Quantification of adducts formed in DNA treated with N-acetoxy-2-acetylaminofluorene or N-hydroxy-2-aminofluorene: comparison of trifluoroacetic acid and enzymatic degradation

Abstract

Two methods of preparation of DNA adducts from .vphi.X174 RF DNA modified by [3H]N-acetoxy-2-acetylaminofluorene ([3H]NA-AAF) or N-hydroxy-2-aminofluorene ([3H]N-OH-AF) were examined. Hydrolysis by enzymes (DNase I, snake venom phosphodiesterase and alkaline or acid phosphatase) and subsequent reverse phase h.p.l.c. of .vphi.X174 RF DNA treated with [3H]NA-AAF yielded 73% N-(deoxyguanosine-8-yl)-2-acetylaminofluorene (dG-C8-AAF), 7% 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF), and a peak of unidentified radioactivity (13%). When [3H]N-OH-AF modified .vphi.X174 DNA was analyzed, both N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and a large percentage of the imidazole ring-opened derivative and unidentified products were found. When anhydrous trifluoroacetic acid (TFA) was used to degrade these DNA, the [3H]NA-AAF modified DNA yielded 86% N-(guanin-8-yl)-2-acetylaminofluorene (G-C8-AAF) and 6% 3-(guanin-N2-yl)-2-acetylaminofluorene (G-N2-AAF), while for [3H]N-OH-AF modified DNA only the N-(guanin-8-yl)-2-aminofluorene (G-C8-AF) was found. When DNA was prepared from human fibroblasts treated with [3H]NA-AAF, only the G-C8-AF product was obtained. Anhydrous TFA solvolysis followed by reverse phase h.p.l.c. is a rapid and convenient method of obtaining quantitative yields of DNA adducts formed with acetylaminofluorene and related compounds: quantification by this method prevents loss of G-N2-AAF adducts, the conversion of AAF adducts to AF adducts, and the production of ring opened products in guanine residue.