical interaction of protein cysteine residues
with reactive metabolites of methyleugenol
Yukun Feng, Hui Wang, Qian Wang, Wenlin Huang, Ying Peng, and Jiang Zheng
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00290 • Publication Date (Web): 20 Jan 2017
Downloaded from http://pubs.acs.org on January 23, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Chemical Research in Toxicology is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 39
Chemical Research in Toxicology
1
2
3
4
5
Chemical interaction of protein cysteine residues with reactive metabolites of
methyleugenol
Yukun Feng†, Hui Wang†, Qian Wang†, Wenlin Huangθ, Ying Peng†*, and Jiang Zheng†¶‡*
6
7
8
9
†Wuya College of Innovation , Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P.
R. China
θDepartment of Biochemistry, University of Washington, Seattle, WA 98195, USA
¶Key Laboratory of Pharmaceutics of Guizhou Province, ‡State Key Laboratory of Functions and
Applications of Medicinal Plants, Guizhou Medical University, Guiyang, Guizhou, 550004, P. R. China
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Running title:
Interaction of cysteine residues with methyleugenol
Correspondence authors
Jiang Zheng
Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, P. R.
China;
Key Laboratory of Pharmaceutics of Guizhou Province, State Key Laboratory of
Functions and Applications of Medicinal Plants, Guizhou Medical University, Huayan
Rd., Guiyang, Guizhou, 550004, P. R. China
Eꢀmail: zhengneu@yahoo.com
Tel: +86ꢀ24ꢀ23986361
Fax: +86ꢀ24ꢀ23986510
Ying Peng
Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Rd.,
Shenhe Qu, Shenyang, Liaoning, 110016, P. R. China
Eꢀmail: yingpeng1999@163.com
Tel: +86ꢀ24ꢀ23986361
Fax: +86ꢀ24ꢀ23986510
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 2 of 39
1
2
3
4
TOC Graphic
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OCH3
OCH3
Cysteine-based protein adduction
ACS Paragon Plus Environment

Page 3 of 39
Chemical Research in Toxicology
1
2
3
4
ABSTRACT
5
6
7
8
Methyleugenol (ME), an alkenylbenzene compound, is a natural ingredient of several
herbs and is used as flavoring agent in foodstuffs and fragrance in cosmetics. The
hepatotoxicity, cytotoxicity, and carcinogenesis of ME have been well documented,
and metabolic activation has been suggested to involve in MEꢀinduced toxicities.
The objective of this study was to identify chemical identity of interactions of protein
with reactive metabolites of ME. Modification of cysteine residues of protein was
observed in microsomal incubations and mice after exposure to ME. Three types of
protein modification derived from the corresponding epoxide, α,βꢀunsaturated
aldehyde, and carbonium ion of ME were detected in vitro and in vivo. The protein
adduction took place in timeꢀ and doseꢀdependent manners. Dexamethasone,
ketoconazole, and Lꢀbuthionine sulfoximine increased the protein modification
induced by ME, which was proportional to the hepatotoxicity of ME. The findings
facilitate the understanding of mechanism action of ME toxicities.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 4 of 39
1
2
3
4
INTRODUCTION
5
6
7
8
Methyleugenol (ME, 4ꢀallylꢀ1,2ꢀdimethoxybenzene), an allylalkoxybenzene
compound, is a natural ingredient of many herbs and is also used as a flavoring
substance in a wide variety of dietary products.1ꢀ3 On given its widespread use by
industry and in herbs and spices, it is probable that humans are exposed to ME on a
daily basis. Thus, any health risks associated with exposure to ME should be
carefully evaluated. Based on the results of a National Toxicology Program (NTP)
rodent bioassay in 2000, ME was found to show a carcinogenic effect in both F344
rats and B6C3F1 mice.4 In 2001, the European Union’s Scientific Committee on
Food (SCF) announced ME to be a genotoxic carcinogen, and reductions in exposure
and restrictions in use levels were indicated.5
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Numbers of studies have demonstrated that the toxicities of ME required
bioactivation to electrophilic intermediates which can react with cellular
macromolecules such as DNA and protein.6ꢀ8 Three possible metabolic pathways
available for ME include 1) Oꢀdemethylation of the methoxy moieties on the phenyl
ring; 2) 2’,3’ꢀepoxidation of the allylic side chain; and 3) 1’ꢀhydroxylation of ME.
OꢀDemethylation of the methoxy substituents of ME yields the corresponding
phenolic derivatives, providing an efficient detoxification option for conjugation and
elimination.9 The epoxides of the allylalkoxybenzenes have been shown to be
DNAꢀreactive and form adducts in vitro. However, these epoxides also showed to
be detoxified by epoxide hydrolases and/or glutathione Sꢀtransferases in vivo.10ꢀ12
1’ꢀHydroxyl ME can be subsequently oxidized to the corresponding ketone which is
efficiently conjugated with GSH by Michael addition. In an alternative pathway,
1’ꢀhydroxylation, followed by sulfate conjugation and desulfonation, results in a
reactive carbocation intermediate.13,14 Herrmann and coworkers reported the
ACS Paragon Plus Environment

Page 5 of 39
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
detection of DNA adducts derived from the carbocation intermediate in surgical
human liver samples from 30 subjects.15 Several studies demonstrated that the
reactive intermediate carbocation was the ultimate carcinogenic metabolite by virtue
of DNA adduct formation.16,17
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The formation of DNA adduction and DNA damage induced by ME have been
the focus of intensive investigations.18 However, relatively less effort has been
directed toward the understanding of the molecular details involved in MEꢀderived
protein covalent binding and possible toxicological significance. Since protein
adduct formation has been implicated in a mechanism of toxicity of various drugs and
chemicals,19,20 it is conceivable that protein adduction may contribute to the toxicity
of ME. Gardner and coworkers developed antibody approaches, such as ELISA and
immunoblot, and reportedly detected protein adduction in livers of rats treated with
ME.7 Unfortunately, the antibodyꢀbased method they developed is unable to address
the chemical identity of proteinꢀreactive metabolite interactions. Recently, we
detected urinary GSH and related cysteine conjugates derived from electrophilic
metabolites of ME.21 This led us to reason that the free thiol groups of protein
cysteine residues may be the target of the reactive metabolites of ME. The major
objectives of the present study were to determine cysteineꢀbased protein modification
by reactive metabolites of ME and to define the correlation of the protein
modification with MEꢀinduced injury.
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 6 of 39
1
2
3
4
EXPERIMENTAL PROCEDURES
5
6
7
8
Chemicals. Methyleugenol (≥ 99.0%) and eugenol (≥ 99.0%) were purchased from
Tokyo Chemical Industry (Shanghai) Development Co., Ltd. (Shanghai, China).
Ketoconazole (KTC), cysteine, Pronase E, chymotrypsin, DLꢀdithiothreitol (DTT),
Lꢀbuthionine sulfoximine (BSO), reduced nicotinamide adenine dinucleotide
phosphate (NADPH), and Sꢀhexylglutathione were purchased from SigmaꢀAldrich (St.
Louis, MO). CD3I (≥ 99.0%) was purchased from Shanghai Bodi chemical
technology Co., Ltd. (Shanghai, China). Dexamethasone (DXM) was purchased
from the National Institute for the Control of Pharmaceutical and Biologic Products
(Shenyang, China). Formic acid was from Fisher Scientific (Springfield, NJ).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Nꢀbromosuccinimide,
mꢀchloroperbenzoic
acid
(mꢀCPBA)
and
2,3ꢀdichloroꢀ5,6ꢀdicyanoquine (DDQ) were purchased from Aladdin Reagent Co., Ltd
(Shanghai, China). All organic solvents were obtained from Fisher Scientific
(Springfield, NJ). All reagents and solvents were either analytical or HPLC grade.
Chemical synthesis of d3-ME. d3ꢀME was synthesized by the methylation of
phenolic hydroxyl of eugenol using CD3I as a methylating agent. Briefly, 160 mg
eugenol dissolved in DMSO was added to 4 ml of NaOH (4 N), followed by dropwise
addition of 0.5 mL CD3I. The reaction mixture was stirred at 70 °C for 4 h, cooled
down to room temperature, and then extracted with CH2Cl2. The remaining CH2Cl2
layer was concentrated in vacuum. Purification by a preparative HPLC system
offered d3ꢀME. ME including equimolar unlabeled d0ꢀME and stable isotope labeled
d3ꢀME was used in all subsequent experiments.
Reactive metabolite trapping. Male Kunming mouse liver microsomes were
prepared, according to a procedure reported by our laboratory.22 The incubation
ACS Paragon Plus Environment

Page 7 of 39
Chemical Research in Toxicology
1
2
3
4
mixture contained 500
µM ME, 1.0 mg/mL MLM protein, 3.2 mM MgCl2 and 40.0
5
6
7
8
mM cysteine in the presence or absence of 1.0 mM NADPH, which were mixed in
potassium phosphate buffer (pH 7.4) with a total volume of 500 L . The reactions
µ
9
were initiated by addition of NADPH and quenched by adding an equal volume of
iceꢀcold acetonitrile after incubation for 60 min at 37 °C. The resulting mixtures
were votexed for 3 min at room temperature, and the precipitated protein was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
removed by centrifuging at 16,000 g for 10 min at 4 °C. A 5
µL aliquot of the
resulting supernatants was injected into an LC–MS/MS system for analysis.
Protein modification and digestion. ME (100
µM) was mixed with mouse liver
microsomes (1.0 mg protein/mL, final volume: 500
µL), and the reaction was initiated
by addition of NADPH (1.0 mM). After 60 min of incubation at 37 °C, the protein
samples were denatured by heating in a water bath at 60 °C for 30 min, followed by
centrifugation at 16,000 g for 10 min. The resulting pallets were reconstituted in 50
mM ammonium bicarbonate (pH 8.0) (final volume: 200 µL). The resultant samples
were mixed with DTT (5.0 mM). After 1 h incubation at 60 °C, the protein samples
were digested with a mixture of chymotrypsin (2.5 mg/mL) and Pronase E (2.5
mg/mL) in the presence of 5.0 mM CaCl2 with continuous incubation at 37 °C for 15
h. The digested mixtures were centrifuged at 16,000 g for 10 min, and the
supernatants were subjected to the LC–MS/MS system for analysis.
Animals and treatment. Male Kunming mice (18ꢀ20 g) were purchased from the
Animal Center of Shenyang Pharmaceutical University (Shenyang, China). Mice
had free access to food and water and were housed in a temperatureꢀcontrolled (22 ±
4 °C) facility with a 12ꢀh dark/light cycle for at least 5 days after receipt and before
treatment. All animal studies were performed according to procedures approved by
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 8 of 39
1
2
3
4
5
6
the Ethics Review Committee for Animal Experimentation of Shenyang
Pharmaceutical University.
7
8
9
Mice were treated intraperitoneally with ME dissolved in corn oil at doses of 0,
100, 200, or 300 mg/kg (n = 4). The blood and livers were harvested 1 h after the
treatment (doseꢀdependent experiment) or the livers were collected after 0.25, 0.5, 1,
2, 4, and 6 h (timeꢀdependent experiment conducted at the dosage of 200 mg/kg) (n =
4). In a separate study, the animals were randomly divided into four groups, and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
each group contained four mice.
One group was pretreated (i.p.) with
dexamethasone (DXM) (50 mg/kg) for 5 consecutive days using the same procedure.
At the sixth day, the animals were treated with ME (i.p.) dissolved in corn oil at 200
mg/kg. Another group of mice was intraperitoneally treated with ketoconazole
(KTC) (100 mg/kg) suspended in corn oil 1.5 h before ME administration at 200
mg/kg (i.p.). The third group was intraperitoneally administered with Lꢀbuthionine
sulfoximine (BSO) (666 mg/kg) dissolved in saline 1 h before ME administration at
200 mg/kg (i.p.). The fourth group as control was only treated with ME (i.p.) at 200
mg/kg. The blood and livers from the mice in each group were harvested 1 h after
the administration.
The liver tissues (0.2 g) were homogenized in 2 mL of 0.1 M phosphate buffer
(pH 7.4), followed by centrifuging at 4,000 g for 5 min. The resulting supernatants
(400
µL) were collected for proteolytic digestion as described above. The blood
samples collected above were allowed to clot in test tubes at room temperature for 1 h,
followed by centrifuging at 8,000g for 10 min. The resulting sera were harvested for
ALT and AST assays. Serum ALT and AST activities were measured with a
VITROS 5600 integrated system (OrthosꢀClinical Diagnostics, Rochester, NY).
Chemical
synthesis
of
cysteine
adducts.
The
synthesis
of
ACS Paragon Plus Environment

Page 9 of 39
Chemical Research in Toxicology
1
2
3
4
3’ꢀ(cysteineꢀSꢀyl)ꢀ2’ꢀhydroxyꢀ2’,3’ꢀdihydromethyleugenol
(A1)
started
with
5
6
7
8
epoxidation of ME, followed by reaction with cysteine. The epoxy intermediate was
prepared using a reported method.9 Briefly, ME (0.15g, 0.84 mmol) dissolved in
CH2Cl2 (5 mL) was mixed with a saturated sodium bicarbonate solution (2 mL),
followed by addition of mꢀchloroperbenzoic acid (80%, 1.26 mmol) dissolved in
CH2Cl2 (5 mL). The resulting mixture was vigorously stirred at room temperature
for 4 h. The organic layer was collected, and the aqueous phase was extracted three
times with CH2Cl2, and the combined organic phases were concentrated to dryness
under vacuum. Purification by a preparative HPLC system offered the epoxy
product. The purified ME epoxide (0.1 g, 0.5 mmol) was dissolved in THF (2 mL)
and mixed with a solution of cysteine (0.06 g, 0.5 mmol, dissolved in 2 mL water).
After 2 h stirring at 37 °C, the mixture was concentrated to dryness, and the resulting
samples were reconstituted with acetonitrile/water (50/50, v/v) and centrifuged. The
supernatants were submitted to HPLC for purification. The purified product was
characterized by mass spectrometry and NMR.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of 1’ꢀ(cysteineꢀSꢀyl)ꢀ3’ꢀoxoꢀ2’,3’ꢀdihydromethyleugenol (A2) was
achieved by initial oxidation of ME to 3’ꢀoxomethylisoeugenol using
2,3ꢀdichloroꢀ5,6ꢀdicyanoquine (DDQ), according to a reported method.23 A solution
of ME (35.6 mg, 0.2 mmol) in CH2Cl2 (8.0 mL) was sequentially mixed with DDQ
(100.0 mg, 0.44 mmol) and 2.0 mL water, and the resulting mixture was stirred for 60
min at room temperature, followed by addition of a solution of ascorbic acid (77.4 mg,
0.44 mmol) in 2.0 mL water. After 60 min stirring, the organic layer was washed
with brine until the aqueous layer was colorless. The organic phase was
concentrated to dryness, dissolved in 2.0 mL acetonitrile/water (50/50, v/v), and
submitted
to
HPLC
for purification.
The
fractions
containing
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 10 of 39
1
2
3
4
5
6
7
8
9
3’ꢀoxomethylisoeugenol were pooled and concentrated to dryness for NMR analysis.
The purified 3’ꢀoxomethylisoeugenol (3.8 mg, 0.02 mmol) was dissolved in THF (0.8
mL), mixed with a cysteine solution (3.6 mg, 0.03 mmol) in 0.2 mL water, and stirred
for 2 h at 37 °C. The resulting mixture was concentrated to dryness, reconstituted
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
with 200 µL acetonitrile/water (50/50, v/v), centrifuged, and analyzed by LCꢀMS/MS.
1’ꢀ(CysteineꢀSꢀyl)methyleugenol (A3) was synthesized by bromination of ME,
followed by conjugation with cysteine. Briefly, a solution of Nꢀbromobutanimide
(0.15 g, 0.86 mmol) in CCl4 (4 mL) was slowly added to a solution of ME (0.19 g,
1.07 mmol) in 4 mL CCl4. After 4 h stirring at room temperature, the organic layer
was concentrated to dryness, reconstituted in 2.0 mL acetonitrile/water (50/50, v/v),
mixed with a cysteine solution (0.12 g, 1 mmol, dissolved in 2 mL water), and stirred
for 2 h at room temperature. The resulting mixture was concentrated to dryness,
reconstituted with acetonitrile/water (50/50, v/v), centrifuged, and submitted to HPLC
for purification. The purified product was submitted to mass spectrometry and NMR
for characterization.
Preparation of standard solutions. Appropriate amounts of A1 and A3 were
weighed and dissolved in methanol to produce the mix stock solution, the
concentrations of which were 225.0 ng/mL and 112.5 ng/mL, respectiveley. A series
of working solutions were prepared by diluting the stocking solution to suitable
concentrations of 7.0–225.0 ng/mL for A1 and 3.5–112.5 mg/mL for A3. The
internal standard solution was prepared by dissolving Sꢀhexylglutathione with
methanol (final concentration: 40 ng/mL). All the solutions were stored in
refrigerator at 4 °C. Assay standard samples for construction of sixꢀpoint calibration
curve were prepared by spiking 80
µL blank hepatic digestion supernatants with mix
standard working solution (10 L) and IS working solution (10
µ
µL), in which the
ACS Paragon Plus Environment

Page 11 of 39
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
concentrations were 0.7, 1.4, 2.8, 5.6, 11.25 and 22.5 ng/mL for A1 and 0.35, 0.7, 1.4,
2.8, 5.6 and 11.25 ng/mL for A3. The typical regression equations and correlation
coefficients (r) of A1 and A3 were y = 0.1479 x + 0.0723 (r = 0.9954) for A1 and y =
0.0478 x + 0.0307 (r = 0.9952) for A3 (where x is the ratio of A1 or A3 peak area over
the internal standard peak area and y is the concentration of A1 or A3 in hepatic
digestion supernatants).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
LC-MS/MS analysis. Metabolites were characterized on an AB SCIEX Instruments
5500 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA)
interfaced online with a 1260 infinity system (Agilent Technologies, Santa Clara, CA).
HPLC separation of incubation samples was performed by gradient elution from a
BDS HYPERSIL C18 ODS column (5.0 µm, 150 mm×4.6 mm; Thermo, San Jose,
CA) with a flow rate of 0.8 mL/min of mobile phases, including 0.1% (v/v) formic
acid in acetonitrile (A) and 0.1% (v/v) formic acid in water (B). Gradient elution
was employed as follows: 10% A at 0ꢀ1 min, 10ꢀ70% A at 1ꢀ11 min, 70ꢀ100% A at
11ꢀ12 min, 100ꢀ10% A at 12ꢀ14 min, and 10% A at 14ꢀ16 min. All the metabolites
were analyzed in positive ion mode by multipleꢀreaction monitoring (MRM) scanning.
The optimized mass spectrometric instrument parameters obtained after tuning were
as follow: curtain gas (CUR), gas 1 (GS1), and gas 2 (GS2) were 35, 50 and 50 psi;
ion source temperature (TEM) was at 650 °C; ion spray voltage (IS) and entrance
potential (EP) were 5,500 and 10 V, respectively. The characteristics of ion pairs
(corresponding to declustering potential DP, collision energy CE, collision cell exit
potential CXP) were m/z 298/151 (115, 45, 10), 301/154 (115, 45, 10), 314/151 (115,
45, 10), 317/154 (115, 45, 10), 316/151 (115, 45, 10), 319/154 (115, 45, 10) for
MEꢀderived cysteine conjugate and m/z 392 /246 (86, 45, 15) for Sꢀhexylglutathione
(internal standard).
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 12 of 39
1
2
3
4
5
6
7
8
9
In addition, AB SCIEX Instruments 4000 QꢀTrapTM (Applied Biosystems, Foster
City, CA) interfaced online with a 1260 infinity system (Agilent Technologies, Santa
Clara, CA) were used to analyze the metabolites, and the informationꢀdependent
acquisition (IDA) method was employed to trigger the enhanced product ion (EPI)
scans by analyzing MRM. IDA was used to trigger acquisition of EPI spectra for
ions exceeding 500 cps with exclusion of former target ions after three occurrences
for 10 s. The EPI scan was also run in positive mode at a scan range for product ions
from m/z 50 to 400. The collision energy (CE) was set at 40 eV with a spread of 15
eV. Other instrument parameters were equivalent to those described above. All
data were analyzed using the AB SCIEX Analyst 1.6.2 software
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Statistical analysis. All data were presented as means ± SD. The t test was used
to analyze data on ME induced liver injury. p < 0.05 was considered significant
different.
ACS Paragon Plus Environment

Page 13 of 39
Chemical Research in Toxicology
1
2
3
4
RESULTS
5
6
7
8
Mass spectrometric behavior of ME. In terms of chemical structure, parent drugs
and their metabolites always share great similarity. Before characterizing the
metabolites, chromatographic and mass spectrometric fragmentation behaviors of ME
(equimolar unlabeled d0ꢀME and deuterium labeled d3ꢀME) were investigated.
d0ꢀME and d3ꢀME were mixed at approximate 1:1 ratio and analyzed by LCꢀMS/MS.
Both analytes eluted at 11.42 min with equal intensity in both peak height and peak
area (Fig. 1A). The MS/MS spectrum showed a doublet of equal intensity at m/z 179
(d0ꢀME) and 182 (d3ꢀME), which gave rise to product ion m/z 151 (d0ꢀME) and 154
(d3ꢀME) by the loss of C2H4, respectively. Fragment ions m/z 138 (d0ꢀME) and 141
(d3ꢀME) were generated by losing C3H5 moiety (Fig. 1B). Detailed tentative
fragment assignments are demonstrated in the inset of Fig. 1CꢀD. The characteristic
product ions from the parent compound can be used to assist the characterization of
the metabolites.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Adduction of cysteine by reactive metabolites of ME. A mixture of d0ꢀME and
d3ꢀME (1:1 molar ratio) was incubated in mouse liver microsomes supplemented with
cysteine as a trapping agent. Three cysteine conjugates, arbitrarily assigned as
A1ꢀA3, were detected (Fig. 2A, 3A, 4A). No such conjugates were detected in the
microsomal incubation system in the absence of NADPH (data not shown). And
each corresponding conjugate exhibited the diagnostic d3ꢀbased double peaks with
approximate equal intensity. The relevant extracted ion chromatograms are
demonstrated in Fig. 2B, 3B, 4B.
Adduct A1 with the retention time at 6.49 min was detected by acquiring ion
pairs m/z 316→151 and m/z 319→154 in positive mode (Fig. 2AꢀB). The MS/MS
spectrum of A1 displayed the indicative fragment ions of parent ME, such as ions m/z
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 14 of 39
1
2
3
4
5
6
7
8
138/141 and 151/154 (Fig. S1A). The fragment assignments are proposed in the
inset of Fig. S1B. The observed molecular and fragment ions led us to propose that
A1 was a cysteine conjugate derived from ME epoxide (Scheme 1).
9
Adduct A2 eluted out at the retention time of 7.11 min (Fig. 3AꢀB). The
MS/MS spectrum of A2 exhibited its protonated molecular ion at m/z 314/317 (Fig.
S2A), which was consistent with the calculated m/z value of
1’ꢀ(cysteineꢀSꢀyl)ꢀ3’ꢀoxoꢀ1’,2’ꢀdihydromethyleugenol (Cysꢀ3’OME) (Scheme 1).
Three fragments at m/z 225, 182, and 270 were assigned to [MHꢀC3H7O2N],
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[MHꢀC3H7O2NꢀC2H3O] and [MHꢀC2H4O], respectively (Fig. S2A).
The
d3ꢀMEꢀderived cysteine conjugate showed the corresponding fragment ions with
similar abundance. The observed molecular ion and fragments indicate that A2 was
Cysꢀ3’OME. Detailed tentative fragments are demonstrated in the inset of Fig. S2B.
The m/z values of A3 (tR = 6.52 min) were 298/301 (Fig. 4AꢀB). The product
ions of A3 included m/z 283 [MHꢀCH3], 266 [MHꢀOCH3], 177 [MHꢀC3H7O2NS], and
209 [MHꢀC3H7O2N] (Fig. S3B), indicating that cysteine was involved in the
formation of A3. The d3ꢀMEꢀderived cysteine conjugate exhibited identical
fragmentation with similar intensity. The observed molecular ion value suggests that
the adduct was a cysteine conjugate derived from an intact molecule of parent ME,
which made us speculate that A3 was Cysꢀ1’ꢀmethyleugenol (Scheme 1). Detailed
tentative fragments are demonstrated in the inset of Fig. S3B.
Modification of cysteine residues of hepatic protein.
ME (a mixture of d0ꢀME
and d3ꢀME) was incubated with mouse liver microsomes. The resulting microsomal
protein was exhaustively digested by proteinases and analyzed by MRM scanning
with ion transition m/z 316/319→151/154, 314/317→151/154, and 298/301→151/154.
Three MEꢀderived cysteine adducts as doublets (316/2319, 314/317, and 298/301)
ACS Paragon Plus Environment

Page 15 of 39
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
were detected (Fig. 2CꢀD, 3CꢀD, 4CꢀD) which shared the same retention time (6.49,
7.11 and 6.52 min) as the three cysteine conjugates generated in the microsomal
incubation of ME complemented with cysteine (Fig. 2AꢀB,3AꢀB,4AꢀB). No such
adducts were detected in the microsomal incubations in the absence of NADPH (data
not shown). Protein covalent binding study was also conducted in vivo. Hepatic
protein samples obtained from mice given ME were treated with a mixture of
chymotrypsin and Protease E, and the resulting mixtures were analyzed as described
above. Three analytes showed identical chromatographic and mass spectrometric
properties as the three MEꢀderived cysteine adducts obtained from the in vitro study
(Fig. 2EꢀF, 3EꢀF, 4EꢀF).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Characterization of synthetic Cys conjugates. MEꢀderived Cys conjugates A1ꢀA3
were chemically synthesized, and the synthetic Cys conjugates showed the same
chromatographic and MS identities (Fig. 2G, 3G, 4G) as that for the corresponding
adducts generated in microsomal incubations. We succeeded in characterizing A1
and A3 by 1HꢀNMR and mass spectrometry. The high resolution mass spectra of A1
and A3 showed [M + H]+ ions at m/z 316.1226 and 298.1104, corresponding to
formula C14H22NO5S (calculated: 316.1213) and C14H20NO4S (calculated: 298.1108),
respectively. Unfortunately, we failed to obtain enough amounts of A2 for NMR
characterization, due to extremely low yield of the chemical reaction. However,
reaction intermediate 3’ꢀoxomethylisoeugenol was chemically synthesized and
characterized by NMR, which is helpful to the identification of A2.
1
NMR characterization of A1: HꢀNMR (DMSOꢀd6, 600 MHz), δ 2.48 (m, 1H,
H3’a), 2.59 (m, 2H, H1’a, H5’a), 2.70 (m, 1H, H3’b), 2.78 (m, 1H, H1’b), 3.04 (m,
1H, H5’b), 3.31 (m, 1H, H6’), 3.70 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.78 (m, 1H,
H2’), 6.70ꢀ6.83 (m, 3H, HꢀAr).
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 16 of 39
1
2
3
4
1
NMR characterization of A3: HꢀNMR (DMSOꢀd6, 600 MHz), δ 2.08 (m, 2H,
5
6
7
8
H3’a, H4’), 3.16 (m, 2H, H1’, H3’b), 3.70 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 4.11
(m, 1H, H2’’), 5.32 (m, 2H, H3’’), 6.70ꢀ6.83 (m, 3H, HꢀAr).
9
NMR characterization of 3’ꢀoxomethylisoeugenol: 1HꢀNMR (CDCl3, 600 MHz),
δ 3.92 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 6.61 (dd, 1H, H1’, J = 7.74, 15.80 Hz),
6.91 (d, 1H, HꢀAr, J = 8.30 Hz), 7.08 (d, 1H, HꢀAr, J = 1.72 Hz), 7.16 (dd, 1H, HꢀAr,
J = 1.78, 8.29 Hz), 7.42 (d, 1H, H2’, J = 15.81 Hz), 9.66 (d, H, CHO, J = 7.74 Hz).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Time- and dose-dependent ME-derived protein adduction in vivo. Hepatic
protein adduction derived from the reactive metabolites were evaluated by monitoring
the production of cysteineꢀbased protein adduction as described above. The hepatic
protein adduction arising from cysteineꢀbased modification reached their peaks at 1 h
after the administration (Fig. 5). In addition, the protein adduction was found to
elevate with the increase in the doses of ME given in mice (Fig. 6).
Effect of KTC, DXM and BSO on ME-derived protein adduction. To
investigate the involvement of P450 3A in MEꢀderived protein adduction, mice were
pretreated with DXM or KTZ prior to ME administration. Interestingly, the levels of
MEꢀderived protein adduction both increased in mice pretreated with DXM or KTC
after administered with ME, compared with that of animals treated with ME alone
(Fig. 7). In order to investigate the effect of GSH depletion on MEꢀderived protein
adduction, BSO, an inhibitor of γꢀglutamylcysteine synthetase, was applied as a
modulator. As expected, BSO pretreatment was found to potentiate MEꢀderived
protein adduction (Fig. 7).
Liver injury induced by ME. Hepatotoxicity of ME was evaluated in mice by
monitoring serum ALT and AST activities. Intraperitoneal administration of ME at
ACS Paragon Plus Environment

Page 17 of 39
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
100, 200, and 300 mg/kg caused an elevation of serum AST and ALT activities in a
doseꢀdependent manner, which are in agreement with the observed dose dependency
of hepatic protein adduction. Likewise, DXM, KTC, or BSO pretreatment was
found to potentiate MEꢀinduced elevations of serum ALT and AST, compared with
those of ME administration alone at 200 mg/kg.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 18 of 39
1
2
3
4
DISCUSSION
5
6
7
8
Electrophilic intermediates of ME including the corresponding epoxide,
α,βꢀunsaturated aldehyde, and carbonium ion have been documented,24ꢀ26 and
MEꢀinduced toxicities are believed to associated with its metabolic activation. In
addition to forming adducts with DNA, the reactive metabolites of ME and of some
other carcinogenic allylbenzenes have been shown to react with hepatic protein. The
protein covalent binding was detected by immunochemical approaches.7,27 The
present study aimed to define the chemical identity of interactions between the
reactive intermediates of ME and protein in vitro and vivo.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The generation of multiple reactive metabolites made the detection of
MEꢀderived protein modification challenging, due to lack of some authentic standards.
We succeeded in development of stable isotope dilution technique. A 1:1 mixture of
d0ꢀME/d3ꢀME was employed for the protein adduction analysis. The special 1:1
doublets minimized the errors from false positive signals, facilitating the
characterization of the protein adduction. NTP rodent bioassay (Edition 2000)
demonstrated that administration of ME at doses of 100 mg/kg or greater caused
hepatocellular injury.4 Accordingly, we selected doses of 100, 200 and 300 mg/kg
for the present study.
In our recent study, we detected urinary ME metabolites associated with GSH
conjugation in mice given ME.21 This led us to investigate the interaction of
cysteine residues of protein with the reactive intermediates of ME. The protein
covalent binding study started with examination of microsomal protein adduction
after exposure to ME. Three cysteine adducts were detected in MEꢀexposed
microsomal protein after proteolytic digestion. The three adducts showed the same
chromatographic and mass spectrometric fragmentation behaviors as that of A1ꢀA3
ACS Paragon Plus Environment

Page 19 of 39
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
detected in microsomal incubations of ME supplemented with cysteine (Fig. 2ꢀ4).
This indicates that the reactive metabolites, including the corresponding epoxide,
α,βꢀunsaturated aldehyde, and carbonium ion, indeed modified cysteine residues of
microsomal protein. No such protein covalent binding was observed in microsomal
incubations in the absence of NADPH, indicating that P450 enzymes mediated the
protein modification. We were surprised to detect A3 in microsomal proteins after
incubation with ME. This suggests that the formation of the corresponding
carbonium ion did not require sulfation process. However, at present, we do not
know how the carbocation was formed, and further investigation is in need.
We further examined MEꢀinduced hepatic protein covalent binding in vivo. As
expected, A1ꢀA3 were detected in the proteinaseꢀdigested hepatic protein samples
obtained from mice given ME. This provided in vivo evidence for cysteineꢀbased
protein modification by the reactive metabolites of ME, including the epoxide,
α,βꢀunsaturated aldehyde, and carbonium ion.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The observed NADPHꢀdependency indicates the involvement of P450 in
MEꢀinduced protein adduction (Fig. 2ꢀ4).
Our recent metabolism study
demonstrated that P450 3A was the primary enzyme responsible for the metabolic
activation of ME. However, a microsomal study demonstrated that P450s 1A2 and
2C9 are the major enzymes involved in the bioactivation of ME.26 As expected,
increases in the three types of hepatic protein adduction were observed in
DXMꢀpretreated mice given ME (Fig. 7), relative to those of animals treated ME
alone. Surprisingly, unexpected elevation of the protein adduction was observed in
mice pretreated with KTC, a reported inhibitor of P450 3A, after exposure to ME.
However, the presence of KTC in microsomal incubations with ME significantly
attenuated the protein adduction (data not shown). The contradicted findings
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 20 of 39
1
2
3
4
5
6
7
8
9
obtained in vitro and in vivo studies indicate that multiple systems are involved in the
determination of ME toxicity in vivo. Urinary glucuronides derived from oxidative
ME metabolites, such as 1’ꢀhydroxy ME and MEꢀderived phenols, have been
reported.28 The glucuronidation pathway is most likely an ME detoxification
pathway. UDPꢀglucuronosyltransferases (UGTs) responsible for the glucuronidation
were reportedly inhibited by KTC.29 We speculate that the reported inhibition of
UGTs by KTC might play a role in the observed enhancement of protein adduction
induced by ME. In light of the findings, drugꢀdrug interactions involved by KTC
may need reinterpretation, and the effect of KCT on glucuronidation pathway should
also be considered. Our results indicate that the coꢀadministration of ME with KTC
or DXM both increased the MEꢀderived protein covalent binding. Hence, a special
attention needs to be paid to potentiating effects of both inhibitors and inducers of
cytochrome P450 3A on MEꢀinduced toxicities.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
GSH conjugation is usually considered as a detoxification mechanism and plays
an important role against toxicity of electrophilic agents.30 Our previous study has
detected GSH conjugates derived from the reactive metabolites of ME in vivo.21 The
present study found that pretreatment with BSO potentiated the formation of the
hepatic protein adducts in mice (Fig. 7). BSO is known to be an inhibitor of
γꢀglutamylcysteine synthetase, a rateꢀlimiting enzyme involved in GSH synthesis, and
can deplete hepatic GSH contents. BSOꢀinduced GSH depletion possibly made the
nucleophilic centers of protein more exposed to the electrophilic intermediates of ME.
Serum AST and ALT activities increased with the increase of doses of ME
administered in mice (Fig. 8A), along with similar doseꢀdependency of the protein
modification (Fig. 6). DXM, KTC or BSO pretreatment was found to potentiate not
only MEꢀinduced elevations of serum ALT and AST activities but also the protein
ACS Paragon Plus Environment

Page 21 of 39
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
modification resulting from metabolic activation of ME accordingly. Given together,
the findings suggest a possible correlation of the protein covalent binding with
MEꢀinduced hepatotoxicity.
9
It needs to mention that the 100 to 300 mg/kg doses of ME applied in the animal
experiments are much higher than that of the anticipated human exposure. Present
exposure to ME resulting from consumption of food, mainly herbs and food, may not
pose a significant hepatotoxicity risk. Nevertheless, further studies are needed to
define correlation of the protein covalent binding with MEꢀinduced hepatotoxicity at
low levels of exposure to ME, particularly in human experimental models.
In conclusion, cysteine residues of hepatic protein were modified by the
electrophilic intermediates of ME, including the corresponding epoxide,
α,βꢀunsaturated aldehyde, and carbonium ion. The formation of hepatic protein
adducts was associated with the activity of P450 3A and occurred in timeꢀ and
doseꢀdependent manners. The findings provided clear evidence for a good
correlation between MEꢀinduced protein modification and hepatotoxicity induced by
ME.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 22 of 39
1
2
3
4
Supporting Information Available
5
6
7
8
MS/MS spectrum and proposed fragment assignments of adduct A1, A2 and A3.
This material is available free of charge via the Internet at http://pubs.acs.org.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Funding sources
This work was supported in part by the National Natural Science Foundation of China
(No. 81430086 and 81373471).
Notes
The authors declare no competing financial interest.
ACS Paragon Plus Environment

Page 23 of 39
Chemical Research in Toxicology
1
2
3
4
ABBREVIATIONS:
5
6
7
8
ME, methyleugenol; KTC, ketoconazole; DXM, dexamethasone; DTT,
DLꢀdithiothreitol; BSO, Lꢀbuthionine sulfoximine; NADPH, reduced nicotinamide
adenine dinucleotide phosphate; DDQ, 2,3ꢀdichloroꢀ5,6ꢀdicyanoquine; GSH,
glutathione; ALT, alanine transaminase; AST, aspartate transaminase; ESI,
electrospray ionization; CE, collision energy; CXP, cell exit potential; DP,
declustering potential; EP, entrance potential; MRM, multipleꢀreaction monitoring.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 24 of 39
1
2
3
4
REFERENCE
5
6
7
8
(1) Robison, S. H., and Barr, D. B. (2006) Use of biomonitoring data to evaluate
methyl eugenol exposure. Environ. Health Perspect. 114, 1797–1801.
(2) Smith, R. L., Adams, T. B., Doull, J., Feron, V. J., Goodman, J. I., Marnett, L.
J., Portoghese, P. S., Waddell, W. J., Wagner, B. M., Rogers, A. E., Caldwell, J., and
Sipes, I. G. (2002) Safety assessment of allylalkoxybenzene derivatives used as
flavouring substances—methyl eugenol and estragole. Food Chem. Toxicol. 40, 851–
870.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3) Smith, B., Cadby, P., Leblanc, J. C., and Setzer, R. W. (2010) Application of
the margin of exposure (MoE) approach to substances in food that are genotoxic and
carcinogenic: example: methyleugenol, CASRN: 93ꢀ15ꢀ2. Food Chem. Toxicol. 48,
89–97.
(4) NTP, National Toxicology Program (2000) NTP Toxicology and
Carcinogenesis Studies of Methyleugenol (CAS No. 93ꢀ15ꢀ2) in F344/N Rats and
B6C3F1 Mice (Gavage Studies), NTP Report Series 491, National Toxicology
Program, Research Triangle Park, NC.
(5) SCF, European Commission (2001) Opinion of the Scientific Committee on
Food on Methyleugenol (4ꢀAllylꢀ1,2ꢀdimethoxybenzene). Health and Consumer
Protection DirectorateꢀGeneral. Directorate C, Scientific Opinions, Brussels, Belgium.
Available at http://ec.europa.eu/food/fs/sc/scf/out102_en.pdf.
(6) Chan, V. S., and Caldwell, J. (1992) Comparative induction of unscheduled
DNA synthesis in cultured rat hepatocytes by allylbenzenes and their 1’ꢀ hydroxy
metabolites. Food Chem. Toxicol. 30, 831–836.
(7) Gardner, I., Bergin, P., Stening, P., Kenna, J.G., and Caldwell, J. (1996)
Immunochemical detection of covalently modified protein adducts in livers of rats
ACS Paragon Plus Environment

Page 25 of 39
Chemical Research in Toxicology
1
2
3
4
treated with methyleugenol. Chem. Res. Toxicol. 9, 713–721.
5
6
7
8
(8) Gardner, I., Wakazono, H., Bergin, P., Waziers, I., Beaune, P., Kenna, J. G.,
and Caldwell, J. (1997) Cytochrome P450 mediated bioactivation of methyleugenol to
1′ꢀhydroxymethyleugenol in Fischer 344 rat and human liver microsomes.
Carcinogenesis 18, 1775–1783.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(9) Cartus, A. T., Herrmann, K., Weishaupt, L. W., Merz, K. H., Engst, W., Glatt,
H., and Schrenk, D. (2012) Metabolism of methyleugenol in liver microsomes and
primary hepatocytes: pattern of metabolites, cytotoxicity, and DNAꢀadduct formation.
Toxicol. Sci. 129, 21–34.
(10) Luo, G., Qato, M. K., and Guenthner, T. M. (1992) Hydrolysis of the
2′,3′ꢀallylic epoxides of allylbenzene, estragole, eugenol, and safrole by both
microsomal and cytosolic epoxide hydrolases. Drug Metab. Dispos. 20, 440−445.
(11) Luo, G., and Guenthner, T. M. (1995) Metabolism of allylbenzene 2′,3′ꢀoxide
and estragole 2′,3′ꢀoxide in the isolated perfused rat liver. J. Pharmacol. Exp. Ther.
272, 588–596.
(12) Guenthner, T. M., and Luo, G. (2001) Investigation of the role of the
2′,3′ꢀepoxidation pathway in the bioactivation and genotoxicity of dietary
allylbenzene analogs. Toxicology 160, 47−58.
(13) Randerath, K., Haglund, R. E., Phillips, D. H., and Reddy, M. V. (1984)
32Pꢀpostꢀlabelling analysis of DNA adducts formed in the livers of animals treated
with safrole, estragole and other naturallyꢀoccurring alkenylbenzenes. I. Adult female
CDꢀ1 mice. Carcinogenesis 5, 1613−1622.
(14) Phillips, D. H., Reddy, M. V., and Randerath, K. (1984) 32Pꢀpostꢀlabelling
analysis of DNA adducts formed in the livers of animals treated with safrole,
estragole and other naturallyꢀoccurring alkenylbenzenes. II. Newborn male B6C3F1
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 26 of 39
1
2
3
4
mice. Carcinogenesis 5, 1623−1628.
5
6
7
8
(15) Herrmann, K., Schumacher, F., Engst, W., Appel, K. E., Klein, K., Zanger, U.
M., and Glatt, H. (2013) Abundance of DNA adducts of methyleugenol, a rodent
hepatocarcinogen, in human liver samples. Carcinogenesis 34, 1025−1030.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(16) Burkey, J. L., Sauer, J. M., McQueen, C. A., and Sipes, I. G. (2000)
Cytotoxicity and genotoxicity of methyleugenol and related congenersꢀꢀ A
mechanism of activation for methyleugenol. Mutat. Res. 453, 25–33.
(17) Herrmann, K., Engst, W., Appel, K. E., Monien, B. H., and Glatt, H. (2012)
Identification of human and murine sulfotransferases able to activate hydroxylated
metabolites of methyleugenol to mutagens in Salmonella typhimurium and detection
of associated DNA adducts using UPLCꢀMS/MS methods. Mutagenesis 27, 453−462.
(18) Groh, I. A., Rudakovski, O., Gründken, M., Schroeter, A., Marko, D., Esselen，
M. (2015) Methyleugenol and oxidative metabolites induce DNA damage and interact
with human topoisomerases. Arch. Toxicol. DOI: 10.1007/s00204ꢀ015ꢀ1625ꢀ3.
(19) Hinson, D. H., and Roberts, D. W. (1992) Role of covalent and noncovalent
interactions in cell toxicity: effects on proteins. Annu. Rev. Pharmacol. Toxicol. 32,
471–450.
(20) Bolesterli, U. A. (1993) Specific targets of covalent drugꢀprotein interactions
in hepatocytes and their toxicological significance in drugꢀinduced liver injury. Drug
Metab. Rev. 25, 395–451.
(21) Yao, H., Peng, Y., and Zheng, J. (2016) Identification of glutathione and
related cysteine conjugates derived from reactive metabolites of methyleugenol in rats.
Chem. Biol. Interact. 253, 143–52.
(22) Lin, G., Tang, J., Liu, X. Q., Jiang, Y., and Zheng, J. (2007) Deacetylclivorine:
a genderꢀselective metabolite of clivorine formed in female SpragueꢀDawley rat liver
ACS Paragon Plus Environment

Page 27 of 39
Chemical Research in Toxicology
1
2
3
4
microsomes. Drug Metab. Dispos. 35, 607–613.
5
6
7
8
(23) Iliefski, T., Li, S.M., and Lundquist, K. (1998) Synthesis of cinnamaldehydes,
esters of cinnamic acids, and acylals of cinnamaldehydes by oxidation of arylpropenes
with 2,3ꢀdicyanoꢀ5,6ꢀdichlorobenzoquinone (DDQ). Tetrahedron Lett. 39, 2413–
2416.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(24) AlꢀSubeihi, A. A., Spenkelink, B., Rachmawati, N., Boersma, M. G., Punt, A.,
Vervoort, J., van Bladeren, P. J., Rietjens, I. M. (2011) Physiologically based
biokinetic model of bioactivation and detoxification of the alkenylbenzene
methyleugenol in rat. Toxicol. In Vitro 25, 267–285.
(25) Guenthner, T. M., and Luo, G. (2001) Investigation of the role of the
2′,3′ꢀepoxidation pathway in the bioactivation and genotoxicity of dietary
allylbenzene analogs. Toxicology 160, 47–58.
(26) Jeurissen, S. M., Bogaards, J. J., Boersma, M. G., ter Horst, J. P., Awad, H. M.,
Fiamegos, Y. C., van Beek, T. A., Alink, G. M., Sudhölter, E. J., Cnubben, N. H., and
Rietjens, I. M. (2006) Human cytochrome P450 enzymes of importance for the
bioactivation of methyleugenol to the proximate carcinogen 1′ꢀhydroxymethyleugenol.
Chem. Res. Toxicol. 19, 111–116.
(27) Wakazono, H., Gardner, I., Eliasson, E., Coughtrie, M. W., Kenna, J. G., and
Caldwell, J. (1998) Immunochemical identification of hepatic protein adducts derived
from estragole. Chem. Res. Toxicol. 11, 863–872.
(28) AlꢀSubeihi, A. A., Spenkelink, B., Punt, A., Boersma, M. G., van Bladeren, P.
J., and Rietjens, I. M. (2012) Physiologically based kinetic modeling of bioactivation
and detoxification of the alkenylbenzene methyleugenol in human as compared with
rat. Toxicol. Appl. Pharmacol. 260, 271–284.
(29) Yong, W. P., Ramirez, J., Innocenti, F., and Ratain, M. J. (2005) Effects of
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 28 of 39
1
2
3
4
5
6
Ketoconazole on Glucuronidation by UDPꢀGlucuronosyltransferase Enzymes. Clin.
Cancer Res. 11, 6699–6704.
7
8
9
(30) Rinaldi, R., Eliasson, E., Swedmark, S., and Morgenstern, R. (2002) Reactive
intermediates and the dynamics of glutathione transferasesnsferases. Drug Metab.
Dispos. 30, 1053–1058.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 29 of 39
Chemical Research in Toxicology
1
2
3
4
Scheme Legend
5
6
7
8
Scheme 1. Proposed pathways for the formation of adduct A1 (m/z 316), A2 (m/z 314),
and A3 (m/z 298).
9
Figure Legends
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 1. Chromatogram (A) and MS/MS spectrum (B) of ME. C: unlabeled d0ꢀME (m/z
179). D: deuteriumꢀlabeled d3ꢀME (m/z 182).
Fig. 2. Representative MRM (m/z 316→151 in black; m/z 319→154 in red)
chromatograms of adduct A1. Extracted ion chromatograms obtained from the
analysis of the incubation of ME with cysteine in the presence of NADPH (AꢀB).
Extracted ion chromatograms obtained from the analysis of the proteolytic digestion
of microsomal protein exposed to ME in the presence of NADPH (CꢀD). Extracted
ion chromatograms obtained from an analysis of the proteolytic digestion of hepatic
protein of mice treated with ME (EꢀF). Extracted ion chromatograms obtained from
an analysis of chemical synthetic A1 (G).
Fig. 3. Representative MRM (m/z 314→151 in black; m/z 317→154 in red)
chromatograms of adduct A2. Extracted ion chromatograms obtained from the
analysis of microsomal incubation of ME fortified with cysteine in the presence of
NADPH (AꢀB). Extracted ion chromatograms obtained from the analysis of the
proteolytic digestion of microsomal protein exposed to ME in the presence of
NADPH (CꢀD). Extracted ion chromatograms obtained from the analysis of
proteolytic digestion of hepatic protein of mice treated with ME (EꢀF). Extracted ion
chromatograms obtained from an analysis of chemical synthetic A2 (G).
Fig. 4. Representative MRM (m/z 298→151 in black; m/z 301→154 in red)
chromatograms of adduct A3. Extracted ion chromatograms obtained from the
analysis of microsomal incubation of ME supplemented with cysteine in the presence
ACS Paragon Plus Environment