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

Abstract

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 (N 7-ethylguanine) in DNA hydrolysates. With the use of isotope internal standards (15N5-N7-MeG and 15N 5-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 μmol/mol of guanine, while N7-EtG was detectable in most of the control fish with a range of 0.05-0.19 μmol/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 through-put required to evaluate the extent of alkylated DNA lesions in small animal models of cancer induced by alkylating agents. © 2007 Biochemical Society.