Localization of DNA adducts induced by N‐acetoxy‐N‐2‐acetylaminofluorene in chinese hamster ovary cells using electron microscopy and colloidal gold

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Abstract
DNA adduct induction by N-acetoxy-N-2-acetylaminofluorene (N-Ac-AAF) has been investigated in Chinese hamster ovary (CHO) cells using immunoelectron microscopy. The major RNA and DNA adducts, N-(guanosin-8-yl)-2-aminofluorene (G-C8-AF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), were localized with a rabbit anti-G-C8-AF antiserum and colloidal gold cytochemistry. Appropriate controls, including incubation of untreated cells with normal rabbit serum and immunogen-absorbed serum, demonstrated that colloidal gold deposits were indicative of the presence of adducts. The localization of gold particles in close association with nuclear chromatin revealed high concentrations of adducts in DNA and RNA of nuclei. Morphometric evaluation of adduct formation in organelles of cells from different carcinogen exposures showed that 85–88% of total adducts were concentrated in nuclei. DNA adducts remaining in nuclei after RNAse treatment appeared to concentrate in heterochromatic areas, and these areas contained 59% of bound gold particles by morphometry. A total of 137–178 particles were found in nuclei of treated cells vs. 15–26 in the surrounding cytoplasm. Treated cells incubated with normal rabbit serum or specific adduct-absorbed serum showed 19–34 particles for all cellular compartments.