Quantitative determination of N7-methyldeoxyguanosine and O6-methyldeoxyguanosine in DNA by LC-UV-MS-MS.
The N-7 and O-6 positions of 2'-deoxyguanosine are the predominant sites of methylation by N-methyl-N-nitrosourea (MNU), which is used to produce a variety of experimental cancers in animal models. Here we report the development of a highly sensitive quantitative assay based on high-performance liquid chromatography-UV-tandem mass spectrometry (LC-UV-MS-MS) to measure N7-methyl-2'-deoxyguanosine (N7-MedG) and O6-methyl-2'-deoxyguanosine (O6-MedG) in DNA hydrolysates. Since this assay was selective for deoxyribonucleosides, potential interference from methylated RNA was eliminated. Isotopically labeled analogues, [2H3]N7-MedG and [2H3]O6-MedG, were synthesized and added to the DNA hydrolysates as internal standards. In-line UV absorbance detection was used for the quantitative analysis of the native deoxyribonucleoside dG, and MS-MS was used for the determination of N7-MedG and O6-MedG. The limits of detection for N7-MedG and O6-MedG were determined to be 64 and 43 fmol, respectively. The limits of quantification were 0.13 pmol for N7-MedG and 0.085 pmol for O6-MedG. The stabilities of N7-MedG and O6-MedG were also investigated. Although O6-MedG was stable at room temperature for at least 11 days, the half-life of N7-MedG at room temperature was 2 days. Both adducts were stable at -20 degrees C. Calf thymus DNA and DNA from the livers of MNU-treated Sprague-Dawley rats were assayed using LC-UV-MS-MS, which was optimized for speed as well as for sensitivity. The levels of N7-MedG and 06-MedG in calf thymus DNA increased with MNU concentration and incubation time. The levels of N7-MedG and O6-MedG in the rat livers 2 h after treatment with a single dose of 50 mg/kg MNU were 95.2 N7-MedG/105 dG and 14.8 O6-MedG/105 dG. This LC-UV-MS-MS assay provides the sensitivity and speed required to evaluate the extent of methylated DNA lesions in animal models of cancer induced by the methylating agent MNU.
Yang
Yanan
Y
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 60612, USA.
Nikolic
Dejan
D
Swanson
Steven M
SM
van Breemen
Richard B
RB
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D004247:DNA / Q000032:analysis*; D018736:DNA Adducts / Q000032:analysis; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis*; D006868:Hydrolysis; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry; D013058:Mass Spectrometry; D008770:Methylnitrosourea / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013056:Spectrophotometry, Ultraviolet
D018736:DNA Adducts; D007202:Indicators and Reagents; C048746:7-methyl-2-deoxyguanosine; D008770:Methylnitrosourea; D004247:DNA; C015558:O(6)-methyl-2'-deoxyguanosine; D003849:Deoxyguanosine