A sample preparation protocol for quantification of radiolabeled nucleoside incorporation into DNA by accelerator mass spectrometry

A general protocol is described for measuring the incorporation of radiocarbon-labeled 2′-deoxynucleosides into DNA using accelerator mass spectrometry (AMS). This technology provides attomole (10^-18 mol) sensitivity, with detection limits for DNA analysis in the range of one ¹⁴C atom per 10¹¹-10¹² total carbons. In practice this corresponds to approximately 1 labeled nucleoside per 10¹¹ normal bases. A key aspect of the method is the use of precautions aimed at prevention of artifactual DNA oxidation during the sample preparation by the use of antioxidants and chaotropic salts during the DNA isolation. In principle, any type of appropriately labeled nucleoside derivative can be studied using the described protocol, provided that there is incorporation of the deoxynucleoside into DNA. We demonstrated this protocol using MCF-7 human breast cancer cells and a mouse model for mammary carcinoma, which we dosed with ¹⁴C-labeled 2′-deoxyguanosine (dG) and ¹⁴C-labeled 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). The nucleoside 8-oxodG is a ubiquitous compound that forms in cells by the reaction of dG with reactive oxygen species which has been associated with numerous disease, carcinogenesis and aging. DNA from cells treated with ¹⁴C-labeled nucleosides was isolated and prepared for analysis by AMS in order to measure the DNA-bound radioactivity. The method allows the generation of reliable and sufficient yields of pure DNA from human cells and animal tissues for analysis of radiocarbon levels. Ultimately, this protocol will be applied to understanding the role of modified nucleoside incorporation into DNA in cancer initiation and progression, but could also be used to study any DNA metabolism process where ¹⁴C-labeled nucleosides are used.