Title page Urinary methyleugenol-deoxyadenosine adduct as potential

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Urinary methyleugenol-deoxyadenosine adduct as potential biomarker of methyleugenol exposure in rats Yukun Feng, Saide Wang, Hui Wang, Ying Peng, and Jiang Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05186 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 13, 2018

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Title page Urinary methyleugenol-deoxyadenosine adduct as potential biomarker of methyleugenol exposure in rats † Yukun Feng , Saide Wang†, Hui Wang†, Ying Peng†*, and Jiang Zheng†θ* †

Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China θ State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province and Guizhou Medical University, Guiyang, Guizhou, 550004, P. R. China

Corresponding Authors: Jiang Zheng, PhD 1 State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, 550004, P. R. China; 2Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China E-mail: [email protected] Tel: +86-24-23984215 Fax: +86-24-23986510 1, 2:

The two corresponding units contributed equally to this work.

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

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ABSTRACT Methyleugenol (ME), a natural ingredient of several herbs and spices used in the

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human diet, is hepatocarcinogenic in rodents.

Following metabolic activation to

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reactive carbocation intermediate, ME can bind covalently to DNA, which is directly

35

associated with its carcinogenicity.

36

determine ME exposure was established by monitoring urinary ME-dA adduct.

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developed method entails liquid-liquid extraction enrichment of the urinary ME-dA,

38

incorporation of deuterated ME-dA as internal standard, and analysis by liquid

39

chromatography coupled tandem mass spectrometry.

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180-200 g) were treated (p.o.) with ME, and ME-dA was excreted in urine in a dose-

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and time-dependent manner.

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determine exposure to ME-containing herbs and spices.

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ME-dA can potentially serve as an effective biomarker of ME exposure in rats.

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expected that the developed approach of detecting urinary ME-dA will facilitate the

45

investigation of ME carcinogenesis.

In this work, a non-invasive approach to The

Male rats (10-12 weeks,

The noninvasive approach enabled us to successfully These results suggest that It is

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Keywords: methyleugenol-deoxyadenosine adduct, biomarker, urine, LC-MS/MS,

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spices 2


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INTRODUCTION Methyleugenol (ME, 4-allyl-1,2-dimethoxybenzene), an allylalkoxybenzene

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compound, is a natural ingredient present in a variety of herbs and spices.1

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has been found in blackberry essence, bananas, black pepper, bilberries and walnuts.2

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And essential oils and extracts of spices rich in ME have been approved for

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commercial use as fragrance and ﬂavoring agent.

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flavouring food and beverages, the general population is exposed to ME at a certain

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level ranging from 14 mcg/kg bw/day to 217 mcg/kg bw/day mainly through

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ingestion of dietary products, herbs, spices and their essential oils on a daily basis.3

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However, based on sufficient evidence of carcinogenicity from studies in

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experimental animals, ME was classified by the National Toxicology Program (NTP)

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as a ‘‘reasonably anticipated to be human carcinogen’’.4

It also

Due to its widespread use in

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ME-induced toxicities are believed to be associated with its metabolic

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activation.5-7 Many studies demonstrated that after hydroxylation and sulphation,

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ME bioactivated to the corresponding carbocation intermediate could result in the

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formation of ME-DNA adducts in liver and extrahepatic tissues of rodents (primarily

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caecum, kidney and stomach).8-10

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hepatic ME-DNA adducts was directly associated with its carcinogenicity.11,12

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general, ME-DNA adducts involved the binding of the 3’-C atom of ME to the

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exocyclic

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N2-(Methylisoeugenol-3’-yl)-2’-deoxyguanosine

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N6-(methylisoeugenol-3’-yl)-2’-deoxyadenosine (ME-dA) as ME-DNA adducts

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formed in vivo have been identified by mass spectrometry and nuclear magnetic

79

resonance (NMR) spectroscopy.13

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tissue samples after exposure to ME.

amino

It has been confirmed that the abundance of

groups

of

In

purine

bases.

(ME-dG)

and

ME-DNA adducts are also abundant in human Tremmel and co-workers reported the 3



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detection of ME-DNA adducts in surgical human liver samples from 121 subjects and

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Monien et al. detected these adducts in 10 out of 10 human lung samples.14,15

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ME-DNA adducts can been considered as effective biomarkers for DNA damage

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induced by ME.

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Hence,

Currently, studies on ME-DNA adducts focus intensively on detection of DNA

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isolated from liver, lung and kidney tissues.

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involves sacrificing the laboratory rodents and these tissues are not typically available

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for human biomonitoring studies.

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be repaired by nucleotide excision repair (NER) mechanism.16-19

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deoxynucleosides as degradation products of DNA adducts were eliminated via

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urination, and the deoxynucleoside adduct level in urine is an indicator of the balance

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between adduct formation and the repair system in response to the chemical damage

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at a certain point in time.

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biomarkers for carcinogen exposure and evaluation of cancer risk.20

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that ME-DNA adducts would be repaired and the corresponding deoxynucleoside

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adducts would be excreted in urine, and urinary ME-dA may be employed as a

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biomarker of ME exposure.

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However, the tissue sampling process

Evidence has showed that the DNA adducts may Modified

Certain deoxynucleoside adducts in urine have served as We reasoned

The major objective of the present study was to define the correlation of ME or ME-containing herbs and spices exposure with the urinary excretion of ME-dA.

It is

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anticipated that ME-dA can serve as an effective biomarker of ME exposure and that

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the developed approach will facilitate the understanding of carcinogenicity of ME.

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Materials and methods Chemicals

and

reagents.

ME

(≥99.0%),

eugenol

(≥99.0%)

and

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2'-deoxyadenosine (dA) (≥99.0%) were purchased from Tokyo Chemical Industry

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(Shanghai) Development Co., Ltd. (Shanghai, China).

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from Shanghai Bodi chemical technology Co., Ltd. (Shanghai, China).

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was acquired from Fisher Scientific (Springfield, NJ).

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was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).

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tatarinowii rhizome, and myristicae semen were obtained from Tong-Ren-Tang

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pharmacy (Shenyang, China).

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Shouyi Shi San Xiang Multi-flavoured Spice Group Co., Ltd. (Henan, China).

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organic solvents were obtained from Fisher Scientific (Springfield, NJ).

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and solvents were either analytical or HPLC grade.

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Chemical Synthesis.

CD3I (≥99.0%) was obtained Formic acid

N-Bromosuccinimide (NBS) Asari radix, acori

Shi San Xiang was purchased from Zhumadian Wang All

All reagents

ME-dA was synthesized by bromination of ME, followed

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by reaction with dA.

Briefly, a solution of NBS (0.15 g, 0.85 mmol) in CH2Cl2 (4.0

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mL) was slowly added to a solution of ME (0.14 g, 0.77 mmol) in 4.0 mL of CH2Cl2.

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After 2 h stirring at 40 °C, the solution was concentrated to dryness, reconstituted in

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1.0 mL of methanol, mixed with a dA solution (0.25 g, 1.0 mmol, dissolved in 10 mL

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water), and stirred for 2 h at 60 °C.

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dryness, reconstituted with acetonitrile/water (50:50, v/v), centrifuged, and submitted

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to HPLC for purification.

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and NMR for characterization.

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standard (IS) was started by preparation of d3-ME.

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methylation of phenolic hydroxyl of eugenol using CD3I as a methylating agent,

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according to our reported method.21

The resulting mixture was concentrated to

The purified product was submitted to mass spectrometry Synthesis of deuterium labeled d3-ME-dA as internal d3-ME was prepared by

5



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HPLC-MS/MS instrumentation and analytical conditions.

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The analysis was

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performed on an AB SCIEX Instruments 5500 triple quadrupole mass spectrometer

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(Applied Biosystems, Foster City, CA) interfaced online with a 1260 infinity system

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(Agilent Technologies, Santa Clara, CA).

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gradient elution from a BDS HYPERSIL C18 ODS column (5.0 µm, 150 mm×4.6 mm;

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Thermo, San Jose, CA) with a flow rate of 0.8 mL/min of mobile phases, including

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0.1 % (v/v) formic acid in acetonitrile (A) and 0.1 % (v/v) formic acid in water (B).

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Gradient elution was employed as follows: 20 % A at 0-1 min, 20-40 % A at 1-10 min,

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40-90 % A at 10-12 min, 90-20 % A at 12-14 min, and 10 % A at 14-17 min. ME-dA

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was analyzed in positive ion mode by multiple-reaction monitoring (MRM) scanning.

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The optimized mass spectrometric instrument parameters obtained after tuning were

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as follow: curtain gas (CUR), gas 1 (GS1), and gas 2 (GS2) were 35, 50 and 50 psi;

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ion source temperature (TEM) was at 650 °C; ion spray voltage (IS) and entrance

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potential (EP) were 5,500 and 10 V, respectively.

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(corresponding to declustering potential DP, collision energy CE, collision cell exit

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potential CXP) were m/z 428.2→177.1 (80, 35, 10) for ME-dA and m/z 431.2→180.1

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(80, 35, 10) for IS.

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Animals and Treatment.

HPLC separation was carried out by

The characteristics of ion pairs

Male Sprague-Dawley rats (180-200 g) were

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purchased from the Animal Center of Shenyang Pharmaceutical University (Shenyang,

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China).

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temperature-controlled (22 ± 4 °C) facility with a 12 h dark/light cycle for at least 5

147

days after receipt and before treatment.

148

according to procedures approved by the Ethics Review Committee for Animal

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Experimentation of Shenyang Pharmaceutical University.

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Rats had free access to food and water and were housed in a

All animal studies were performed

Propanediol as a 20 % aqueous solution was used as the vehicle for ME.

Rats 6


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were treated with ME at doses of 1, 5, or 25 mg/kg by gavage (n = 4).

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study, rats (n = 4) were administered with the dose of 5 mg/kg ME 12 h per intervals

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in three consecutive days.

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groups, and each group contained four animals.

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a single dose of vehicle, asari radix extracts (0.3 g/kg), acori tatarinowii rhizoma

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extracts (1.0 g/kg), myristicae semen extracts (1.0 g/kg), or Shi San Xiang extracts

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(2.0 g/kg), respectively.

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and 12 h intervals post-dosing were collected over ice.

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urine was filtered through a gauze filter to remove pieces of feces and crumbs of food

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pellets.

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washes were added to the main portion of collected urine.

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samples were processed immediately for LC–MS/MS quantification of urinary

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ME-dA.

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In another

In a separate study, rats were randomly divided into five Each group of rats was treated with

In all studies, urine samples from the day prior to dosing During sample collection the

The walls of the metabolic cages were rinsed with distilled water and the

Standard solutions preparation.

The collected urine

An appropriate amount of ME-dA was

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weighed and dissolved in methanol to produce the stock solution to obtain a

166

concentration of 1.25 mg/mL.

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to obtain working solutions at six concentration levels.

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(450 ng/mL) was prepared by dissolving d3-ME-dA with methanol.

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samples for construction of six-point calibration curve were prepared by spiking blank

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rat urine with the corresponding working solutions (100 µL) and IS (100 µL), and the

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final concentrations of the calibration standards were 0.25, 0.5, 1.25, 2.5, 6.25 and

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12.5 ng/mL.

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The stock solution was further diluted with methanol The IS working solution Assay standard

All the solutions were stored in refrigerator at 4 °C.

Urine samples preparation.

The collected urine samples (1.0 mL) were

spiked with 100 µL IS solution, followed by mixing with 2.0 mL methanol, vortexing 7



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for 3 min, and centrifuging at 16,000 g for 5 min at 4 °C.

The supernatants were

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collected and evaporated to dryness under a nitrogen gas stream.

177

residue was reconstituted with 1.0 mL H2O and then extracted with 3.0 mL of ethyl

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acetate.

179

residue was reconstituted with 100 µL of incipient mobile phase, and 10 µL of the

180

supernatants were injected to LC–MS/MS for analysis.

The resulting

The organic phase was collected and evaporated to dryness.

Each dried

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Results and discussion Characterization of Synthetic ME-dA.

The synthetic ME-dA was

184

characterized by MS and 1H NMR.

185

+ H]+ ions at m/z 428.1933, which corresponded to the formula C12H26N5O5

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(calculated: 428.1928).

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were consistent with published results.13

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(DMSO-d6, 600 MHz), δ 2.25 (m, 1H, H2’’’a), 2.59 (dt, 1H, H2’’’b), 3.50 (m, 1H,

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H5’’’a), 3.62 (m, 1H, H5’’’b), 3.70 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.85 (dd, 1H,

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H4’’’, J = 4.0, 1.2 Hz), 4.23 (S, 2H, H3’), 4.39 (m, 1H, H3’’’), 5.30 (m, 1H, OH-5’’’),

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5.41 (dd, 1H, OH-3’’’, J = 5.2, 2.7 Hz), 6.25 (m, 1H, H2’), 6.33 (dd, 1H, H1’’’, J = 6.3,

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7.5 Hz), 6.42 (d, 1H, H1’, J = 15.9 Hz), 6.84 (m, 1H, H6 or H5), 6.87 (m, 1H, H5 or

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H6), 6.98 (d, 1H, H3, J = 1.3 Hz), 8.06 (bs, 1H, H6’’), 8.20 (bs, 1H, H2’’), 8.33 (s, 1H,

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H8’’).

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The high resolution mass spectrum showed [M

The 1H NMR data and MS identities of the synthetic ME-dA NMR characterization of ME-dA: 1H NMR

Optimization of sample preparation.

The performance of sample preparation

196

method was based on the recovery of ME-dA in urine matrix and the loss of signal

197

intensity caused by ion suppression.

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samples were initially precipitated by methanol, and urinary ME-dA was then

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enriched by liquid-liquid extraction (LLE).

200

n-hexane were tested respectively to enrich analytes.

201

extraction efficiency than the others so that ethyl acetate was selected as the extraction

202

solvent.

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investigated to optimize the extraction efficiency.

204

increased with the increase of volume applied until 3.0 mL was employed.

205

In order to reduce the matrix effect, the urine

Dichloromethane, ethyl acetate and Ethyl acetate showed better

Various volumes of ethyl acetate in the range of 1.0-5.0 mL were The extraction efficiency

Calibration curve, lower limit of quantification and recovery.

The typical 9



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chromatograms from LC–MS/MS analysis of ME-dA and d3-ME-dA are displayed in

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Fig. 1.

208

collected from vehicle-treated rats. Calibration curve was established by computing

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regression line of the peak area ratios (Y) of ME-dA to d3-ME-dA versus the

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concentrations (X) of ME-dA using a 1/x2 weighted least-squares linear regression

211

model.

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0.2234X - 0.002 with good linearity (r = 0.9996) (Fig. 2).

213

quantification (LLOQ) determining the sensitivity of the method was assessed as the

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lowest concentration on the calibration curve and as the concentration level with a

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signal-to-noise (S/N) ratio of 10.

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extraction recovery was evaluated by comparing peak areas obtained from extracted

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spiked samples with those of the post extracted spiked samples at corresponding

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concentration.

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replicate samples at final concentration of 2.5 ng/mL.

The average of extraction

220

recovery was 80.0 % and the RSD value was 6.6%.

The data obtained from

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validation procedure suggest that this method exhibited good linearity and acceptable

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recovery.

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No such peak responsible for ME-dA was observed in the urine samples

The typical regression equation and correlation coefficients (r) was Y = The lower limit of

The LLOQ of ME-dA was 0.25 ng/mL.

The

The extraction efficiency of ME-dA was determined by analyzing six

Urinary ME-DNA adduct study in rats.

ME-dA was detected in the urine

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samples of ME-treated animals, and the approach developed in the present study

225

enabled us to quantify urinary ME-dA.

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was found to be proportional to the doses of ME applied in rats (Fig. 3).

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Interestingly, a plateau of urinary ME-dA content was observed in animals after the

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fourth consecutive administration at the dose of 5 mg/kg.

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ME-dA was undetectable (under the limit of quantification) 60 h after the last

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administration (Fig. 4).

The amount of the adduct excreted in urine

The level of urinary

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It has been reported that metabolic activation of ME leads to the formation of

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mutagenic DNA adducts.22 Based on NER mechanisms, ME-DNA adduction can be

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repaired and the corresponding deoxynucleoside adducts were eliminated via

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urination (Scheme 1). Urinary deoxynucleoside or nucleobase adducts derived from

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some important carcinogens, such as aflatoxin B1, benzo[a]pyrene, benzene and

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aristolochic acid have been proposed to be valuable noninvasive biomarkers of

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exposure.23-26

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would be an important contribution to biomarker of exposure to ME.

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important feature of the urinary DNA adduct biomarkers, besides the possibility of

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noninvasive sampling, is their capability to reflect the extent of compound specific

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damage to DNA.

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develop a noninvasive approach for the sensitive assessment of ME exposure by

243

quantifying urinary excreted deoxynucleoside adducts of ME.

As such, a possible finding of urinary deoxynucleoside adducts of ME The most

In order to investigate the carcinogenesis of ME, we sought to

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Human exposure to ME primarily occurs via ingestion of herbs and spices.2

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is anticipated that ME-dA could be employed as a reliable biomarker after

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ME-containing herbs and spices exposure.

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spice used in human diet.

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a very popular multi-flavoured natural seasoning.

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clove, cinnamon, galangal, fennel, pepper and nutmeg.

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tatarinowii rhizome are representative Chinese herbs rich in ME and have a long

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history of use in clinic.

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exposure to those model ME-containing herbal medicines or spices.

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ME in the extracts of Asari radix, Acori tatarinowii rhizome, nutmeg and Shi San

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Xiang tested were 1.01, 0.84, 1.23 and 0.96 mg/g, respectively.

255

administered with the corresponding extracts individually at 0.3 g/kg, 1.0 g/kg, 1.0

It

Nutmeg is a common ME-containing

Shi San Xiang, awarded “China Time-Honored Brand”, is It consists of many spices such as Asari radix and Acori

We examined the urinary excretion of ME-dA in rats after The contents of

Rats were orally

11



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g/kg and 2.0 g/kg, respectively, and urinary ME-dA was analyzed and quantified.

257

expected, ME-dA was detected in the urine samples of all rats after exposure to the

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individual extracts.

259

proportional to the content of ME detected in the extracts administered in the animals

260

(Fig. 5).

261

exposure.

262

As

Additionally, the content of urinary ME-dA was found to almost

These findings indicate that ME-dA would be a potential biomarker for ME

It is worth noting that no urinary ME-dG was detected until oral dose of ME

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reached 25 mg/kg.

Surprisingly, the level of ME-dG was lower than that of ME-dA

264

in the urine, since this is inconsistent with the report that the level of ME-dG was

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50-70 fold higher than that of ME-dA in liver and other tissues of animals given

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ME.27

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DNA adduction at dG.

A possible explanation could be the poor efficiency for repair of ME-derived

268

GSH conjugation is a prominent detoxification pathway of metabolism of

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electrophilic species.28 Our early study demonstrated that a high dose (100 mg/kg)

270

of ME was required to see urinary GSH and related conjugates derived from

271

electrophilic metabolites of ME in rats.29

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of ME-dA in urine of rats given ME at 5 mg/kg.

273

of ME-derived DNA adduction relative to that arising from GSH conjugation.

This

274

observation

soft

275

electrophiles/nucleophiles.30-32

276

react predominantly with hard nucleophiles (amino group of purine bases) rather than

277

soft nucleophiles (sulfhydryl group of cysteine or GSH).

278

is better than the corresponding GSH conjugates as a biomarker for ME exposure.

could

be

interpreted

The present study showed high abundance

by

This could result from high volume

concepts

of

hard

and

Briefly, hard electrophiles (alkyl carbonium ion)

To sum up, urinary ME-dA

279

In conclusion, ME-dA could be excised from ME-DNA adducts by NER

280

mechanism and the urinary excretion of ME-dA occurred in time- and dose-dependent 12


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manners.

Results from this study indicate that it is possible to monitor ME exposure

282

noninvasively by quantifying urinary excreted ME-dA.

283

urinary ME-dA may be a valuable biomarker for monitoring ME exposure.

284

developed analytical approach was sensitive and selective and will facilitate the

285

investigation of the mechanisms of ME carcinogenesis.

The work suggests that The

13



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286

Funding sources

287

This work was supported in part by the National Natural Science Foundation of China

288

(No. 81430086, 81373471 and 81773813).

289 290 291 292

Notes

293

The authors declare no competing financial interest.

294

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Abbreviations:

296

ME, methyleugenol; dA, 2'-deoxyadenosine; dG, 2’-deoxyguanosine; GSH,

297

glutathione; NER, nucleotide-excision repair; NBS, N-bromosuccinimide; NMR,

298

nuclear magnetic resonance spectroscopy; LLE, liquid-liquid extraction; LLOQ,

299

lower limit of quantification; ESI, electrospray ionization; CE, collision energy; CXP,

300

cell exit potential; DP, declustering potential; EP, entrance potential; MRM,

301

multiple-reaction monitoring.

15



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strong increase by transgenic human SULT. Carcinogenesis 2014, 35, 935–941.

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(10) Herrmann, K.; Engst, W.; Florian, S.; Lampen, A.; Meinl, W.; Glatt, H. The

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influence of the SULT1A status - wild-type, knockout or humanized - on the DNA

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adduct formation by methyleugenol in extrahepatic tissues of mice. Toxicol. Res. 2016,

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5, 808–815.

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(11) Waddell, W. J.; Crooks, N. H.; Carmichael, P. L. Correlation of Tumors with

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DNA Adducts from Methyl Eugenol and Tamoxifen in Rats. Toxicol. Sci. 2004, 79,

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38–40.

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(12) Paini, A.; Scholz, G.; Marin-Kuan, M.; Schilter, B.; O'Brien, J.; van Bladeren,

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P. J.; Rietjens, I. M. Quantitative comparison between in vivo DNA adduct formation

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from exposure to selected DNA-reactive carcinogens, natural background levels of

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DNA adduct formation and tumour incidence in rodent bioassays. Mutagenesis 2011,

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of human and murine sulfotransferases able to activate hydroxylated metabolites of

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methyleugenol. Mutagenesis 2012, 27, 453–462.

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(14) Tremmel, R.; Herrmann, K.; Engst, W.; Meinl, W.; Klein, K.; Glatt, H.; Zanger,

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U. M. Methyleugenol DNA adducts in human liver are associated with SULT1A1

351

copy number variations and expression levels. Arch. Toxicol. 2017, 91, 3329–3339.

352

(15) Monien, B. H.; Schumacher, F.; Herrmann, K.; Glatt, H.; Turesky, R. J.;

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Chesné, C. Simultaneous Detection of Multiple DNA Adducts in Human Lung

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Samples by Isotope-Dilution UPLC-MSMS. Anal. Chem. 2015, 87, 641–648.

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(16) Lukin, M.; Zaliznyak, T.; Johnson, F.; de los Santos, C. Structure and stability

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of DNA containing an aristolactam II-dA lesion: implications for the NER recognition

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of bulky adducts. Nucleic. Acids Res. 2012, 40, 2759–2770.

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(17) Sidorenko, V. S.; Yeo, J. E.; Bonala, R. R.; Johnson, F.; Schärer, O. D.;

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Grollman, A. P. Lack of recognition by global-genome nucleotide excision repair

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accounts for the high mutagenicity and persistence of aristolactam-DNA adducts.

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Nucleic. Acids Res. 2012, 40, 2494–2505.

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(18) Yan, S.; Wu, M.; Buterin, T.; Naegeli, H.; Geacintov, N. E.; Broyde, S. Role of

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Base Sequence Context in Conformational Equilibria and Nucleotide Excision Repair

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of Benzo[a]pyrene Diol Epoxide-Adenine Adducts. Biochemistry. 2003, 42, 2339–

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the nucleotide excision repair machinery. Biopolymers. 2002, 65, 202–210. (20) Vineis, P.; Perera, F. DNA adducts as markers of exposure to carcinogens and risk of cancer. Int. J. Cancer. 2000, 88, 325–328.

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Chem. Res. Toxicol. 2017, 30, 564–573.

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H.; Schrenk, D. Metabolism of methyleugenol in liver microsomes and primary

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hepatocytes: pattern of metabolites, cytotoxicity, and DNA-adduct formation. Toxicol.

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Sci. 2012, 129, 21–34.

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Wogan, G. N.; Groopman, J. D. A follow-up study of urinary markers of aflatoxin

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408

Scheme Legend

409

Scheme 1. Proposed pathway for the formation of urinary ME-dA.

410 411 412

Figure Legends

413

Fig. 1. Representative MRM chromatograms of ME-dA (m/z 428.2→177.1) and IS

414

(m/z 431.2→180.1) obtained from analysis of blank urine sample (A), urine sample

415

collected from a rat 12 h after an oral administration of ME (B), blank urine sample

416

spiked with ME-dA (C), and urine sample spiked with IS (D).

417

and proposed fragment assignments of ME-dA (E) and IS (F).

418

Fig. 2. Calibration curve of ME-dA with IS

419

Fig. 3. Dose-dependent and time-course changes in the levels of urinary ME-dA.

420

Rats were dosed orally with ME at 1, 5, and 25 mg/kg, respectively.

421

of 12 h intervals were collected through the experiment.

422

SD for four rats.

423

Fig. 4. Urinary excretion of ME-dA in rats treated with ME.

424

with ME at 5 mg/kg 12 h per intervals in three consecutive days.

425

12 h intervals were collected through the experiment.

426

for four rats.

427

Fig. 5. Levels of urinary ME-dA in Asari radix (0.3 g/kg), Acori tatarinowii rhizome

428

(1.0 g/kg), Myristicae semen (1.0 g/kg) and Shi San Xiang (2.0 g/kg) dosed rats.

429

Urine samples 12 h post-dosing were collected.

430

four rats.

The MS/MS spectrum

Urine samples

The data represent mean ±

Rats were orally dosed Urine samples of

The data represent mean ± SD

The data represent mean ± SD for

21



CYP 1A, SULT 1A

ME

431 432

NER

ME-DNA adducts

Page 22 of 28

Urine

ME-dA

Scheme 1. Proposed pathway for the formation of urinary ME-dA.

433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 22


Page 23 of 28


148.1

A

1000 500 177.1 0

0

2

4

6

Intensity, cps

B

8 10 Time, min

12

14

1.2e6

8.39

1.2e4 8000.0

312.1

8.0e5

428.2 4.0e5 148.1

4000.0 0.0 0.0

0

2

4

6

C Intensity, cps

312.1

177.1

16

Intensity, cps

Intensity, cps

1500

E

8 10 Time, min

12

14

100

200

300 m/z, Da

16

400

500

148.1

8.38

F

2.0e4 1.5e4 1.0e4 5000.0 0.0

0

2

4

6

8 10 Time, min

12

14

16

8.36

180.1

315.1

180.2 3.0e5

3.0e4

Intensity, cps

Intensity, cps

D 2.0e4 1.0e4

315.1

2.0e5

431.2 1.0e5 148.1

0.0

450

0

2

4

6

8 10 Time, min

12

14

16

0.0

100

200

300 m/z, Da

400

500

451

Fig. 1. Representative MRM chromatograms of ME-dA (m/z 428.2→177.1) and IS

452

(m/z 431.2→180.1) obtained from analysis of blank urine sample (A), urine sample

453

collected from a rat 12 h after an oral administration of ME (B), blank urine sample

454

spiked with ME-dA (C), and urine sample spiked with IS (D).

455

and proposed fragment assignments of ME-dA (E) and IS (F).

The MS/MS spectrum

456 457 458 459 460 461

23



Page 24 of 28

ME-dA Area / d3-ME-dA Area

3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 462 463

2

4

6

8

10

12

14

ng/mL

Fig. 2. Calibration curve of ME-dA with IS

464 465 466 467 468 469 470 471 472 473 474 475 476 477 24



Adduct concentration, ng/mL urine

Page 25 of 28

10 1 mg/kg 8 5 mg/kg 6

25 mg/kg

4 2 0 0-12 h

12-24 h

24-36 h

36-48 h

48-60 h

478 479

Fig. 3. Dose-dependent and time-course changes in the levels of urinary ME-dA.

480

Rats were dosed orally with ME at 1, 5, and 25 mg/kg, respectively.

481

of 12 h intervals were collected through the experiment.

482

SD for four rats.

Urine samples

The data represent mean ±

483 484 485 486 487 488 489 490 491 492 493 494 25




8

Page 26 of 28

withdrawal

6

4

2

0 0

1

2

3

4

5

6

Time elapsed, days

495 496

Fig. 4. Urinary excretion of ME-dA in rats treated with ME.

497

with ME at 5 mg/kg 12 h per intervals in three consecutive days.

498

12 h intervals were collected through the experiment.

499

for four rats.

Rats were orally dosed Urine samples of

The data represent mean ± SD

500

501

502

503

504

505

506

507

508

26



0.8

1.4 Adduct concentration Doses of ME

0.4

0.7

0

0 Vehicle

509

Doses of ME, mg/kg


Page 27 of 28

Asari radix

Acori tatarinowii rhizome

Nutmeg

Shi San Xiang

510

Fig. 5. Levels of urinary ME-dA in Asari radix (0.3 g/kg), Acori tatarinowii rhizome

511

(1.0 g/kg), Myristicae semen (1.0 g/kg) and Shi San Xiang (2.0 g/kg) dosed rats.

512

Urine samples 12 h post-dosing were collected.

513

four rats.

The data represent mean ± SD for

27



514

Page 28 of 28

TOC Graphic

515

28


Title page Urinary methyleugenol-deoxyadenosine adduct as potential

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