Characterization of nitrogen mustard formamidopyrimidine adduct formation of bis(2-chloroethyl)ethylamine with calf thymus DNA and a human mammary cancer cell line.
A robust, quantitative ultraperformance liquid chromatography ion trap multistage scanning mass spectrometric (UPLC/MS(3)) 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-G) adduct and its cross-link (G-NM-G), the ring-opened formamidopyrimidine (FapyG) monoadduct (NM-FapyG) and cross-links in which one (FapyG-NM-G) or both (FapyG-NM-FapyG) 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 μM) 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 °C, followed by hydrolysis of the FapyG adducts at 95 °C. The limit of quantification values ranged between 0.3 and 1.6 adducts per 10(7) DNA bases when the equivalent of 5 μg of DNA hydrolysate was assayed on column. The principal adduct formed was the G-NM-G cross-link, followed by the NM-G monoadduct; the FapyG-NM-G cross-link adduct; and the FapyG-NM-FapyG was below the limit of detection. The NM-FapyG adducts were formed in CT DNA at a level ∼20% that of the NM-G adduct. NM-FapyG has not been previously quanitified, and the FapyG-NM-G and FapyG-NM-FapyG 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 μM) for 24 h. The major adduct formed was NM-G (970 adducts per 10(7) bases), followed by G-NM-G (240 adducts per 10(7) bases), NM-FapyG (180 adducts per 10(7) bases), and, last, the FapyG-NM-G cross-link adduct (6.0 adducts per 10(7) bases). These lesions are expected to contribute to NM-mediated toxicity and genotoxicity in vivo.
Departments of Chemistry and Biochemistry, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville, Tennessee 37235, United States.
Francesca
F
Gruppi
Masonic Cancer Center and Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota , 2231 Sixth Street South East, Minneapolis, Minnesota 55455, United States.
Leila
L
Hejazi
Departments of Chemistry and Biochemistry, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville, Tennessee 37235, United States.
Plamen P
PP
Christov
Masonic Cancer Center and Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota , 2231 Sixth Street South East, Minneapolis, Minnesota 55455, United States.
Sesha
S
Krishnamachari
Masonic Cancer Center and Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota , 2231 Sixth Street South East, Minneapolis, Minnesota 55455, United States.
Robert J
RJ
Turesky
Departments of Chemistry and Biochemistry, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville, Tennessee 37235, United States.
Carmelo J
CJ
Rizzo
D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D004247:DNA / Q000187:drug effects*; D006801:Humans; D008466:Mechlorethamine / Q000737:chemistry*; D009588:Nitrogen Mustard Compounds / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D013950:Thymus Gland / Q000187:drug effects* / Q000378:metabolism
D009588:Nitrogen Mustard Compounds; D011743:Pyrimidines; D008466:Mechlorethamine; C121482:bis(2-chloroethyl)ethylamine; D004247:DNA; C030986:pyrimidine