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  • Title: Detection and quantitation of acetylated and deacetylated N-2-fluorenylacetamide-DNA adducts by radioimmunoassay.
    Author: Poirier MC, Yuspa SH.
    Journal: Natl Cancer Inst Monogr; 1981 Dec; (58):211-6. PubMed ID: 7341978.
    Abstract:
    Antibody raised in rabbits has been used for the detection of picomole quantities of the major adducts formed upon interaction of activated N-acetoxy-2-fluorenylacetamide (N-AcO-2-FAA) with DNA. By radioimmunoassay the quantitation of N(deoxyguanosin-8-yl)-2-FAA and N(deoxyguanosin-8-yl)-2-fluorenamine and discernment of the proportion of each in a mixture are possible. The antibody does not recognize the minor adduct 3-(deoxyguanosin-N(2)-yl)-2-FAA, 2-FAA, or DNA and is therefore specific for the acetylated and deacetylated C-8 adducts. We used the radioimmunoassay to detect and quantitate these adducts in DNA from several types of cultured cells exposed to 10(-5) M N-AcO-2-FAA. Levels of bound C-8 adducts varied between 100 to 200 fmol/micrograms DNA for all cells investigated. In all cells except primary rat hepatocytes, 95-97% of the C-8 adducts were in the deacetylated form, but in the rat hepatocytes, 80% of the C-8 adducts were acetylated. Our attempts to manipulate the amount and proportion of C-8 adducts bound to the DNA of primary BALB/c epidermal cells met with success in two areas. When cells were exposed to the carcinogen in the absence of serum, total binding and the percentage of acetylation were increased twofold to threefold. Also, in the presence of paraoxon, 99% of the binding and the formation of all deacetylated C-8 adducts were inhibited. We also used the radioimmunoassay to monitor repair of C-8 adducts from the DNA of BALB/c epidermal cells and normal human fibroblasts (YDF line) for 24 hours after removal of the carcinogen-containing culture medium. During this interval, the BALB/c epidermal cells and YDF cells removed approximately 40 and 50%, respectively, of the C-8 adducts from the DNA. These studies demonstrated that carcinogen-DNA adduct antibodies are useful for determining specific adducts in investigations related to aromatic amine carcinogenesis.
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