Molecular Pharmacology, Vol 18, 581-587, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Effect of Culture Conditions, Cell Type, and Species of Origin on the Distribution of Acetylated and Deacetylated Deoxyguanosine C-8 Adducts of N-Acetoxy-2-Acetylaminofluorene

¹ Laboratory of Experimental Pathology, National Cancer Institute, Building 37, Room 3A19, Bethesda, Maryland 20205
² Naylor Dana Institute, American Health Foundation, Valhalla, New York 10595

Antibodies directed against guanosin-8-yl-acetylaminofluorene have been employed in a sensitive radioimmunoassay able to distinguish between the acetylated and deacetylated deoxyguanosine C-8 adducts of 2-acetylaminofluorene. Most cultured cells (primary BALB/c and Sencar epidermal and fibroblast cells and primary rat epidermal cells and fibroblasts) form 90% of the total C-8 adducts as the deacetylated adduct after a 1-h exposure to 10^-5 M N-acetoxy-2-acetylaminofluorene. However, primary rat hepatocytes, exposed to either the activated derivative of the carcinogen for 1 h or the parent compound 2-acetylaminofluorene for 5-24 h, form 80% of the C-8 adducts as the acetylated adduct. Treatment conditions were varied in order to alter the levels of binding and proportion of acetylated and deacetylated C-8 adducts formed in primary BALB/c epidermal cells. Cells exposed in medium 199 with 1.2 mM Ca²⁺ in the presence of 10% fetal calf serum (standard culture conditions) bind 100-200 fmol/µg DNA and form 3% of the C-8 adducts as acetylated. Exposure to carcinogen in the absence of serum increased binding levels two- to fivefold with 8% of the C-8 adducts in the acetylated form. The addition of 10^-5 M ethidium bromide (a DNA intercalating agent), 10^-3 M sodium butyrate (a deacetylase inhibitor), or 10^-5 M harman (which augments 2-acetylaminofluorene mutagenesis) failed to change the level or pattern of binding. Carcinogen exposure under culture conditions which select for the growing fraction of epidermal cells (0.09 mM Ca²⁺) also failed to alter binding. However, a 20-min pretreatment with 10^-5 M paraoxon under standard culture conditions resulted in the inhibition of 99% of the binding and formation of deacetylated adduct. The small amount of C-8 adduct formed was acetylated. These results indicate that the extent and pattern of 2-acetylaminofluorene DNA binding vary in different cell types and can be altered by variations in exposure conditions. By these means it should be possible to investigate the biological importance of individual adducts.

Note:
ACKNOWLEDGMENTS Our continuing appreciation is extended to Drs. I. B. Weinstein and S. Blobstein for having supplied the original antigen for these studies, and for helpful discussions. Dr. I. C. Hsu performed the USERIA assays. Dr. R. J. Connor is responsible for the linear regression analysis, resulting equation, and confidence limits. The paraoxon was a gift of Dr. S. Thorgiersson. The technical assistance of Marc A. Dubin, Brigitte Former, and Theresa L. Ben was also appreciated.

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