Huitfeldt H S, Spangler E F, Baron J, Poirier M C
Cancer Res. 1987 Apr 15;47(8):2098-102.
The intensities of immunofluorescence in nuclei stained by an antiserum specific for the DNA adduct N-deoxyguanosin(8-yl)aminofluorene (dG-8-AF), were quantified by microfluorometry in frozen liver sections from male Fischer rats fed 2-acetylaminofluorene (AAF). Results of previous studies demonstrated that dG-8-AF is the predominant adduct (80-100%) formed in livers of rats fed AAF continuously, and that nuclei of hepatocytes and bile duct epithelial cells in rats fed AAF exhibit an adduct-specific immunofluorescence. In the present investigation, nuclear staining for dG-8-AF was quantified by microfluorometry in liver sections from male Fischer rats fed 0.02% AAF continuously for 2, 4, 8, 12, 16, 20, and 28 days. Microfluorometric determinations of the intensities of nuclear immunofluorescence staining within periportal, midzonal, and centrilobular hepatocytes and bile duct epithelial cells revealed that levels of the dG-8-AF adduct increased in these cells during AAF feeding, reaching a plateau by 12 days. However, significant differences were detected in dG-8-AF levels within cells of each lobular area. Nuclei of periportal hepatocytes exhibited the most intense immunofluorescence, nuclei of centrilobular hepatocytes and bile duct epithelial cells emitted the least intense fluorescence, and nuclei of midzonal hepatocytes exhibited an intermediate fluorescence intensity. Quantitation of whole-liver levels of the dG-8-AF adduct by RIA, after extraction of DNA, also revealed that adduct accumulation reached a plateau by 12 days of AAF feeding. Thus, similar profiles of adduct accumulation were obtained by microfluorometric analysis of immunofluorescence staining within frozen liver sections, and by RIA analysis of DNA extracted from whole livers. The periportal concentration of DNA adducts in livers of rats continuously fed a carcinogenic dose of AAF may be an important early event in AAF-induced liver tumorigenesis.
用对DNA加合物N - 脱氧鸟苷（8 - 基）氨基芴（dG - 8 - AF）具有特异性的抗血清对细胞核进行免疫荧光染色，通过显微荧光测定法对连续喂食2 - 乙酰氨基芴（AAF）的雄性Fischer大鼠的冰冻肝切片中的免疫荧光强度进行定量。先前研究结果表明，dG - 8 - AF是连续喂食AAF的大鼠肝脏中形成的主要加合物（80 - 100%），并且喂食AAF的大鼠肝细胞和胆管上皮细胞核呈现加合物特异性免疫荧光。在本研究中，通过显微荧光测定法对连续2、4、8、12、16、20和28天喂食0.02% AAF的雄性Fischer大鼠肝切片中dG - 8 - AF的核染色进行定量。对门周、中区和中央小叶肝细胞及胆管上皮细胞核免疫荧光染色强度的显微荧光测定表明，在AAF喂食期间这些细胞中的dG - 8 - AF加合物水平升高，到12天时达到平台期。然而，在每个小叶区域的细胞内检测到dG - 8 - AF水平存在显著差异。门周肝细胞的核呈现最强的免疫荧光，中央小叶肝细胞和胆管上皮细胞的核发出最弱的荧光，中区肝细胞的核呈现中等荧光强度。在提取DNA后，通过放射免疫分析（RIA）对全肝dG - 8 - AF加合物水平进行定量，结果还表明，在AAF喂食12天时加合物积累达到平台期。因此，通过对冰冻肝切片内免疫荧光染色进行显微荧光分析以及对从全肝提取的DNA进行RIA分析，获得了相似的加合物积累情况。连续喂食致癌剂量AAF的大鼠肝脏中门周DNA加合物浓度可能是AAF诱导肝脏肿瘤发生过程中的一个重要早期事件。
