Simultaneous determination of N7-alkylguanines in DNA by isotope-dilution LC-tandem MS coupled with automated solid-phase extraction and its application to a small fish model.
In the present study, we report the development of a sensitive and selective assay based on LC (liquid chromatography)-MS/MS (tandem MS) to simultaneously measure N7-MeG (N7-methylguanine) and N7-EtG (N7-ethylguanine) in DNA hydrolysates. With the use of isotope internal standards (15N5-N7-MeG and 15N5-N7-EtG) and on-line SPE (solid-phase extraction), the detection limit of this method was estimated as 0.42 fmol and 0.17 fmol for N7-MeG and N7-EtG respectively. The high sensitivity achieved here makes this method applicable to small experimental animals. This method was applied to measure N7-alkylguanines in liver DNA from mosquito fish (Gambusia affinis) that were exposed to NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) alone or their combination over a wide range of concentrations (1-100 mg/l). Results showed that the background level of N7-MeG in liver of control fish was 7.89+/-1.38 mmol/mol of guanine, while N7-EtG was detectable in most of the control fish with a range of 0.05-0.19 mmol/mol of guanine. N7-MeG and N7-EtG were significantly induced by NDMA and NDEA respectively, at a concentration as low as 1 mg/l and increased in a dose-dependent manner. Taken together, this LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of alkylated DNA lesions in small animal models of cancer induced by alkylating agents.
Chao
Mu-Rong
MR
Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan. mrchao@csmu.edu.tw
Wang
Chien-Jen
CJ
Yen
Cheng-Chieh
CC
Yang
Hsi-Hsien
HH
Lu
Yao-Cheng
YC
Chang
Louis W
LW
Hu
Chiung-Wen
CW
D000478:Alkylation; D000818:Animals; D001331:Automation; D002853:Chromatography, Liquid / Q000379:methods*; D003532:Cyprinodontiformes; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D005030:Ethylenes / Q000737:chemistry; D006147:Guanine / Q000032:analysis* / Q000737:chemistry*; D007201:Indicator Dilution Techniques; D007554:Isotopes; D008099:Liver / Q000378:metabolism; D008110:Liver Extracts / Q000378:metabolism; D023421:Models, Animal; D053719:Tandem Mass Spectrometry / Q000379:methods*
D005030:Ethylenes; D007554:Isotopes; D008110:Liver Extracts; D006147:Guanine; D004247:DNA; C036216:ethylene