Analysis of DNA adducts of acetaldehyde by liquid chromatography–mass spectrometry

Abstract

A highly sensitive method using liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) was developed for the analysis of DNA adducts of acetaldehyde (AA). AA, which is the primary oxidative metabolite of ethanol, is considered to possess carcinogenic activity. AA reacts with the exocyclic amino group of guanine in DNA to form N 2-ethylguanine (Et-Gua) and 1, N 2-propanoguanine (Pr-Gua) adducts. With the present method, such adducts were detected as the base forms from DNA chains using depurination in the pretreatment process. In our measurement with LC–ESI-MS, the limits of detection (LODs) of the Et-Gua and Pr-Gua adducts of the base forms were 3.0·10 −10 and 1.0·10 −9 M, respectively, and the LODs are about two orders of magnitude lower than those of the nucleoside forms. Calf thymus DNA samples treated with AA and NaBH 3CN were analyzed by this method. Et-Gua was clearly detected and, in the absence of NaBH 3CN, Pr-Gua was detected predominantly. Furthermore, the method was also applied to study whether or not these two adducts are formed in DNA of cultured HL-60 cells during exposure to AA for 24 h. Pr-Gua was clearly detected and traces of Et-Gua were also detected in the DNA of the cells. Although the sensitivity of this method is lower by at least one order of magnitude than the 32P-postlabeling assay, currently the most sensitive method, our method does not involve complex enzymatic reactions for the postlabeling and the use of troublesome radioactive materials. Furthermore, it enables structural identification of guanine adducts. The present method would be a useful tool for studies of Et-Gua and Pr-Gua adducts in connection with carcinogenesis.