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 acti vation, two main types of DNA adducts in animal tissue, i.e., (deoxygu anine-8-yl)-aminofluorene (dG-C8-AF) and N-(deoxyguanine-8-yl)-acetyla minofluorene (dG-C8-AAF). Quantification of the frequency of these add ucts usually relies on the use of radioactively labeled 2-acetylaminof luorene. Here, we report the development of a sensitive, non-radioacti ve method for the quantification of dG-C8-AF and dG-C8-AAF, Essentiall y, the modified DNA bases are separated by high-performance liquid chr omatography (HPLC) and quantified by electrochemical detection. We est ablished 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 eth yl acetate, separated by HPLC, and detected electrochemically, This pr ocedure has been applied in the analysis of dG-C8-AAF in N-acetoxy-2-a cetylaminofluorene-modified calf thymus DNA and in the detection of dG -C8-AAF and dG-C8-AF in liver DNA of mice injected intraperitoneally w ith 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 ad duct/10(6) nucleotides) in mouse liver DNA demonstrates the sensitivit y of this electrochemical detection procedure, The detection limit of the method is 1 adduct per 10(6) nucleotides for both adducts using 20 mu g of DNA and 4 adducts per 10(8) nucleotides using 500 mu g DNA. ( C) 1997 Academic Press.