Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of Bis(2-chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

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• 作者：Francesca Gruppi ; Leila Hejazi ; Plamen P. Christov ; Sesha Krishnamachari ; Robert J. Turesky ; Carmelo J. Rizzo
• 刊名：Chemical Research in Toxicology
• 出版年：2015
• 出版时间：September 21, 2015
• 年：2015
• 卷：28
• 期：9
• 页码：1850-1860
• 全文大小：481K
• ISSN：1520-5010

文摘

A robust, quantitative ultraperformance liquid chromatography ion trap multistage scanning mass spectrometric (UPLC/MS³) method was established to characterize and measure five guanine adducts formed by reaction of the chemotherapeutic nitrogen mustard (NM) bis(2-chloroethyl)ethylamine with calf thymus (CT) DNA. In addition to the known N7-guanine (NM鈥揋) adduct and its cross-link (G鈥揘M鈥揋), the ring-opened formamidopyrimidine (FapyG) monoadduct (NM鈥揊apyG) and cross-links in which one (FapyG鈥揘M鈥揋) or both (FapyG鈥揘M鈥揊apyG) guanines underwent ring-opening to FapyG units were identified. Authentic standards of all adducts were synthesized and characterized by NMR and mass spectrometry. These adducts were quantified in CT DNA treated with NM (1 渭M) as their deglycosylated bases. A two-stage neutral thermal hydrolysis was developed to mitigate the artifactual formation of ring-opened FapyG adducts involving hydrolysis of the cationic adduct at 37 掳C, followed by hydrolysis of the FapyG adducts at 95 掳C. The limit of quantification values ranged between 0.3 and 1.6 adducts per 10⁷ DNA bases when the equivalent of 5 渭g of DNA hydrolysate was assayed on column. The principal adduct formed was the G鈭扤M鈭扜 cross-link, followed by the NM鈭扜 monoadduct; the FapyG鈭扤M鈭扜 cross-link adduct; and the FapyG鈭扤M鈭扚apyG was below the limit of detection. The NM鈥揊apyG adducts were formed in CT DNA at a level 鈭?0% that of the NM鈥揋 adduct. NM鈥揊apyG has not been previously quanitified, and the FapyG鈥揘M鈥揋 and FapyG鈥揘M鈥揊apyG adducts have not been previously characterized. Our validated analytical method was then applied to measure DNA adduct formation in the MDA-MB-231 mammary tumor cell line exposed to NM (100 渭M) for 24 h. The major adduct formed was NM鈥揋 (970 adducts per 10⁷ bases), followed by G鈥揘M鈥揋 (240 adducts per 10⁷ bases), NM鈥揊apyG (180 adducts per 10⁷ bases), and, last, the FapyG鈥揘M鈥揋 cross-link adduct (6.0 adducts per 10⁷ bases). These lesions are expected to contribute to NM-mediated toxicity and genotoxicity in vivo.