Chemical Interaction of Protein Cysteine Residues with Reactive

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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

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Chemical Research in Toxicology

Chemical interaction of protein cysteine residues with reactive metabolites of methyleugenol Yukun Feng†, Hui Wang†, Qian Wang†, Wenlin Huangθ, Ying Peng†*, and Jiang Zheng†¶‡*

†

Wuya College of Innovation , Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China θ Department 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

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 E-mail: [email protected] Tel: +86-24-23986361 Fax: +86-24-23986510 Ying Peng Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Rd., Shenhe Qu, Shenyang, Liaoning, 110016, P. R. China E-mail: [email protected] Tel: +86-24-23986361 Fax: +86-24-23986510

ACS Paragon Plus Environment


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TOC Graphic

OCH3 OCH3

Cysteine-based protein adduction


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ABSTRACT 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 ME-induced 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,β-unsaturated aldehyde, and carbonium ion of ME were detected in vitro and in vivo. adduction took place in time- and dose-dependent manners.

The protein

Dexamethasone,

ketoconazole, and L-buthionine 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.



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INTRODUCTION Methyleugenol (ME, 4-allyl-1,2-dimethoxybenzene), 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.1-3

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 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.6-8

Three possible metabolic pathways

available for ME include 1) O-demethylation of the methoxy moieties on the phenyl ring; 2) 2’,3’-epoxidation of the allylic side chain; and 3) 1’-hydroxylation of ME. O-Demethylation 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

DNA-reactive and form adducts in vitro.

However, these epoxides also showed to

be detoxified by epoxide hydrolases and/or glutathione S-transferases in vivo.10-12 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


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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 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 ME-derived 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 antibody-based method they developed is unable to address the chemical identity of protein-reactive 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 cysteine-based protein modification by reactive metabolites of ME and to define the correlation of the protein modification with ME-induced injury.



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EXPERIMENTAL PROCEDURES 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, DL-dithiothreitol (DTT), L-buthionine sulfoximine (BSO), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and S-hexylglutathione were purchased from Sigma-Aldrich (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).

N-bromosuccinimide,

m-chloroperbenzoic

acid

(m-CPBA)

and

2,3-dichloro-5,6-dicyanoquine (DDQ) were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). (Springfield, NJ).

All organic solvents were obtained from Fisher Scientific All reagents and solvents were either analytical or HPLC grade.

Chemical synthesis of d3-ME.

d3-ME 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. layer was concentrated in vacuum. offered d3-ME.

The remaining CH2Cl2

Purification by a preparative HPLC system

ME including equimolar unlabeled d0-ME and stable isotope labeled

d3-ME 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

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mixture contained 500 µM ME, 1.0 mg/mL MLM protein, 3.2 mM MgCl2 and 40.0 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 µL .

The reactions

were initiated by addition of NADPH and quenched by adding an equal volume of ice-cold 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 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). were mixed with DTT (5.0 mM).

The resultant samples

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 (18-20 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 temperature-controlled (22 ± 4 °C) facility with a 12-h dark/light cycle for at least 5 days after receipt and before treatment.

All animal studies were performed according to procedures approved by



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the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University. 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 (dose-dependent experiment) or the livers were collected after 0.25, 0.5, 1, 2, 4, and 6 h (time-dependent experiment conducted at the dosage of 200 mg/kg) (n = 4).

In a separate study, the animals were randomly divided into four groups, and

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 L-buthionine

sulfoximine (BSO) (666 mg/kg) dissolved in saline 1 h before ME administration at 200 mg/kg (i.p.). mg/kg.

The fourth group as control was only treated with ME (i.p.) at 200

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. ALT and AST assays.

The resulting sera were harvested for

Serum ALT and AST activities were measured with a

VITROS 5600 integrated system (Orthos-Clinical Diagnostics, Rochester, NY). Chemical

synthesis

of

cysteine

adducts.


The

synthesis

of

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3’-(cysteine-S-yl)-2’-hydroxy-2’,3’-dihydromethyleugenol epoxidation of ME, followed by reaction with cysteine. prepared using a reported method.9

(A1)

started

with

The epoxy intermediate was

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 m-chloroperbenzoic acid (80%, 1.26 mmol) dissolved in CH2Cl2 (5 mL). for 4 h.

The resulting mixture was vigorously stirred at room temperature

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. product.

Purification by a preparative HPLC system offered the epoxy

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. supernatants were submitted to HPLC for purification.

The

The purified product was

characterized by mass spectrometry and NMR. Synthesis of 1’-(cysteine-S-yl)-3’-oxo-2’,3’-dihydromethyleugenol (A2) was achieved

by

initial

oxidation

of

ME

to

3’-oxomethylisoeugenol

2,3-dichloro-5,6-dicyanoquine (DDQ), according to a reported method.23

using

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


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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

with 200 µL acetonitrile/water (50/50, v/v), centrifuged, and analyzed by LC-MS/MS. 1’-(Cysteine-S-yl)methyleugenol (A3) was synthesized by bromination of ME, followed by conjugation with cysteine.

Briefly, a solution of N-bromobutanimide

(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 S-hexylglutathione with methanol (final concentration: 40 ng/mL). refrigerator at 4 °C.

All the solutions were stored in

Assay standard samples for construction of six-point calibration

curve were prepared by spiking 80 µL blank hepatic digestion supernatants with mix standard working solution (10 µL) and IS working solution (10 µL), in which the


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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). 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 0-1 min, 10-70% A at 1-11 min, 70-100% A at 11-12 min, 100-10% A at 12-14 min, and 10% A at 14-16 min.

All the metabolites

were analyzed in positive ion mode by multiple-reaction 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 ME-derived cysteine conjugate and m/z 392 /246 (86, 45, 15) for S-hexylglutathione (internal standard).



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In addition, AB SCIEX Instruments 4000 Q-TrapTM (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 information-dependent 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 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.


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RESULTS 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 d0-ME and deuterium labeled d3-ME) were investigated. d0-ME and d3-ME were mixed at approximate 1:1 ratio and analyzed by LC-MS/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

(d0-ME) and 182 (d3-ME), which gave rise to product ion m/z 151 (d0-ME) and 154 (d3-ME) by the loss of C2H4, respectively.

Fragment ions m/z 138 (d0-ME) and 141

(d3-ME) were generated by losing C3H5 moiety (Fig. 1B).

Detailed tentative

fragment assignments are demonstrated in the inset of Fig. 1C-D.

The characteristic

product ions from the parent compound can be used to assist the characterization of the metabolites. Adduction of cysteine by reactive metabolites of ME.

A mixture of d0-ME and

d3-ME (1:1 molar ratio) was incubated in mouse liver microsomes supplemented with cysteine as a trapping agent.

Three cysteine conjugates, arbitrarily assigned as

A1-A3, 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 d3-based 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. 2A-B).

The MS/MS

spectrum of A1 displayed the indicative fragment ions of parent ME, such as ions m/z



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138/141 and 151/154 (Fig. S1A). inset of Fig. S1B.

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The fragment assignments are proposed in the

The observed molecular and fragment ions led us to propose that

A1 was a cysteine conjugate derived from ME epoxide (Scheme 1). Adduct A2 eluted out at the retention time of 7.11 min (Fig. 3A-B).

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’-(cysteine-S-yl)-3’-oxo-1’,2’-dihydromethyleugenol (Cys-3’OME) (Scheme 1). Three fragments at m/z 225, 182, and 270 were assigned to [MH-C3H7O2N], [MH-C3H7O2N-C2H3O]

and

[MH-C2H4O],

respectively

(Fig.

S2A).

The

d3-ME-derived cysteine conjugate showed the corresponding fragment ions with similar abundance. Cys-3’OME.

The observed molecular ion and fragments indicate that A2 was

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. 4A-B).

The product

ions of A3 included m/z 283 [MH-CH3], 266 [MH-OCH3], 177 [MH-C3H7O2NS], and 209 [MH-C3H7O2N] (Fig. S3B), indicating that cysteine was involved in the formation of A3.

The d3-ME-derived 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 Cys-1’-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 d0-ME

and d3-ME) 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 ME-derived cysteine adducts as doublets (316/2319, 314/317, and 298/301)


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were detected (Fig. 2C-D, 3C-D, 4C-D) 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. 2A-B,3A-B,4A-B).

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 ME-derived cysteine adducts obtained from the in vitro study (Fig. 2E-F, 3E-F, 4E-F). Characterization of synthetic Cys conjugates.

ME-derived Cys conjugates A1-A3

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. and A3 by 1H-NMR and mass spectrometry.

We succeeded in characterizing A1

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. NMR characterization of A1: 1H-NMR (DMSO-d6, 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’), 6.70-6.83 (m, 3H, H-Ar).



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Page 16 of 39

NMR characterization of A3: 1H-NMR (DMSO-d6, 600 MHz), δ 2.08 (m, 2H, 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’’), 6.70-6.83 (m, 3H, H-Ar). NMR characterization of 3’-oxomethylisoeugenol: 1H-NMR (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, H-Ar, J = 8.30 Hz), 7.08 (d, 1H, H-Ar, J = 1.72 Hz), 7.16 (dd, 1H, H-Ar, J = 1.78, 8.29 Hz), 7.42 (d, 1H, H2’, J = 15.81 Hz), 9.66 (d, H, CHO, J = 7.74 Hz). 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 cysteine-based protein adduction as described above.

The hepatic

protein adduction arising from cysteine-based 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 ME-derived protein adduction, mice were pretreated with DXM or KTZ prior to ME administration.

Interestingly, the levels of

ME-derived 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 ME-derived protein

adduction, BSO, an inhibitor of γ-glutamylcysteine synthetase, was applied as a modulator.

As expected, BSO pretreatment was found to potentiate ME-derived

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


Page 17 of 39

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100, 200, and 300 mg/kg caused an elevation of serum AST and ALT activities in a dose-dependent manner, which are in agreement with the observed dose dependency of hepatic protein adduction.

Likewise, DXM, KTC, or BSO pretreatment was

found to potentiate ME-induced elevations of serum ALT and AST, compared with those of ME administration alone at 200 mg/kg.



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DISCUSSION Electrophilic intermediates of ME including the corresponding epoxide, α,β-unsaturated aldehyde, and carbonium ion have been documented,24-26 and ME-induced 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. The generation of multiple reactive metabolites made the detection of ME-derived protein modification challenging, due to lack of some authentic standards. We succeeded in development of stable isotope dilution technique.

A 1:1 mixture of

d0-ME/d3-ME 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 ME-exposed

microsomal protein after proteolytic digestion. The three adducts showed the same chromatographic and mass spectrometric fragmentation behaviors as that of A1-A3


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detected in microsomal incubations of ME supplemented with cysteine (Fig. 2-4). This indicates that the reactive metabolites, including the corresponding epoxide, α,β-unsaturated 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. incubation with ME.

We were surprised to detect A3 in microsomal proteins after 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 ME-induced hepatic protein covalent binding in vivo.

As

expected, A1-A3 were detected in the proteinase-digested hepatic protein samples obtained from mice given ME.

This provided in vivo evidence for cysteine-based

protein modification by the reactive metabolites of ME, including the epoxide, α,β-unsaturated aldehyde, and carbonium ion. The observed NADPH-dependency indicates the involvement of P450 in ME-induced protein adduction (Fig. 2-4).

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 DXM-pretreated 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



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Page 20 of 39

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 ME-derived phenols, have been reported.28 pathway.

The glucuronidation pathway is most likely an ME detoxification UDP-glucuronosyltransferases (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, drug-drug interactions involved by KTC

may need reinterpretation, and the effect of KCT on glucuronidation pathway should also be considered.

Our results indicate that the co-administration of ME with KTC

or DXM both increased the ME-derived protein covalent binding.

Hence, a special

attention needs to be paid to potentiating effects of both inhibitors and inducers of cytochrome P450 3A on ME-induced toxicities. 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

γ-glutamylcysteine synthetase, a rate-limiting enzyme involved in GSH synthesis, and can deplete hepatic GSH contents.

BSO-induced 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 dose-dependency of the protein modification (Fig. 6).

DXM, KTC or BSO pretreatment was found to potentiate not

only ME-induced elevations of serum ALT and AST activities but also the protein


Page 21 of 39

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modification resulting from metabolic activation of ME accordingly.

Given together,

the findings suggest a possible correlation of the protein covalent binding with ME-induced hepatotoxicity. 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 ME-induced 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

α,β-unsaturated aldehyde, and carbonium ion.

the

corresponding

epoxide,

The formation of hepatic protein

adducts was associated with the activity of P450 3A and occurred in time- and dose-dependent manners.

The findings provided clear evidence for a good

correlation between ME-induced protein modification and hepatotoxicity induced by ME.



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Supporting Information Available 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.

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.


Page 22 of 39

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ABBREVIATIONS: ME,

methyleugenol;

KTC,

ketoconazole;

DXM,

dexamethasone;

DTT,

DL-dithiothreitol; BSO, L-buthionine sulfoximine; NADPH, reduced nicotinamide adenine

dinucleotide

phosphate;

DDQ,

2,3-dichloro-5,6-dicyanoquine;

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, multiple-reaction monitoring.



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Page 24 of 39

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mice. Carcinogenesis 5, 1623−1628. (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. (16) Burkey, J. L., Sauer, J. M., McQueen, C. A., and Sipes, I. G. (2000) Cytotoxicity and genotoxicity of methyleugenol and related congeners-- 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 UPLC-MS/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/s00204-015-1625-3. (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 drug-protein interactions in hepatocytes and their toxicological significance in drug-induced 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 gender-selective metabolite of clivorine formed in female Sprague-Dawley rat liver


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microsomes. Drug Metab. Dispos. 35, 607–613. (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 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ). Tetrahedron Lett. 39, 2413– 2416. (24) Al-Subeihi, 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) Al-Subeihi, 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



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Ketoconazole on Glucuronidation by UDP-Glucuronosyltransferase Enzymes. Clin. Cancer Res. 11, 6699–6704. (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.


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Scheme Legend Scheme 1. Proposed pathways for the formation of adduct A1 (m/z 316), A2 (m/z 314), and A3 (m/z 298). Figure Legends Fig. 1. Chromatogram (A) and MS/MS spectrum (B) of ME. C: unlabeled d0-ME (m/z 179). D: deuterium-labeled d3-ME (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 (A-B). Extracted ion chromatograms obtained from the analysis of the proteolytic digestion of microsomal protein exposed to ME in the presence of NADPH (C-D).

Extracted

ion chromatograms obtained from an analysis of the proteolytic digestion of hepatic protein of mice treated with ME (E-F).

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.


analysis of microsomal incubation of ME fortified with cysteine in the presence of NADPH (A-B).

Extracted ion chromatograms obtained from the analysis of the

proteolytic digestion of microsomal protein exposed to ME in the presence of NADPH (C-D).

Extracted ion chromatograms obtained from the analysis of

proteolytic digestion of hepatic protein of mice treated with ME (E-F).

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.


analysis of microsomal incubation of ME supplemented with cysteine in the presence



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of NADPH (A-B).

Page 30 of 39


proteolytic digestion of microsomal protein exposed to ME in the presence of NADPH.

Extracted ion chromatograms obtained from an analysis of proteolytic

digestion of hepatic protein of mice treated with ME (E-F).

Extracted ion

chromatograms obtained from an analysis of chemical synthetic A3 (G). Fig. 5. Time-course changes in protein modification represented by the levels of A1 (A), A2 (B), and A3 levels (C). Mice were treated with ME (i.p.) at 200 mg/kg. Cysteine-based protein adduction was examined at various time points after the administration (n = 4). Fig. 6. Dose-dependent changes in protein modification represented by the levels of A1 (A), A2 (B), and A3 (C).

Mice were treated with ME (i.p.) at various dosages.

Cysteine-based protein adduction was examined after 1 h administration (n = 4). Fig. 7. Changes in protein modification represented by the levels of A1 (A), A2 (B), and A3 (C) in mice pretreated with KTC, DXM or BSO. (i.p.) ME at 200 mg/kg body weight.

Mice were administered

Cysteine-based protein adduction was

examined after 1 h administration (n = 4). Fig. 8. Serum ALT and AST activities in mice after treatment with ME.

A:

Dose-dependent changes in serum AST and ALT activities in mice 1 h following i.p. administration of ME at 0, 100, 200, or 300 mg/kg (mean ± SD, n = 4). DXM (B), KTC (C) and BSO (D) on ME-induced hepatotoxicity. < 0.01 were considered significantly different.

*p < 0.05 and **p

The vehicle was corn oil.


Effects of

Page 31 of 39

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OCH3

OCH3 OCH3

OCH3

OH O S

OH

O NH2

A1 OCH3 OCH3

OH O

ME

S NH2

O

A2 OCH3 OCH3

-

O3SO

A3

Scheme 1 Proposed pathways for the formation of adduct A1 (m/z 316), A2 (m/z 314), and A3 (m/z 298).



A

C

11.42

6.0e4

Intensity, cps

164

4.0e4

149 138

2.0e4 151 0.0 0

2

4

6 8 Time, min

10

12

121

14

B

D 151.1

167

4.0e5

Intensity, cps

1 2 3 4 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

Page 32 of 39

154.4

149

138.4 124.2 121.1

2.0e5 95.1

141.2 149.0

107.4

179.0 182.3

77.1 91.3

0.0 20

40

60

80

141 164.0

100 120 m/z, Da

154

167.4

140

160

180

200

124

Fig. 1. Chromatogram (A) and MS/MS spectrum (B) of ME. C: unlabeled d0-ME (m/z 179). D: deuterium-labeled d3-ME (m/z 182).


Page 33 of 39

A

Intensity, cps

2.0e5

0

0

C

6

8

10

12

6.47

0

2

4

6

8

10

12

E

6000

6.49 2000 0

0

2

4

6

8 6.46

10

12

14 Time, min

2

4

6

8

10

12

14 Time, min

G

6.0e5

0

5000

2

4

D

6

8

10

12

14 Time, min

8

10

12

14 Time, min

8

10

12

14 Time, min

6.47

2000

0

14 Time, min

6.49

2.0e5

0

14 Time, min

2000

0

Intensity, cps

4

Intensity, cps

Intensity, cps

5000

2

B

4.0e5

6.49

Intensity, cps

Intensity, cps

4.0e5

Intensity, cps

1 2 3 4 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


0

2

4

6

F

6000

6.49 2000 0

0

2

4

6

3.0e5

0

0

Fig. 2. Representative MRM (m/z 316→151 in black; m/z 319→154 in red) chromatograms of adduct A1.


analysis of the incubation of ME with cysteine in the presence of NADPH (A-B). Extracted ion chromatograms obtained from the analysis of the proteolytic digestion of microsomal protein exposed to ME in the presence of NADPH (C-D).

Extracted

ion chromatograms obtained from an analysis of the proteolytic digestion of hepatic protein of mice treated with ME (E-F).

Extracted ion chromatograms obtained from

an analysis of chemical synthetic A1 (G).


Intensity, cps

3000

0

2

4

6

8

10

12

C

2320

14 Time, min

1500 7.10

2

4

6

8

10

12

E

14 Time, min

3000

0

2

4

6

8

10

12

14 Time, min

10

12

14 Time, min

10

12

14 Time, min

D

2320 1500

7.10

2000 7.11

0

3.5e4

2

4

6

4

6

G

8 10 7.09

12

14 Time, min

12

14 Time, min

0 Intensity, cps

Intensity, cps

0

4000

0

7.11

500

500 0

Page 34 of 39

B

7000

0 Intensity, cps

Intensity, cps

7.11

A

7000

0

Intensity, cps

1 2 3 4 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

Intensity, cps


0

2

4

6

8

F

4000

2000 7.11 0

0

2

4

6

8

2.0e4

0 0

2

8

10



analysis of microsomal incubation of ME fortified with cysteine in the presence of NADPH (A-B).

Extracted ion chromatograms obtained from the analysis of the

proteolytic digestion of microsomal protein exposed to ME in the presence of NADPH (C-D).


proteolytic digestion of hepatic protein of mice treated with ME (E-F). chromatograms obtained from an analysis of chemical synthetic A2 (G).


Extracted ion

0

2

4

6

Intensity, cps

8

10

12

C

4620

14 Time, min

2000 6.51

0

2

4

E

1188

6 8 6.52

10

12

14 Time, min

600

0

0

3.0e6

2

4

6

8 6.50

10

12

14 Time, min

2

4

6

8

10

12

14 Time, min

G

B

1.8e5

1.0e5

0.0 Intensity, cps

Intensity, cps

6.52

6.52

0

2

4

6

8

10

12

10

12

D

4620

14 Time, min

2000 6.51 0

Intensity, cps

1.0e5

0 Intensity, cps

A

1.8e5

0.0

Intensity, cps

1 2 3 4 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


Intensity, cps

Page 35 of 39

0

2

4

6

8

F

1188

14 Time, min

6.52

600

0

0

2

4

6

8

10

12

14 Time, min

2.0e6 1.0e6 0.0



analysis of microsomal incubation of ME supplemented with cysteine in the presence of NADPH (A-B).


proteolytic digestion of microsomal protein exposed to ME in the presence of NADPH.

Extracted ion chromatograms obtained from an analysis of proteolytic

digestion of hepatic protein of mice treated with ME (E-F).

Extracted ion

chromatograms obtained from an analysis of chemical synthetic A3 (G).



A

ng, A1/ mg, protein

0.25 0.20 0.15 0.10 0.05 0.00 0

Formation of A2(%)

1

2

3

4

5

6

Time (h)

B

150 120 90 60 30 0 0

1

2

3

4

5

6

Time (h)

C

0.15

ng, A3/ mg, protein

1 2 3 4 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

0.12 0.09 0.06 0.03 0.00 0

1

2

3

4

5

6

Time (h)

Fig. 5. Time-course changes in protein modifications represented by the levels of A1 (A), A2 (B), and A3 levels (C). Mice were treated with ME (i.p.) at 200 mg/kg. Cysteine-based protein adduction was examined at various time points after the administration (n = 4).


Page 36 of 39

Page 37 of 39

ng, A1/ mg, protein

0.32

A

0.24

0.16

0.08

0 100

Formation of A2(%)

120

200

300

Dosage (mg/kg)

B

90

60

30

0 100 0.16

ng, A3/ mg, protein

1 2 3 4 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


200

300

Dosage (mg/kg)

C

0.12

0.08

0.04

0 100

200

300

Dosage (mg/kg)

Fig. 6. Dose-dependent changes in protein modifications represented by the levels of A1 (A), A2 (B), and A3 (C).

Mice were treated with ME (i.p.) at various dosages.

Cysteine-based protein adduction was examined after 1 h administration (n = 4).



ng, A1/ mg, protein

1.00

A

0.80 0.60 0.40 0.20 0.00 ME

Formation of A2(%)

150

KTC+ME

DXM+ME

BSO+ME

B

120 90 60 30 0 ME 0.40

ng, A3/ mg, protein

1 2 3 4 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

Page 38 of 39

KTC+ME

DXM+ME

BSO+ME

C

0.32 0.24 0.16 0.08 0.00 ME

KTC+ME

DXM+ME

BSO+ME

Fig. 7. Changes in protein modification represented by the levels of A1 (A), A2 (B), and A3 (C) in mice pretreated with KTC, DXM or BSO. (i.p.) ME at 200 mg/kg body weight.

Mice were administered

Cysteine-based protein adduction was

examined after 1 h administration (n = 4).


A

AST

**

*

300.0

ALT

*

200.0

100.0

*

*

*

0.0 100

200

AST and ALT activities (U/L)

400.0

0

500.0

B

**

AST

**

*

ALT

400.0

**

300.0 200.0 100.0 0.0

300

Vehicle

DXM

DXM+ME

ME

400.0

C

*

AST

*

ALT

300.0

200.0

100.0

0.0 Vehicle

KTC

KTC+ME


Dosage (mg/kg)


1 2 3 4 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



Page 39 of 39

ME

400.0

D

**

AST

*

ALT

*

300.0

200.0

100.0

0.0 Vehicle

BSO

BSO+ME

ME

Fig. 8. Serum ALT and AST activities in mice after treatment with ME.

A:

Dose-dependent changes in serum AST and ALT activities in mice 1 h following i.p. administration of ME at 0, 100, 200, or 300 mg/kg (mean ± SD, n = 4). DXM (B), KTC (C) and BSO (D) on ME-induced hepatotoxicity. < 0.01 were considered significantly different.

*p < 0.05 and **p

The vehicle was corn oil.


Effects of

Chemical Interaction of Protein Cysteine Residues with Reactive

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