Article pubs.acs.org/crt
Synthesis, Characterization, and Identification of New in Vitro Covalent DNA Adducts of Divinyl Sulfone, an Oxidative Metabolite of Sulfur Mustard Shanshan Lv,§ Yajiao Zhang,§ Bin Xu, Hua Xu, Yumei Zhao, Jia Chen, Zhongcai Gao, Jianfeng Wu,* and Jianwei Xie* State Key Laboratory of Toxicology and Medical Countermeasures and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, Beijing 100850, China S Supporting Information *
ABSTRACT: Divinyl sulfone (DVS) is an important oxidative metabolic product of sulfur mustard (SM) in vitro and in vivo. Although DVS is not a classical blister agent, its high reactivity and toxicity induced by vinyl groups can also cause blisters like SM upon contact with the skin, eyes, and respiratory organs. The purpose of this paper was to identify whether DVS could covalently bind to DNA bases to form new DNA adducts in cells in vitro. A series of adducts were synthesized and characterized using purine, nucleoside, or DNA, separately, as starting materials. The covalent site, pattern, and relative reactivity of adduct formation were identified and discussed in detail. The results showed that five high abundance site-specific DNA adducts, including two monoadducts and three cross-linked adducts, were obtained when DNA was used as a substrate. When HaCaT cells were exposed to 30 μM of DVS, four new DNA adducts containing monoadducts and cross-linked adducts were found and identified in cells, including N3-A monoadduct, N7-G monoadduct, N7G-N7G bis-adduct, and N3A-N7G cross-linked adduct. Among them, the abundance of N3-A monoadduct was 10 times higher than that of the other three adducts. DNA adduct formation with DVS showed significant differences from that observed with SM. The observation of these new DNA adduct in vitro cells revealed that DNA damage could be also induced by DVS.
1. INTRODUCTION Sulfur mustard (SM) is a banned chemical warfare agent (CWA) that is controlled by the Organization for the Prohibition of Chemical Weapons (OPCW). SM is one of the most hazardous CWAs and can induce severe injury to the skin, eyes, respiratory system, etc. SM still poses a threat to public safety due to its facile synthesis, hypertoxicity, and recent use as CWA. In addition, SM could undergo many complicated metabolic processes after entering into the body. Our preliminary results showed that most SM in vivo would be oxidized to mustard sulfoxide (SMO). SMO can be further oxidized to mustard sulfone (SMO2).1,2 SMO2 is extremely unstable and is spontaneously converted into DVS via a twostep elimination reaction under alkaline conditions.3 More importantly, SMO2 and DVS have been demonstrated to retain a vesicant property similar to SM, which can cause respiratory and allergic reactions. The vesicant effect of DVS may be attributed to its high reactive activity from vinyl groups that can © 2017 American Chemical Society
participate as electrophiles in a nucleophilic Michael addition reaction.4,5 However, it seems certain that DVS is even more toxic than SM or SMO2. In previous publications, results showed that DVS has an IC50 (the concentration at which cell viability was inhibited by 50%) of 34 μM,6 which is lower than that of SM (100 μM).7 The oral LD50 value for DVS of 32 mg/ kg in rats is half that of SM.8,9 Moreover, rats that were subcutaneously exposed to SMO2 show more toxicity than those that were intravenously exposed. The reasons may be attributed to the transformation of SMO2 into the more toxic DVS in body fluids after subcutaneous injection.10 DVS is a reactive divinyl compound that can irreversibly bind to various types of compounds, including functional proteins, amino acids, and glutathione (GSH), all of which have lone pair electrons in their molecular structures.6,11−17 Among them, Received: July 16, 2017 Published: September 29, 2017 1874
DOI: 10.1021/acs.chemrestox.7b00196 Chem. Res. Toxicol. 2017, 30, 1874−1882
Article
Chemical Research in Toxicology GSH depletion or oxidative stress may severely reduce the protecting capabilities of the cell and lead to an accumulation of DVS, which subsequently might evoke cellular reactions.3 Several cellular effects of DVS have been reported. For instance, DVS could inhibit the degradation of proteins targeted to the proteasome and/or stimulate large-scale protein aggregation within cells owing to its cross-linking characteristics. 6 Furthermore, previous reports have evaluated the mutagenicity of DVS for its ability to undergo a Michael reaction with a nucleophilic molecule. The results indicated that genotoxicity tests with DVS were positive for mutagenesis in mouse lymphoma cells.18 However, the toxicological mechanism of DVS on DNA is still not very clear. To the best of our knowledge, there is still no report on the genetic toxicology of DVS. Consequently, it is necessary to carry out a detailed investigation of the DNA damage of DVS, especially the DVSDNA adducts. In this study, we designed and optimized synthetic routes for the preparation of a series of new DVS-DNA adducts. The structure, exact addition site, and purity of the adducts were characterized using HPLC-MS and NMR. Furthermore, a highly sensitive LC-MS/MS method was developed to characterize DNA adducts based on the synthesized DNA adducts. Lastly, a series of new covalent DNA-adducts in the cells exposed to DVS were identified and investigated in detail. The results showed that DVS can form five types of high abundance site-specific adducts with salmon sperm DNA in vitro. However, only four of the five DVS-DNA adducts could be identified from HaCaT cells exposed to DVS, and N3HESVA was identified as the major product. DNA adducts are important biomarkers of genotoxicity. The identification of a series of DVS-DNA adducts in cells demonstrated the DNA damage originating from DVS, which is an important oxidative metabolite of SM in vivo. Therefore, the results will play an important role in clarifying the damage mechanism of SM.
Figure 1. Chemical structures of the five DVS-DNA adducts, including DVS-A, DVS-G, A-DVS-A, G-DVS-G, and A-DVS-G.
Table 1. MS/MS Parameters for Five DVS-DNA Adducts DP (V)
CE(V)
CXP(V)
EP (V)
N3-HESVA
type of adduct
80
16
10
N7-HESVG
80
16
10
N7-GHESEHG-N7 N3-AHESEHA-N3
80 80
16 16
10 10
N3-AHESEHG-N7
80
35 27 30 28 34 25 45 25 27
16
10
transition 254 254 270 270 421 389 389 405 405
→ → → → → → → → →
135 162 151 178 270 254 136 254 270
obtained white solid was directly used in the next reaction without further purification. 2.3. Synthesis of Mustard Sulfone. The mustard sulfone (SMO2) was synthesized as by previously reported method.16 SMO (3 g, 17.1 mM) was dissolved in 5 mL of acetonitrile and added dropwise to a stirred mixture of glacial acetic acid (5 mL) and hydrogen peroxide (30%, 10 mL), which was stirred at room temperature for 4 h until the SMO disappeared (monitored by GCMS). The reaction solution was extracted with dichloromethane three times (3 × 20 mL), and the organic solution was washed (2 × 20 mL) with deionized water, dried with anhydrous Na2SO4, then evaporated to dryness, yielding crude SMO2 without further purification. 2.4. Synthesis of Divinyl Sulfone. The crude SMO2 solid was dissolved using a proper amount of acetonitrile and then added dropwise into an aqueous solution (3%, weight %). The mixture was refluxed for 4 h. The progress of the reactions was monitored by GC/ MS analysis until SMO2 reacted almost completely. Then, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure. The residue was dissolved in CH2Cl2 (10 mL) and washed twice with deionized water (2 × 10 mL). The organic phase was dried with anhydrous NaSO4 and then the solvent was evaporated, and 0.82 g of colorless liquid was obtained (yield was 79%). Product was characterized using 1H NMR combined with 13C NMR (Figures S2 and S3) and the purity was more than 98% according to GC-MS analysis (Figure S4). 2.5. Reaction of DVS with Adenine or Guanine. DVS (112 mg, 1.12 mmol) was mixed with adenine (7.5 mg, 0.056 mmol) or guanine (8.4 mg) in 8 mL of 50 mM phosphate buffer solutions at 37 °C and pH 7.4 for 12 h. The progress of the reactions was monitored by
2. MATERIAL AND METHODS Caution: SM and DVS are all highly reactive alkylating vesicant and cytotoxic agents. These agents should be handled only in well-ventilated f ume hoods. The use of gloves and stringent protective measures should be adopted. 2.1. Chemicals and Reagents. Salmon sperm DNA was purchased from Sagan Corporation (Shanghai, China). Guanosine, adenosine, guanine, and adenine were all obtained from Innochem Co., Ltd. (Beijing, China). SM with purity higher than 95% was provided by the Institute of Chemical Defense (China). SMO2 and DVS were synthesized in our laboratory. HPLC grade methanol and acetonitrile were purchased from Dunsan Pure Chem. Co., Ltd. (Ansan-si, Korea). Analytical grade formic acid was obtained from Sinopharm Chemical Reagents Co., Ltd. (Beijing, China). Phenol was supplied by HaoYang Biological Co., Ltd. (Tianjin, China). All other reagents were of analytical grade and were purchased from Beijing Chemical Works and Sinopharm Chemical Reagents Co. Ltd. (Beijing, China). Water was obtained from a Milli-Q Academic water purification system from Millipore (Billerica, MA, USA). 2.2. Synthesis of Mustard Sulfoxide. Mustard sulfoxide (SMO) was prepared as in Figure S1.19 SM (5 g, 31 mM) was first dissolved in acetonitrile (3 mL) and then added dropwise into HNO3 (10 mL) in an ice bath for 30 min. The mixture solution was stirred at 0−5 °C for 4 h and allowed to warm to room temperature for an additional 2 h until SM disappeared (monitored by GC-MS). The crude product was extracted by CH2Cl2 three times (3 × 20 mL), and the organic phase was washed with distilled water (2 × 20 mL). The organic phase was dried with anhydrous Na2SO4 and evaporated to dryness, and the 1875
DOI: 10.1021/acs.chemrestox.7b00196 Chem. Res. Toxicol. 2017, 30, 1874−1882
Article
Chemical Research in Toxicology
N7-GHESEHG-N7. ESI+-MS/MS, m/z 421 [M + H]+, m/z 389 [M + H − 2O]+, m/z 270 [M + H − Guo]+, m/z 211 [Guo + CH2CH2S]+, m/z 151 [Guo]+. lH NMR (DMSO-d6, 400 MHz) δ 8.21 (s, 2H, −CH), 4.46(s, 4H, −NH2), 3.78 (m, 4H, −CH2). HRMS (Q-TOF), obsd m/z 421.1154, calcd for C14H16N10O4S, m/z 421.1155 [M + H]+. Retention time is 4.0 min. N3-AHESEHG-N7. ESI+-MS/MS, m/z 405 [M + H]+, m/z 203 [Guo + CH2CH2 + Na]2+. lH NMR (DMSO-d6, 400 MHz) δ 8.18, (s, 2H, -NH2), 8.11 (s, 2H, −NH2), 4.46 (m, 2H, −CH), 3.97 (m, 2H, −CH), 3.76 (s, 2H, −CH), 3.52 (m, 2H, −CH2). HRMS (Q-TOF), obsd m/z 405.1165, calcd for C14H16N10O3S, m/z 405.1206 [M + H]+. Retention time is 4.5 min. 2.8. Reaction of DVS with dsDNA. Incubations of DVS with salmon sperm DNA were performed in a final volume of 5 mL containing 112 mg of DVS, 10 mg salmon sperm DNA, and 50 mM phosphate buffer at pH 7.4. Reactions were performed at 55 °C for 24 h in the water bath. The unreacted DVS was removed by repetitive extraction with cold anhydrous ethanol (2.5 volumes) and washed three times with 70% ethanol (v/v). Then, the DNA pellet was precipitated from the aqueous layer and subsequently hydrolyzed by 15% formic acid at 75 °C for 30 min. The supernatant was diluted and subjected to the analysis by HPLC-QTOF/MS. 2.9. Cell Culture. The human keratinocyte cell line HaCaT was provided by the Shanghai Cell Bank of the Chinese Academy of Sciences and all maintained in a standard culture medium. HaCaT cells were maintained in complete DMEM culture medium with 4.5 g/ L glucose supplemented with 10% FBS (Gibco, NY, USA). All cultures were maintained at 37 °C in a humidified incubator in 5% CO2. HaCaT cells were individually plated at 3 × 105 cells per 10 cm dish and incubated overnight. DVS was initially diluted with DMSO and subsequently diluted to 30 μM with culture medium for cell exposure, in which the final concentration of DMSO was strictly restricted to
