Electrochemical detection and quantification of the acetylated and deacetylated C8-deoxyguanosine DNA adducts induced by 2-acetylaminofluorene.

The genotoxic agent 2-acetylaminofluorene induces, upon metabolic activation, two main types of DNA adducts in animal tissue, i.e., (deoxyguanine-8-yl)-aminofluorene (dG-C8-AF) and N-(deoxyguanine-8-yl)-acetylaminofluorene (dG-C8-AAF). Quantification of the frequency of these adducts usually relies on the use of radioactively labeled 2-acetylaminofluorene. Here, we report the development of a sensitive, non-radioactive method for the quantification of dG-C8-AF and dG-C8-AAF. Essentially, the modified DNA bases are separated by high-performance liquid chromatography (HPLC) and quantified by electrochemical detection. We established that both modified bases guanine-C8-aminofluorene and guanine-C8-acetylaminofluorene are electrochemically active. Subsequently, a procedure was developed to quantify dG-C8-AF and dG-C8-AAF in genomic DNA. Following DNA hydrolysis the adducted bases were extracted by ethyl acetate, separated by HPLC, and detected electrochemically. This procedure has been applied in the analysis of dG-C8-AAF in N-acetoxy-2-acetylaminofluorene-modified calf thymus DNA and in the detection of dG-C8-AAF and dG-C8-AF in liver DNA of mice injected intraperitoneally with 150-450 mg N-hydroxy-2-acetylaminofluorene/kg. The quantification of relatively low dG-C8-AF and dG-C8-AAF adduct levels (i.e., 0.1-1 adduct/10(6) nucleotides) in mouse liver DNA demonstrates the sensitivity of this electrochemical detection procedure. The detection limit of the method is 1 adduct per 10(6) nucleotides for both adducts using 20 micrograms of DNA and 4 adducts per 10(8) nucleotides using 500 micrograms DNA.