Quantification of adducts formed in DNA treated with N-acetoxy-2-acetylaminofluorene or N-hydroxy-2-aminofluorene: comparison of trifluoroacetic acid and enzymatic degradation
We have examined two methods of preparation of DNA adducts from phi X174 RF DNA modified by (/sup 3/H)N-acetoxy-2-acetylaminofluorene ((/sup 3/H)NA-AAF) or N-hydroxy-2-aminofluorene ((/sup 3/H)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 phi X174 RF DNA treated with (/sup 3/H)NA-AAF yielded 73% N-(deoxyguanosin-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 (/sup 3/H)N-OH-AF modified phi 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 (/sup 3/H)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 (/sup 3/H)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 (/sup 3/H)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.
- Research Organization:
- Department of Pathology, Washington University School of Medicine, St. Louis, MO
- OSTI ID:
- 5532942
- Journal Information:
- Carcinogenesis (N.Y.); (United States), Vol. 4:8
- Country of Publication:
- United States
- Language:
- English
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DNA
CHEMICAL BONDS
FLUORENE
METABOLIC ACTIVATION
BACTERIOPHAGES
CELL CULTURES
DNA ADDUCTS
DNA-ASE
FIBROBLASTS
IMIDAZOLES
LIQUID COLUMN CHROMATOGRAPHY
MAN
MUTAGENS
PHOSPHATASES
PHOSPHODIESTERASES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ADDUCTS
ANIMAL CELLS
ANIMALS
AROMATICS
AZOLES
CHROMATOGRAPHY
CONDENSED AROMATICS
CONNECTIVE TISSUE CELLS
ENZYMES
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HETEROCYCLIC COMPOUNDS
HYDROCARBONS
HYDROLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MICROORGANISMS
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ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PARASITES
PRIMATES
SEPARATION PROCESSES
SOMATIC CELLS
VERTEBRATES
VIRUSES
560301* - Chemicals Metabolism & Toxicology- Cells- (-1987)