Detection of DNA adducts by high-performance liquid chromatography with electrochemical detection

Abstract

A method for the detection of rare adducts in DNA has been developed by combining the resolution of high-performance liquid chromatography with the specificity and sensitivity of electrochemical detection. Many adducts are electrochemically active, while the normal bases, except for guanine, are not. Enzymatic hydrolysis of DNA is used to obtain the deoxynucleosides for analysis, or where appropriate, acid hydrolysis or thermal depurination of DNA is used to free the adduct base for analysis. Various types of DNA damage have been induced by in vitro exposure of DNA to acrolein, dimethyl sulfate, sodium nitrite, ascorbate/Cu2+ and gamma-irradiation. Several adducts are detected at a level of one adduct in 10(5)-10(6) normal bases in micrograms of DNA. The method is also useful for measuring O6-methylguanine (O6MeGua) in DNA from rats treated with N-nitrosodimethylamine and 8-hydroxydeoxyguanosine (oh8dG), and O6-MeGua in DNA from bacteria treated with hydrogen peroxide and dimethyl sulfate. oh8dG, which appears to be the most suitable marker for measuring the steady-state level of oxidative DNA damage, can be measured at fmol levels in DNA from normal rat tissues. The method is applicable to the analysis of DNA base damage caused by major endogenous processes relevant to aging, such as deamination, methylation and oxidation. The analysis of DNA adducts with this simple assay also may be potentially useful for studies on carcinogenesis and as a tool in studies on the epidemiology of cancer.