We have examined two methods of preparation of DNA adducts from ∅X174 RF DNA modified by [3H]N-acetoxy-2-acetylaminofluorene ([3H]N-AAF) or N-hydroxy-2-aminofluorene ([3H]N-OH-AF). Hydrolysis by enzymes (DNase I, snake venom phosphodiesterase and alkaline or acid phosphatase) and subsequent reverse phase h.p.l.c. of ∅X174 RF DNA treated with [3H]NA-AAF yielded 73% N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), 7% 3-(deoxyguanosin-N2-yl)-21-acetylaminofluorene (dG-N2-AAF), and a peak of unidentified radiaoctivity (13%). When [3H]N-OH-AF modified ∅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. In contrast, when anhydrous trifluoroacetic acid (TFA) was used to degrade these DNAs, we found for the [3H]NA-AAF modified DNA 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. Thus, anhydrous TFA solvolysis followed by reverse phase h.p.l.c. is a rapid and convenient method to obtain 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.
ASJC Scopus subject areas
- Cancer Research