TY - JOUR
T1 - Quantification of adducts formed in DNA treated with N-acetoxy-2-acetylaminofluorene or N-hydroxy-2-aminofluorene
T2 - Comparison of trifluoroacetic acid and enzymatic degradation
AU - Tang, Moon shong
AU - Lieberman, Michael W.
N1 - Funding Information:
We thank Charles M.King for modifying 0X174 RF DNA with ['HJN-OH-AF, Craig MacArthur for a gift of human DNA modified in vivo with PH]NA-AAF, F.A.Beland for authentic samples of dG-C8-AAF and dG-N2-AAF and M.F.MacFresh for n.m.r. analysis of our standards. We are grateful to Bonnie J.Gowans and Darel J.Hunting for many helpful suggestions about h.p.l.c. and Mitchell R.Smith for critical reading of this manuscript. This work was supported by NIH grants CA-20513 and ESO2740-CA and by the following companies: Brown and Williamson Tobacco Corporation, Philip Morris Incorporated, R.J.Reynolds Tobacco Company and United States Tobacco Company. During part of these studies Moon-shong Tang was supported by CA 09118.
Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.
PY - 1983/8
Y1 - 1983/8
N2 - 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.
AB - 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.
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U2 - 10.1093/carcin/4.8.1001
DO - 10.1093/carcin/4.8.1001
M3 - Article
C2 - 6307542
AN - SCOPUS:0020961425
SN - 0143-3334
VL - 4
SP - 1001
EP - 1006
JO - Carcinogenesis
JF - Carcinogenesis
IS - 8
ER -