Microfluorometric determination of DNA adducts in immunofluorescent-stained liver tissue from rats fed 2-acetylaminofluorene.
Microfluorometric determination of DNA adducts in immunofluorescent-stained liver tissue from rats fed 2-acetylaminofluorene.
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发表时间：
1987-04
期刊：
影响因子：
11.2
通讯作者：
H. Huitfeldt;E. F. Spangler;J. Baron;M. Poirier
中科院分区：
文献类型：
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作者：
H. Huitfeldt;E. F. Spangler;J. Baron;M. Poirier
通过显微荧光法对喂食 2-乙酰氨基芴 (AAF) 的雄性 Fischer 大鼠的冰冻肝脏切片中的 DNA 加合物 N-脱氧鸟苷 (8-基)氨基芴 (dG-8-AF) 特异性抗血清染色的细胞核中的免疫荧光强度进行定量。先前的研究结果表明，dG-8-AF是连续喂养AAF的大鼠肝脏中形成的主要加合物（80-100%），并且喂养AAF的大鼠的肝细胞和胆管上皮细胞的细胞核表现出加合物特异性免疫荧光。在本研究中，通过显微荧光法对连续喂食 0.02% AAF 2、4、8、12、16、20 和 28 天的雄性 Fischer 大鼠的肝脏切片中的 dG-8-AF 核染色进行定量。对汇管周围、中区、小叶中心肝细胞和胆管上皮细胞内核免疫荧光染色强度的微荧光测定显示，在 AAF 喂养期间，这些细胞中 dG-8-AF 加合物的水平增加，并在 12 天时达到平台期。然而，每个小叶区域细胞内的 dG-8-AF 水平存在显着差异。汇管区肝细胞核的免疫荧光最强，小叶中心肝细胞和胆管上皮细胞核发出的荧光最弱，中区肝细胞核的荧光强度中等。提取 DNA 后，通过 RIA 对全肝 dG-8-AF 加合物水平进行定量，结果也表明加合物积累在 AAF 喂养 12 天时达到稳定水平。因此，通过冷冻肝切片内免疫荧光染色的显微荧光分析以及从整个肝脏提取的 DNA 的 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 DNA 加合物的浓度可能是 AAF 诱导的肝脏肿瘤发生的重要早期事件。
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.