Chromosome site-specific immunohistochemical detection of DNA adducts in N-acetoxy-2-acetylaminofluorene-exposed chinese hamster ovary cells

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Abstract
In these studies a polyclonal antiserum elicited against a carcinogen‐DNA adduct was used to explore the localization of DNA adducts in metaphase chromosomes of cultured cells. Morphological visualization of the adduct N‐(deoxyguanosin‐8‐yl)‐2‐aminofluorene (dG‐C8‐AF) in Chinese hamster ovary (CHO) cells exposed to the direct‐acting carcinogen N‐acetoxy‐2‐acetylaminofluorene (N‐Ac‐AAF) was accomplished by indirect immunofluorescence with an anti‐G‐C8‐AF antiserum. At the same time the pattern of chromosomal DNA replication was determined by replicative incorporation of bromodeoxyuridine (BrdUrd) and chromosomal staining with anti‐BrdUrd. Visualization of DNA in chromosomes was accomplished with Hoechst 33258 dye. When synchronized CHO cells were exposed to N‐Ac‐AAF for 0.5 h during early S phase, the chromosomal pattern of dG‐C8‐AF adduct formation was not random. Metaphase chromosome spreads from cells exposed to N‐Ac‐AAF in different experiments contained certain chromosome regions that had a consistently high adduct concentration. The regions of high DNA damage corresponded to the regions active in DNA synthesis when BrdUrd and the carcinogen were given simultaneously in early S phase. In addition, the patterns of high adduct concentration and replicative synthesis shifted when the carcinogen and BrdUrd were given simultaneously during late S phase. Thus, the stage of cell cycle in which adducts are induced is an important factor in the specific location of the highest concentrations of this type of DNA lesion.

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