Olivero O A, Semino C, Poirier M C
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, MD 20892.
Genes Chromosomes Cancer. 1990 Jul;2(2):130-6. doi: 10.1002/gcc.2870020209.
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 concentration of adducts in DNA and RNA of nuclei. Morphometric evaluation of adduct formation in organelles of 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.
利用免疫电子显微镜技术，在中国仓鼠卵巢（CHO）细胞中研究了N - 乙酰氧基 - N - 2 - 乙酰氨基芴（N - Ac - AAF）诱导的DNA加合物。主要的RNA和DNA加合物，即N - （鸟苷 - 8 - 基）- 2 - 氨基芴（G - C8 - AF）和N - （脱氧鸟苷 - 8 - 基）- 2 - 氨基芴（dG - C8 - AF），通过兔抗G - C8 - AF抗血清和胶体金细胞化学进行定位。适当的对照，包括用正常兔血清和免疫原吸附血清孵育未处理的细胞，表明胶体金沉积指示加合物的存在。与核染色质紧密相关的金颗粒定位显示，细胞核的DNA和RNA中加合物浓度很高。对不同致癌物暴露后细胞器中加合物形成的形态计量学评估表明，总加合物的85 - 88%集中在细胞核中。RNA酶处理后细胞核中残留的DNA加合物似乎集中在异染色质区域，通过形态计量学，这些区域含有59%的结合金颗粒。在处理过的细胞的细胞核中总共发现137 - 178个颗粒，而周围细胞质中为15 - 26个。用正常兔血清或特异性加合物吸附血清孵育的处理过的细胞，所有细胞区室显示有19 - 34个颗粒。
