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.
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.
State Key Laboratory of Antitoxic Drugs and Toxicology, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, No. 27 Taiping Road, Haidian District 100850, Beijing, China.
Lijun
L
Yue
Yuxia
Y
Wei
Jia
J
Chen
Huiqin
H
Shi
Qin
Q
Liu
Yajiao
Y
Zhang
Jun
J
He
Lei
L
Guo
Tingfen
T
Zhang
Jianwei
J
Xie
Shuangqing
S
Peng
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000187:drug effects*; D018736:DNA Adducts / Q000032:analysis*; D007554:Isotopes; D057230:Limit of Detection; D008297:Male; D009151:Mustard Gas / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry / Q000379:methods*
D018736:DNA Adducts; D007554:Isotopes; D004247:DNA; D009151:Mustard Gas