Accepted Manuscript Title: Simultaneous determination of four sulfur mustard-DNA adducts in rabbit urine after dermal exposure by isotope-dilution liquid chromatography-tandem mass spectrometry Author: Yajiao Zhang LijunYue Zhiyong Nie Jia Chen Lei Guo Bidong Wu Jianlin Feng Qin Liu Jianwei Xie PII: DOI: Reference:

S1570-0232(14)00294-3 http://dx.doi.org/doi:10.1016/j.jchromb.2014.04.050 CHROMB 18919

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

19-2-2014 24-4-2014 28-4-2014

Please cite this article as: Y. Zhang, Z. Nie, J. Chen, L. Guo, B. Wu, J. Feng, Q. Liu, J. Xie, Simultaneous determination of four sulfur mustardDNA adducts in rabbit urine after dermal exposure by isotope-dilution liquid chromatography-tandem mass spectrometry, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.04.050 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Simultaneous determination of four sulfur mustard-DNA adducts in rabbit urine after dermal exposure by isotope-dilution liquid

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chromatography-tandem mass spectrometry

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Qin Liu*, Jianwei Xie*

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Yajiao Zhang, LijunYue, Zhiyong Nie, Jia Chen, Lei Guo, Bidong Wu, Jianlin Feng,

State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory

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of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military

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medical Sciences, 27 Taiping Road, Haidian District, Beijing 100850 PR China

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*Corresponding authors. Dr. Jianwei Xie, State Key Laboratory of Toxicology and Medical

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Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military medical Sciences. Tel.(or Fax): +86 10 68225893. E-mail:

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[email protected]; Dr. Qin Liu, State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military medical Sciences. Tel.: +86 10 66930621, Fax: +86 10 68225893,

E-mail:

[email protected]

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Abstract Sulfur mustard (SM) is a classic vesicant agent, which has been greatly employed in

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several wars or military conflicts. The most lesion mechanism is its strong alkylation

of DNAs in vivo. Until now there are four specific DNA adducts of SM identified for

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further retrospective detection, i.e. N7-(2-hydroxyethylthioethyl)-2’-guanine (N7-

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HETEG), bis(2-ethyl-N7-guanine)thioether (Bis-G), N3-(2-hydroxyethylthioethyl)-2’adenine (N3-HETEA) and O6-(2-hydroxyethylthioethyl)-2’-guanine (O6-HETEG),

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respectively. Here, a novel and sensitive method of isotope-dilution ultrahigh performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS)

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combining with solid phase extraction was reported for the simultaneous determination of four SM-DNA adducts. A lower limit of detection of 2-5 ng·L-1, and

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a lower limit of quantitation of 5-10 ng·L-1 were achieved, respectively, and the

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recoveries ranged from 87% to 116%. We applied this method in the determination of four SM-DNA adducts in rabbit urine after dermal exposure by SM in three dose

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levels (2, 5, 15 mg·kg-1), so as to investigate the related metabolic behavior in vivo.

For the first time, in SM exposed rabbit urine, our results revealed the relative accumulation abundance of four SM-DNA adducts, i.e., 67.4% for N7-HETEG, 22.7%

for Bis-G, 9.8% for N3-HETEA, 0.1% for O6-HETEG, and significant dose and time dependent responses of these SM-DNA adducts. The four adducts were detectable after 8 hours, afterwards, their contents continuously increased, achieved maximum in the first two or three days and then gradually decreased till the end of one month. Meanwhile, the amounts of SM-DNA adducts were positively correlated with the exposure doses. Keywords: Sulfur mustard; DNA adducts; Isotope-dilution ultrahigh performance 2

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liquid

chromatography-tandem

mass

spectrometry;

Solid-phase

extraction;

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Metabolism

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1.

Introduction Sulfur mustard (SM, U.S. Code HD, chemical name bis(2-chloroethyl)sulfide,

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molecular formula C4H8Cl2S), is known as a bifunctional, alkylating, vesicant chemical warfare agent. SM can easily enter into the cells of skin, mucous membrane

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and organs, causing generally pathological changes in skin, eye, alimentary canal and

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respiratory systems and resulting in severe symptoms such as the erosion, degeneration necrosis and inflammation [1]. Since its first synthesis in pure form in

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1886, because of its ease of preparation, low volatility, and lack of antidotes, SM has been used in wars and military conflicts, for example, World War I and Iran-Iraq

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conflict, which continually threatened the public safety. It was reported that ca. 80% of the chemical casualties in WWI was caused by SM, and there were over 90,000

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died of SM caused secondary infection in these 1.3 million SM injured people [2, 3].

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weapons [4].

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Syria regime has also contained large amount of SM in their acclaimed chemical

After administration into body, SM quickly forms sulfonium ion and reacts with

numerous nucleophiles, making the intact SM too difficult to be detected in vivo. However, its metabolites, DNA adducts and protein adducts have relatively longer lifetimes from week(s) to months and can be used as good biomarkers for retrospective analysis. Generally, the biomarkers of SM include four kinds, i.e., hydrolysis/oxidation products [5, 6], β-lyase metabolites of glutathione adducts [7, 8], and alkylated products with proteins [9] and DNAs [10]. DNAs have large susceptibility towards alkylating agents, which is the exact reason for the cytotoxicity of SM [11, 12]. The main positions of DNAs to be alkylated by 4

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SM are the N7, O6 position of guanine and N3 position of adenine. Until now four kinds of SM-DNA adducts have been recognized and can be employed as good retrospective biomarkers, i.e., N7-(2-hydroxyethylthioethyl)-2’-guanine (N7-HETEG),

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bis(2-ethyl-N7-guanine)thioether (Bis-G), which is formed by reacting with each N7 position of two guanines in double-stranded DNA (dsDNA), N3 - (2-

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hydroxyethylthioethyl) - 2’ - adenine (N3-HETEA) [13, 14], and O6-(2hydroxyethylthioethyl)-2’-guanine (O6 - HETEG), respectively [15]. The chemical

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structures of these four SM-DNA adducts were shown in Fig. 1.

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According to multiple independent experiments in vitro using calf thymus DNA or human blood, N7-HETEG has the most abundance, accounting ca. 61% of the total

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SM-DNAs [14], while Bis-G has 16% [16], N3-HETEA has 11% [15], and O6HETEG has the minimum proportion, only ca. 0.1% of total [15]. For N7-HETEG and

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Bis-G, only one or two guanines react with chloroethane group(s) of SM in the N7

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position, no hydrogen bonds of Watson-Crick type are destroyed [14]. For O6HETEG, once formed during alkylation with SM, the hydrogen bonds between the

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bases are cleaved and double-strand DNAs are thus broken down [17]. Therefore, although it occupies the minimum percentage, O6-HETEG is still accepted as the main product responsible for the DNA damages by SM. More abundance data of different four SM-DNA adducts can be seen in Table S1 in support information. Overall, as the material base of cytotoxicity induced by SM, it is of great

importance to fully understand the content, distribution, dose and time relationship of these four kinds of SM-DNA adducts in vivo, and of course a sensitive, reliable method of simultaneous detection is firstly to be chosen. Several

methods

regarding SM-DNA adducts

such

as

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P-postlabeling,

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immunoassay,

gas

chromatography-mass

spectrometry

(GC-MS),

liquid

chromatography-mass spectrometry (LC-MS) have been reported [18-23].

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P-

postlabeling technique has a relatively high sensitivity, but radiochemical handling

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and consequently radioactive contamination cannot be ignored. Immunoassay is not widely applied due to the preparation difficulty of antibodies towards such a toxic

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hapten, and unavoidable cross-reaction with chemicals of similar structures [19]. GCMS technique can usually provide good sensitivity and selectivity, but the

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involvement of derivatization makes the whole work quite tedious and needs more

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elaboration. LC-MS or LC-tandem MS (MS/MS) technique is a preferred choice, because this technique is quite simple, ease of operation, and has good sensitivity and

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selectivity [21-23]. Most current methods are focusing on one or two SM-DNA adducts, simultaneous determination of three or four SM-DNA adducts is still rare

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[22, 23], which is not favorable to the further research on the toxicology of SM.

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Based on an advanced LC-MS/MS method of simultaneous detection of four kinds of SM-DNA adducts [23], this paper is aimed to investigate the time and dose effects

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of SM-DNA adducts in urine towards a SM exposed rabbit model in vivo. Considering that the percutaneous adsorption is the main path for SM exposure, and that rabbit has a similar half lethal dose (LD50) with human [24], a rabbit model with dermal exposure to SM was developed, and the urine samples were collected in schedule and analyzed by LC-MS/MS. Since most of the alkylated DNAs can be repaired in vivo, only the removed alkylated bases, i.e., SM-DNA adducts were

delivered into the circulatory system and then passed out from the body through the urine, this method may serve as a tool to assess the severity of DNA damage and the period required for repair in a convenient, non-destructive way. 2.

Experimental 6

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2.1. Caution SM is a highly reactive alkylation vesicant and cytotoxic agent. Handling of this agent should be carried out in the well-ventilated fume hoods. The uses of gloves and

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stringent protective measures must be adopted.

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2.2. Chemicals and materials

Methanol (HPLC grade) was purchased from J&K Scientific Ltd (Beijing, China).

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Formic acid (analytical grade) was obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). SM (purity is 96%) was supplied by the Institute of Chemical

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Defense of Chinese People’s Liberation Army. N7-HETEG, O6-HETEG, Bis-G, N3HETEA with purity of more than 96%, 98%, 97% and 98% by LC (with UV detector)

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and NMR, respectively, and deuterated internal standards (ISs, including d4-N7HETEG, d4-O6-HETEG, d4-Bis-G, d4-N3-HETEA) were home-synthesized, and the

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deuterated ratio was greatly than 99% [23]. Bulk sorbent C18 cartridges with high

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capacity for solid phase extraction (SPE) were purchased from W.R. Grace & Co.

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(Albany, OR, USA). Ultrapure water was produced by Milli-Q A10 purification system (Millipore, Billerica, MA, USA). 2.3. Calibration solutions

Standard stock solutions of N7-HETEG, Bis-G, N3-HETEA were prepared in

ultrapure water, while O6-HETEG was in methanol, and all stored at -20 °C. A series

of standard working solutions containing four SM-DNA adducts at concentrations of 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 150, 200 μg·L-1 were prepared by serial dilution of the stock solution with water. Quality control (QC) working solutions were prepared at concentrations of 0.1 (except for Bis-G at 0.2), 2, 200 μg·L-1. The mixing ISs working solution of d4-N7-HETEG, d4-O6-HETEG, d4-Bis-G, d4-N3-HETEA at concentrations 7

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of 21, 14, 62, 37 μg·L-1 was prepared by dilution of the stock solution. An aliquot of 100 μL standard/QC working solution and 50 μL of ISs working solution were spiked into 0.85 mL blank urine, making the final concentrations at 0.005, 0.01, 0.05, 0.1,

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0.5, 1, 5, 10, 15, 20 μg·L-1 to establish standard curves and evaluate precision and accuracy. The lower limit of detection (LLOD) was defined as the lowest

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concentration at which the analysis can reliably differentiate signal of the peak of

analyte from background noise (signal to noise ratio, S/N, is greater than 3). The

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lower limit of quantification (LLOQ) was defined as the lowest concentration at S/N

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greater than 10 [25]. 2.4. Animals and treatment

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Adult rabbits (male, 2.2-2.8 kg) were supplied by the Laboratory Animal Center of Beijing. The animal experiment was conducted in the Beijing Center for

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Toxicological Evaluation and Research, in accordance with the protocol approved by

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the Institutional Animal Care and Use Committee of the Center, which is in

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compliance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Twelve adult male rabbits were divided into four groups, i.e., three animals per

group. One was blank control group, and other three groups were rabbits with dermal exposure to SM in fume hoods with concentration of 2, 5 and 15 mg·kg-1 (0.02, 0.05 and 0.15LD50). After adaptable feeding for seven days, these rabbits were shaved first on the back to leave a bare area of 6 cm × 6 cm, and cutaneously cleaned by 8% Na2S 24 hours before exposure. The animals were anesthetized with 20% urethane by peritoneal injection before SM exposure, and the skin exposed to SM was covered with plastic wrap immediately after exposure. After six hours, the plastic wrap was 8

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removed and the residual SM was decontaminated. The urine samples were collected from individual animals every eight hours after exposure, the volume of each sample was ca. 5-50 mL and the exact volumes was recorded, samples were then stored in

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refrigerator at ca. -70 °C. 2.5. Sample preparation

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An aliquot of 1 mL frozen-thawed urine was spiked with 50 μL mixed ISs and

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extracted by SPE. The calibration samples were prepared in the similar way, i.e., 0.85 mL blank urine pooled from three animals unexposed, and spiked with 0.1 mL mixed

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standards solution and 50 μL mixed ISs. The SPE column was self-loaded with filling 1 g C18 packing material into a 6 mL cartridge with the bed depth of 13 mm. The

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packed C18 cartridge was firstly conditioned with 2 mL methanol and 4 mL water, and the urine or calibration sample was then passed through the conditioned cartridge

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without suction. After that the cartridge was washed with 3 mL water and 2 mL of

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20% methanol (v/v), and the retained solution was removed by vacuum. Four SM-

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DNA adducts were eluted by 2 mL of 20% methanol (v/v) in 5% formic acid (v/v) solution and 1.5 mL of 50% methanol (v/v) in 5% formic acid (v/v) solution within 10 min. The elutes were collected together and evaporated to dryness at 65 °C, and the residues were re-dissolved in 50 μL of 50% methanol (v/v). After short mixing, the

solutions were centrifuged at 14000 r·min-1 for 10 min, and the final reconstituted supernatants were transferred into sample vials and ready for analysis. 2.6. UPLC-MS/MS analysis All UPLC separations were performed in an ACQUITY UPLC system (Waters Co., Manchester, UK) consists of a binary pump, an autosampler, and an ACQUITY UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm, Waters Co.) with a gradient elution 9

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mode at 40 °C. Mobile phase A and B was water and methanol, respectively. The gradient elution started with 5% B (v/v), and increased linearly to 35% B (v/v) in 4 min. The flow rate was 0.35 mL·min-1, and the injection volume was 3 μL. The LC

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elute was delivered to a 5500 tandem mass spectrometer (AB Sciex, Framingham, MA, USA) except elutes in the first 0.5 min to avoid possible contamination on the

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turbo-ion spray source.

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In MS/MS analysis, the transitions of the analytes were selected based on their full scan mass spectra obtained from direct electrospray ionization-MS of standard

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solutions without LC separation. MS equipment was operated in a positive MS/MS mode at a unit resolution for both Q1 and Q3 using multiple reaction monitoring

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(MRM) with a dwell time of 100 ms for N7-HETEG, O6-HETEG, Bis-G, N3-HETEA, and ISs. The transition of precursor to product ion was monitored at m/z 256105 for

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N7-HETEG and O6-HETEG, m/z 389210 for Bis-G, m/z 240105 for N3-HETEA

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and m/z 260109 for d4-N7-HETEG and d4-O6-HETEG, m/z 393214 for d4-Bis-G, m/z 244109 for d4-N3-HETEA, respectively. The collision energy was set at 20, 20,

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49 and 21 eV, the declustering potential was 80, 80, 51, and 51 eV, and the collision cell exit potential was 16, 16, 14, 10 eV for N7-HETEG, O6-HETEG, Bis-G, N3-

HETEA and ISs, respectively. The source conditions were, source temperature, 550 C;

°

3.

pressure for gas 1 and gas 2, 50 kPa; and for curtain gas, 20 kPa. Results and Discussion

3.1. Optmization of sample pretreatment method It should be noted that the sample pretreatment method is absolutely necessary in this work, since the content of SM-DNAs in vivo is usually quite low at ng·L-1-μg·L-1 level, and the biomedical samples are so complicated to cause severe interference for 10

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the consequent UPLC-MS/MS analysis. Urine abounds in small molecules and large biomolecules, such as urea, uric acid, inorgnic salt, and small amount of glucose and protein [26], it needs special sample pretreatment method to extract and enrich the

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SM-DNA adducts, for example, SPE used in this work. The systematical optimization was performed due to different pKa, solubility, and other chemical properties of these

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four SM-DNA adducts. The factors included loading capacity, the component of

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elution solution, etc.

For a series of C18 packing with 0.2, 0.5 and 1 g weight, when 1 mL urine was

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loaded onto SPE cartridges, packing of 1 g with the bed depth of 13 mm was suitable, while others were overloading. In this SPE approach, C18 column was used to

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directly adsorb the analytes from urine sample with most hydrophobic interaction, and the matrix of urine was removed or cleaned by 3 mL water and consequent 2 mL of

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20% methanol (v/v). For the choice of elution solution, considering the pH of urine is

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ca. 8, and the pKa values of guanine and adenine are pKa,1 = 3.2-3.2, pKa,2 = 9.2-9.6 for guanine and pKa = 4.1 for adenine [27], the analytes should be transformed to

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ionic forms with low pH and then eluted. It was found that the extraction recovery was obviously improved with the addition of an acidic modifier, formic acid in the elution solution. When the concentration of formic acid was increased from 0 to 5% (v/v), the recovery was increased at least ca. 50%, and slightly decreased if the formic

acid concentration was further increased to 10% (v/v). Moreover, we thoroughly examined the parameter of methanol concentration while the elution was fixed at 3 times, and 2 mL per each time. As shown in Fig. 2a, N7-

HETEG and Bis-G can be easily eluted from C18 cartridge in the first time, while N3HETEA and O6-HETEG need one more elution. The stronger the elution solvents, and the highly extraction recovery is achieved. Elution of 2 mL each, three times with 11

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50% methanol (v/v), 5% formic acid (v/v) can provide best recovery of four SM-DNA adducts. However, higher concentration of methanol in three times elution will coelute more impurity into the UPLC-MS/MS analysis. One example is shown in Fig.

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2b, after three times elution with 50% methanol (v/v) (namely, route A), in the case of MRM chromatogram for N7-HETEG and O6-HETEG, an obvious unknown peak

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appeared near O6-HETEG and cannot fully separated. Thus we combined the elution solutions of different strength and reduced the elution time, and found the final

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adopted elution route (first 2 mL of 20% methanol (v/v) plus 5% formic acid (v/v),

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then 1.5 mL of 50% methanol (v/v) plus 5% formic acid (v/v), namely, route B) can give us a clean signal without apparent loss of recovery. In such an optimized SPE

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condition for 1 mL urine sample, the enrichment factor was 20. By using this SPE procedure, the complexity of the matrix and the resulted matrix effect were

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3.2. Method validation

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significantly reduced.

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Coupled with SPE, a UPLC-MS/MS method was performed to analyze urine samples collected from SM exposed rabbit model. The UPLC-MS/MS condition was adopted according to previous method [23], except a slight modification of gradient elution. The issues of method validation include selectivity, sensitivity, linear range, recovery, accuracy, precision, stability, and matrix effect [25]. The selectivity of this method was assessed by evaluating the unequivocal

separation and identification of analytes in the presence of urine matrix, which might contain interferences. We compared the UPLC-MS/MS chromatograms of blank rabbit urine from three rabbits unexposed to SM with rabbit urine samples, which were collected at 8 h after dermal exposure at 15 mg·kg-1 dose (Fig. 3), and found no 12

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significant interference from endogenous substances at the exact retention times of four SM-DNA adducts. Calibration curve was plotted by the peak area ratios of four SM-DNA adducts to

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the ISs vs. concentrations, and a good linear relationship was obtained over the ranges

from 0.005 (except Bis-G, 0.01) to 20 μg·L-1 in rabbit urine for four SM-DNA adducts

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(Fig. S1). The LLOD was 2 ng·L-1 for N7-HETEG, N3-HETEA, O6-HETEG and 5

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ng·L-1 for Bis-G, respectively, while the LLOQ was 5 ng·L-1 for N7-HETEG, O6HETEG, N3-HETEA, and 10 ng·L-1 for Bis-G in urine, respectively (Table 1).

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Precision, accuracy and recovery were measured at three QC concentrations, 0.01 (with regard to Bis-G, 0.02), 0.2, 20 μg·L-1 mixed SM-DNA adducts spiked in blank

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rabbit urine matrix. The mean extraction recoveries of four SM-DNAs were ranged from 87% to 116%, the mean value of accuracy was within 15%, and all inter-day and

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bioanalytical method.

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intra-day precisions were less than 15% (Table 2), showing good feasibility as a

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To meet the requirement of reliable measurement, the stability of four SM-DNA adducts was investigated from the aspects of freeze and thaw stability (three freeze/thaw cycles at -70 °C and twenty freeze/thaw cycles at -20 °C), short-term

temperature stability (in the mobile phase at 4 °C and 25 °C for 1, 2, 4, 8, 12, 24 h, respectively), long-term stability (-20 °C for 3 month, at -70 °C for 2 month), and stock

solution stability (25 °C for 24 h). All relative error (RE) values between post-storage and original QC samples were within 15%, demonstrating a good stability in use, stock, short-term and long-term storage. Matrix effect was evaluated by comparing the samples in matrix and in mobile phase at three QC concentrations. The values between matrix and mobile phase 13

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samples were ranged from 76% to 116% (Table 3). 3.3. Determination of four SM-DNA adducts in rabbit urine after dermal exposure

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With this established method with good validation, we measured the urine samples

collected from 8 h to 29 d after SM exposure by a dermal administration towards

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rabbits. The time and dose dependent responses were observed for four SM-DNA

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adducts. As shown in Fig. 4, once exposed to SM, the excretion of SM-DNA adducts in urine kept increasing during the initial 8-48 h until reached maximum at 2-4 d.

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Subsequently, the excretion of adducts decreased rapidly, only can be quantifiable at the LLOQ level within the time interval of 7 d to 13 d after exposure, but still

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detectable until 29 days. For the cases of middle and high dosages, both N7-HETEG and Bis-G, as two kinds of SM-DNA adducts with higher abundance, could be

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detected until ca. one month after exposure. This phenomenon is certainly related to

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exposure [28].

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the entire process of DNA damage, repair and excretion to urine in vivo after SM

Dose dependent effect was clearly demonstrated from the results of concentration

vs. dosage. All SM-DNA adducts turned out ascending trend along with the increasing doses, except O6-HETEG showed concentrations around LLOQ level, which cannot

be accurately determined due to the lowest content in vivo. According to the statistical

results, the abundance order of these four SM-DNA adducts in urine from highest to lowest was, N7-HETEG, Bis-G, N3-HETEA, and O6-HETEG. N7-HETEG occupied the most content of all four SM-DNAs (67.4%), almost three times of Bis-G (22.7%) and ca. seven times of N3-HETEA (9.8%), while O6-HETEG had the lowest content, i.e., just ca. 0.1% of all (Table 4). Comparing with previous results summarized in a 14

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review[17], in which human blood and calf thymus DNA were used as model in vitro, the abundances of Bis-G and N3-HETEA have the significant differences, the crosslinked adduct Bis-G is much higher while N3-HETEA is much lower. This elucidation

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is similar to that of one report of 2013 on human THP1 monocytes [22] and our former report on Sprague-Dawley rat derma [23]. The results provide high reliability

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and advantage of our method for its simultaneous determination of all four SM-DNA adducts, while the conclusion of previous review was deduced from a series of

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different articles, of which all the determinations were performed on individual or two

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SM-DNAs. The ratios of accumulation content for SM-DNA adducts in the earlier 13 days at three dose levels were about 0.05-0.14‰ for N7-HETEG, 0.02-0.06‰ for Bis-

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G, 0.003-0.03‰ for N3-HETEA, and 0.0001-0.0003‰ for O6-HETEG, respectively (Table 4).

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In general, these data indicated that the SM-DNA adducts in urine are suitable

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biomarkers for assessing SM exposure. Employing with this simultaneous quantification method with good sensitivity and selectivity, more reliable and

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informative results have been obtained from the urines of SM-exposed rabbit model. 4.

Conclusion

In this work, we developed an isotope-dilution UPLC-MS/MS method coupled

with SPE pretreatment for simultaneous quantification of four SM-DNA adducts in rabbit urine with a good sensitivity lower to 5-10 ng·L-1, and further applied to

investigate the time and dose dependent relationships in the rabbit model with dermal exposure to SM. The content of SM-DNA adducts had good correlation with time or doses. SM-DNA adducts can be detected even at 29 days later after SM exposure in the cases of middle and high dosages, and among all four SM-DNA adducts, N715

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HETEG was the most abundant, Bis-G and N3-HETEA were the less, while O6HETEG was the least. Given the similar LD50 values of rabbit and human, we suggested that this method has a good feasibility to human urines if exposed to SM,

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and expected the related results of SM-DNA adducts between rabbit and human have good correlations. Since SM-DNA adducts are the material basis and origination of

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SM toxicity, our results provided reliable evidence on better understanding of

metabolic process of adducts and DNA damage and repair towards SM exposure, and

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would be helpful for the related toxicology and clinical treatment.

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Acknowledgment

This work was supported by National Science & Technology Major Project of the

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Ministry of Science and Technology of China (Grant No.2012ZX09301003-001-010).

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References

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[2] M. Wattana, T. Bey, Mustard gas or sulfur mustard: an old chemical agent as a new terrorist threat, Prehospital. Disast. Med. 24 (2009) 19-29.

[3] K. Kehe, L. Szinicz, Medical aspects of sulphur mustard poisoning, Toxicology 214 (2005) 198209.

[4] Organisation for the Prohibit of Chemical Weapons, Decision: Detailed requirements for the destruction of Syrian chemical weapons and Syrian chemical weapons production facilities, http://www.opcw.org/index.php?eID=dam_frontend_push&docID=16875. Accessed 22 Jan 2014. [5] J. Riches, R.W. Read, R.M. Black, Analysis of the sulphur mustard metabolites thiodiglycol and thiodiglycol sulphoxide in urine using isotope-dilution gas chromatography-ion trap tandem mass spectrometry, J. Chromatogr. B 845 (2007) 114-120. [6] C. Li, J. Chen, Y. Zhong, Y. Zhong, J. Xie, H. Li, Simultaneous quantification of thioglycol and 16

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[8] J.R. Barr, C.L. Pierce, J.R. Smith, B.R. Capacio, A.R. Woolfitt, M.I. Solano, J.V. Wooten, S.W.

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Lemire, J.D. Thomas, D.H. Ash, D.L. Ashley, Analysis of urinary metabolites of sulfur mustard in

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two individuals after accidental exposure, J. Anal. Toxicol. 32 (2008) 10-16.

[9] D. Noort, A. Fidder, H.P. Benschop, L.P.A. de Jong, J.R. Smith, Procedure for monitoring

an

exposure to sulfur mustard based on modified Edman degradation of globin. J. Anal. Toxicol. 28 (2004) 311-315.

M

[10] D. Noort, A. Fidder, Sample collection, preparation and analytical methods, in: M. Mesilaakso (Ed.), Chemical weapons convention chemicals analysis, John Wiley & Sons, Ltd., New York,

d

2005, pp. 433-451.

te

[11] P.V. Lakshmana-Rao, R. Vijayaraghavan, A.S.B. Bhaskar, Sulphur mustard induced DNA damage in mice after dermal and inhalation exposure, Toxicology 139 (1999) 39-51.

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[12] P.A. Jowsey, F.M. Williams, P.G. Blain, DNA damage, signaling and repair after exposure of cells to the sulphur mustard analogue 2-chloroethyl ethyl sulphide, Toxicology 257 (2009) 105112.

[13] M. Kameswara-Rao, P.S. Bhadury, M. Sharma, R.S. Dangi, A.S. Bhaskar, S.K. Raza, D.K. Jaiswal, A facile methodology for the synthesis and detection of N7-guanine adduct of sulfur

mustard as a biomarker. Can. J. Chem. 80 (2002) 504-509.

[14] D.B. Ludlum, P. Austin-Ritchie, M. Hagopian, T. Niu, D. Yu, Detection of sulfur mustardinduced DNA modifications, Chem-biol. Interact. 91 (1994) 39-49. [15] D.B. Ludlum, S. Kent, Formation of O6-ethylthioethylguanine in DNA by reaction with the sulfur mustard, chloroethyl sulfide, and its apparent lack of repair by O6-alkylguanine-DNA alkyltransferase, Carcinogenesis 7 (1986) 1203-1206. 17

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[16] A. Fidder, G.W.H. Moes, A.G. Scheffer, G.P. van der Schans, R.A. Baan, L.P.A. de Jong, H.P. Benschop, Synthesis, characterization, and quantitation of the major adducts formed between sulfur mustard and DNA of calf thymus and human blood, Chem. Res. Toxicol. 7 (1994) 199-204.

induced cutaneous inflammation and blistering, Toxicology 263 (2009) 12-19.

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[17] K. Kehe, F. Balszuweit, D. Steinritz, H. Thiermann, Molecular toxicology of sulfur mustard-

[18] T.Q. Niu, Z. Matijasevic, P. Austin-Ritchie, A. Stering, D.B. Ludlum, A 32P-postlabeling method

cr

for the detection of adducts in the DNA of human fibroblasts exposed to sulfur mustard, Chem-

us

Biol. Interact. 100 (1996) 77-84.

[19] G.P. van der Schans, R. Mars-Groenendijk, L.P.A. de Jong, H.P. Benschop, D. Noort, Standard

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operating procedure for immunuslotblot assay for analysis of DNA/Sulfur Mustard Adducts in human blood and skin, J. Anal. Toxicol. 28 (2004) 316-319.

M

[20] A. Fidder, D. Noort, L.P.A. de Jong, H.P. Benschop, A.G. Hulst, N7-(2-hydroxyethylthioethyl)guanine: a novel urinary metabolite following exposure to sulphur mustard, Arch. Toxicol. 70

d

(1996) 854-855.

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[21] Y. Wei, L. Yue, Q. Liu, J. Chen, J. Xie, A sensitive high performance liquid chromatographypositive electrospray tandem mass spectrometry method for N7-[2-[(2-hydroxyethyl)thio]-

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ethyl]guanine determination, J. Chromatogr. B 879 (2011) 1707-1712. [22] M. Batalab, I. Boudryb, C. Cléry-Barraudb, S. Mouretb, T. Doukia, Relative yields of monomeric and dimeric adducts induced by sulphur mustard in isolated and cellular DNA as determined by HPLC/tandem mass spectrometry, Toxicol. Environ. Chem. 95 (2013) 260-276.

[23] L. Yue, Y. Wei, J. Chen, H. Shi, Q. Liu, Y. Zhang, J. He, L. Guo, T. Zhang, J. Xie, S. Peng, Abundance of four sulfur mustard-DNA adducts ex vivo and in vivo revealed by simultaneous quantification in stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry, Chem. Res. Toxicol. (2014) doi. 10.1021/tx4003403. [24] L.M. Robert, Mustard gas, in: T.C. Marrs, R.L. Maynard, F.R. Sidell (Eds.), Chemical warfare agents toxicology and treatment, John Wiley & Sons, Ltd., New York, 2007, pp. 375-407.

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[25] US Food and Drug Administration, Guidance for industry: bioanalytical method validation, http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidance/ucm36 8107.pdf. Accessed 22 Jan 2014. [26] P. Mikeša, M. Koříneka, I. Linhartb, J. Krouželkab, E. Frantíkc, L. Vodičkovác, L. Neufussovác,

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Excretion of urinary N7 guanine and N3 adenine DNA adducts in mice after inhalation of styrene, Toxicol. Lett. 184 (2009) 33-37.

cr

[27] V. Verdolino, R. Cammi, B.H. Munk, H.B. Schlegel, Calculation of pK values of nucleobases and

the guanine oxidation products guanidinohydantoin and spiroiminodihydantoin using density

us

functional theory and a polarizable continuum model, J. Phys. Chem. B 112 (2008) 16860-16873. [28] J.H.J. Hoeijmakers, Genome maintenance mechanisms for preventing cancer, Nature 411 (2001)

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te

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366-374.

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Page 19 of 29

Figures caption Figure 1 Chemical structures of four SM-DNA adducts. Figure 2 a The recovery of four SM-DNA adducts with different concentration of

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methanol in elution solvents. b The recovery of four SM-DNA adducts with

cr

different elution routes.

Route A is 2 mL of 50% methanol (v/v), 5% formic acid (v/v) for 3 times, and route B is 2 mL of

us

20% methanol (v/v), 5% formic acid (v/v), and followed with 1.5 mL 50% methanol (v/v) with 5% formic acid (v/v).

an

Figure 3 UPLC tandem MS analysis of blank urine samples and SM exposed urine

M

samples in MRM mode.

Fragmentation m/z 240→105 is used for the quantification of N3-HETEA (I), m/z 256→105 for

d

N7-HETEG and O6-HETEG (II), and m/z 389→210 for Bis-G (III) (b), and m/z 244→109 for d4-

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N3-HETEA, m/z 260→109 for d4-N7-HETEG, m/z 260→109 for d4-O6-HETEG, and m/z 393→214 for d4-Bis-G (c), respectively. Blank urine sample (a) was pooled from 3 rabbits

Ac ce p

unexposed, while SM exposed urine sample was collected from 8 h in rabbit after dermal intoxication at 15 mg kg-1 dose.

Figure 4 Time-dependent relationship of the four SM-DNA adducts after dermal exposure to SM at doses of 2.0 mg·kg-1 (a), 5.0 mg·kg-1 (b) and 15.0 mg·kg-1 (c), respectively.

21

Page 20 of 29

Highlights 1. We developed a simultaneous UPLC-MS/MS determination method combining with SPE.

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2. SM-DNAs status directly reveals DNA lesion extent for urine as excretion

cr

endpoint.

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3. We found significant dose and time dependent responses of four SM-DNA adducts.

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4. Accumulation abundance follows the order N7-HETEG, Bis-G, N3-HETEA, and

M

O6-HETEG.

Ac

ce pt

ed

5. Most abundant N7-HETEG still appeared in the end of a month at high dosages.

Page 21 of 29

CH2CH2SCH2CH2OH O

N

HN

H2 N

CH2CH2SCH2CH2OH

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O

te

d

M

an

us

cr

ip t

Figure

N

N

N7-HETEG

N

N H

N

H2 N

HN H2 N

N

CH2CH2SCH2CH2 N

N

N

N

Bis-G

N

CH2CH2SCH2CH2OH

N3-HETEA

O6-HETEG

O

N

N

N

N

NH2

O

N

NH2

Page 22 of 29

Figure

150

a 6

O -HETEG

3 N -HETEA 7

N -HETEG

d 3rd elution

s

us

2n

cr

ip t

Bi -G

an

50

0 20

30

40

50

20

30

40

M

1st

Recovery (%)

100

50

20

30

40

50

20

30

40

50

d

Concentration of methanol in elution solvents (%)

te

200

Route A

Intensity

Route A

Route B 0 0.0

0.5

1.0

1.5

2.0

2.5

Time (min)

2n

100

50000

d 3rd elution

Route B

O6-HETEG

N7-HETEG

100000

50

1st

Recovery (%)

150

Ac ce p

b

m/z 256>105

0 N7-HETEG

Bis-G

N3-HETEA

SM-DNA adduct

O6-HETEG

Page 23 of 29

Figure 6.0x10

6

a 6

2.0x10

ip t

Intensity

4.0x10

6

cr

III II

0.0

0.5

1.0

1.5

2.0

Time (min)

6.0x10

6

Bis-G

4

4

6.0x10

4

7 -HETEG

6

Intensity

N

M

3.0x10

4.0x10

2.5

an

9.0x10

b

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I 0.0

O6-HETEG

0.0

6

te

2.0x10

III

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II

2.0

2.2

2.4

2.6

2.8

0.0

Bis-G

N3-HETEA

I

0.0

3x10

1.8

d

1.6

0.5

1.0

1.5

2.0

2.5

Time (min)

5

c

N3-HETEA

d 4

Intensity

2x10

5

N7-HETEG

d 4

4

1x10

O6-HETEG

d -

Bis-G

d -

5

4

0 0.0

0.5

1.0

1.5 Time (min)

2.0

2.5

Page 24 of 29

Figure

a

6

0.5

4

0.0 5

10

2

0

5

10

15

25

30

us

0

Time after exposure (d)

7

an

N -HETEG Bis-G 3 N -HETEA 6 O -HETEG

b 0.05

M

12

0.00

6

5

10

0

0

Ac ce p

te

d

0

C

oncentration of SM-DNA adducts (ng/m L urine)

18

cr

ip t

0

C

oncentration of SM-DNA adducts (ng/m L urine)

7

N -HETEG Bis-G 3 N -HETEA 6 O -HETEG

5

10

15

25

30

Time after exposure (d)

C

oncentration of SM-DNA adducts (ng/m L urine)

180

7

N -HETEG Bis-G 3 N -HETEA 6 O -HETEG

5

c 150

60

0

0

5

10

15

25

30

0

0

5

10 Time after exposure (d)

15

25

30

Page 25 of 29

Tables

Table 1 Linearity and sensitivity of the method (n = 5)

Linear range (μg·L-1)

Calibration curve

r2

LLOD (μg·L-1)

LLOQ (μg·L-1)

N7-HETEG

0.005-20

Y= 2.05x+0.14

0.990

0.002

0.005 0.01

Bis-G

0.01-20

Y=6.94x+0.33

0.997

0.005

3

N -HETEA

0.005-20

Y=0.50x+0.01

0.991

0.002

6

0.005-20

Y=3.62x-0.01

0.999

0.002

0.005 0.005

Ac

ce pt

ed

M

an

us

cr

O -HETEG

ip t

Analyte

Page 26 of 29

Table 2 Inter-, intra-day precision and accuracy of the method

N3-HETEA

Intra-day (n=5)

(%)

RSD (%)

Accuracy (%)

RSD (%)

Accuracy (%)

0.01 0.2 20 0.02 0.2 20 0.01 0.2 20 0.01 0.2 20

104 ± 4 100 ± 9 107 ± 5 87 ± 6 90 ± 14 101 ±12 100 ± 9 88 ± 9 94 ± 11 116 ± 6 103 ± 5 111 ± 8

2.6 6.1 3.1 7.5 4.8 4.0 9.1 12.7 3.2 8.4 13.6 7.2

106 102 104 102 105 103 101 103 105 109 103 101

14.8 8.3 3.8 9.9 8.1 6.9 5.5 9.8 13.3 9.2 5.9 7.9

108 105 110 111 107 106 111 106 107 105 106 106

Ac

ce pt

ed

M

an

O6-HETEG

Inter-day (n=5)

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Bis-G

Recovery (n=3)

cr

N7-HETEG

Concentration (μg·L-1)

us

Analyte

Page 27 of 29

Table 3 Matrix effect of the method Matrix effect (%) (n=3) Analyte

Concentration (μg·L-1)

0.01 (0.02)

a

0.2

20 89 ± 0.4

116 ± 13

Bis-G

105 ± 12

87 ± 11

95 ± 3

N3-HETEA

110 ± 10

80 ± 3

89 ± 8

O6-HETEG

108 ± 10*

82 ± 9

88 ± 1

The matrix effect of Bis-G is calculated in the case of 0.02 μg·L-1.

Ac

ce pt

ed

M

an

us

cr

a

ip t

N -HETEG

76 ± 9

7

Page 28 of 29

Table 4 Percentage of SM-DNA adducts on SM exposed dose or total DNAs (n = 5)

Bis-G

3

N -HETEA

6

a

2.0

33.7

1204.7

0.014

5.0

79.1

2828.4

0.014

15.0

218.6

2955.4

0.005

2.0

33.7

322.3

0.002

5.0

79.1

2026.5

0.006

15.0

218.6

2890.6

0.003

2.0

33.7

273.1

0.003

5.0

79.1

580.3

0.003

15.0

218.6

151.0

0.0003

2.0

33.7

2.3

5.0

79.1

6.7

15.0

218.6

3.3

Percentage of each adduct on total adducts (%)

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Percentage of SM-DNA adducts on SM dose b (%)

67.4

22.7

9.8

0.00003 0.00003

0.141

0.00001

M

O -HETEG

Total excretion amount a (ng)

cr

N -HETEG

SM dose (μmol)

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7

SM dose (mg·kg-1)

an

Analyte

Total excretion amount was calculated by the concentration of SM-DNA adducts in urine multiplies the

b

ed

volume of excreted urine of rabbits.

Percentage of SM-DNA adducts on exposed SM dose were derived from the formula, P=m/M/N, where P is

ce pt

the ratio of SM-DNA adducts to exposed SM dose (%), m is the total excretion amount of adducts (ng), M is

Ac

the molar mass of adducts (g·mol-1), and N is the mole of exposed SM (μmol), respectively.

Page 29 of 29