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

Abstract

Sulfur mustard (SM) is a highly reactive alkylating vesicant and causes blisters upon contact with skin, eyes, and respiratory organs. It covalently links with DNAs by forming four mono- or cross-link adducts. In this article, the reference standards of SM-DNA adducts and deuterated analogues were first synthesized with simplified procedures containing only one or two steps and using less toxic chemical 2-(2-chloroethylthio)ethanol or nontoxic chemical thiodiglycol as starting materials. A sensitive and high-throughput simultaneous quantification method of N(7)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N(7)-HETEG), O(6)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (O(6)-HETEG), N(3)-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N(3)-HETEA), and bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G) in the Sprague-Dawley rat derma samples was developed by stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) with the aim of revealing the real metabolic behaviors of four adducts. The method was validated, the limit of detection (S/N ratio greater than 10) was 0.01, 0.002, 0.04, and 0.11 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively, and the lower limit of quantification (S/N ratio greater than 20) was 0.04, 0.01, 0.12, and 0.33 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively. The accuracy of this method was determined to be 76% to 129% (n = 3), and both the interday (n = 6) and intraday (n = 7) precisions were less than 10%. The method was further applied for the quantifications of four adducts in the derma of adult male Sprague-Dawley rats exposed to SM ex vivo and in vivo, and all adducts had time- and dose-effect relationships. To the best of our knowledge, this is the first time that the real presented status of four DNA adducts was simultaneously revealed by the MS-based method, in which Bis-G showed much higher abundance than the result previously reported and N(3)-HETEA showed much less. It should be noted that since the interstrand cross-linked adduct is believed to stall DNA replication and finally induce a double-strand break, the higher abundance of Bis-G is a great indication of a more serious DNA lesion by SM alkylation.

Distribution of DNA Adducts and Corresponding Tissue Damage of Sprague–Dawley Rats with Percutaneous Exposure to Sulfur Mustard

“…The animal experiments were conducted in the Beijing Center for Toxicological Evaluation and Research, in accordance with the protocol approved by the Institutional Animal Care and Use Committee of the Center, which is in compliance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAA-LAC). Experiment 1: The animals described in the previous paper were treated carefully, 11 and the body weights, tissue coefficient and histopathologic examination were monitored. Liver, kidney, lung, spleen, skin, and blood samples were collected at the first, second, fourth, seventh, and 14th day after exposure, respectively.…”

“…ID-UPLC-MS/MS Analysis. The analysis of DNA adducts of SM was performed as previously described 11 with minor modification. The UPLC-MS/MS analyses were carried out in a 5500 UPLC-MS/MS instrument (AB SCIEX, Framingham, MA, USA) with a Waters ACQUITY UPLC BEH C 18 column (2.1 mm ID × 50 mm, 1.7 μm, Waters Co., MA, USA).…”

Analysis of Different Fates of DNA Adducts in Adipocytes Post-sulfur Mustard Exposure in Vitro and in Vivo Using a Simultaneous UPLC-MS/MS Quantification Method

“…Standards (N 7 -HETEG and Bis-G, N 3 -HETEA) and deuterated internal standards (ISs) (d 4 -N 7 -HETEG, d 4 -Bis-G, d 4 -N 3 -HETEA) were synthesized with purity above 95%, and deuterated ratios were more than 99%. 12 purchased from Solar Bio S&T Co., Ltd. (Beijing, China). Ultrapure water was produced by a Milli-Q A10 purification system from EMD Millipore Co. (MA, USA).…”

Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences

“…In addition, bi functional mustards, such as SM, HN2, or chlorambucil form biadducts between two guanine N7 positions to yield bis(N7guanine ethyl) sulfide (N7 Gua ETE N7 Gua) DNA crosslinks. Quantitative analysis estimated the relative distribution of these adducts to be $60 88% dG monoadducts, $10 42 % dG crosslinks, and $1 10% dA monoadducts depending on the methods used, the target material/tissue exposed, and the dose applied (Batal et al, 2013;Fidder et al, 1994;Yue et al, 2014). Crosslinks can be detected as early as 30 min after exposure in human lymphocytes (Debiak et al, 2011) and persist in SM exposed mouse tissue for up to several weeks (Batal et al, 2013).…”