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

Biochem J. 2007 Mar 15;402(3):483-90. doi: 10.1042/BJ20061447.

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 (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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylation
  • Animals
  • Automation
  • Chromatography, Liquid / methods*
  • Cyprinodontiformes*
  • DNA / analysis*
  • DNA / chemistry*
  • Ethylenes / chemistry
  • Guanine / analysis*
  • Guanine / chemistry*
  • Indicator Dilution Techniques
  • Isotopes
  • Liver / metabolism
  • Liver Extracts / metabolism
  • Models, Animal
  • Tandem Mass Spectrometry / methods*

Substances

  • Ethylenes
  • Isotopes
  • Liver Extracts
  • Guanine
  • DNA
  • ethylene