Menkveld G J, Van der Laken C J, Hermsen T, Kriek E, Scherer E, Den Engelse L
Carcinogenesis. 1985 Feb;6(2):263-70. doi: 10.1093/carcin/6.2.263.
Antibodies raised in rabbits against the bovine serum albumin conjugates of O6-ethylguanosine (O6-EtGuo) and N-(guanosin-8-yl)-N-acetyl-2-aminofluorene (Guo-8-AAF) have been used in a double peroxidase-antiperoxidase staining assay to visualize the localization of DNA-O6-ethyldeoxyguanosine (O6-EtdGuo) and some of the interaction products of N-acetyl-2-aminofluorene (AAF) with DNA guanine in liver sections of rats treated with diethylnitrosamine (DEN), ethylnitrosourea (ENU), or AAF respectively. O6-EtdGuo could be detected in nuclei of parenchymal cells after injection of DEN (12-50 mg/kg) or ENU (140 mg/kg). Clear time and dose dependencies were observed. The lowest dose of DEN which, at 5 h after injection, resulted in immunohistochemically detectable levels of O6-EtdGuo, was 12 mg/kg; 5 h after 6 mg/kg no consistent difference between treated and untreated rats could be observed. A striking heterogeneity in staining pattern was observed after DEN: centrilobular regions were stained much more than peripheral zones. At 7 days after a single DEN injection of 50 mg/kg small rims of significantly stained hepatocytes could still be observed around the central veins. No heterogeneity of staining pattern was observed 2 h after ENU. ENU, in contrast with DEN, also resulted in a significant staining of nonparenchymal cells. At 24 h after ENU no significant staining of hepatocytes could be detected, but positive staining was still present in bile duct cells, vascular endothelial cells and sinusoidal cells. The results indicate a detection level of approximately 5 mumol O6-EtdGuo/mol DNA-P, i.e., 5 X 10(4) O6-EtdGuo residues per diploid genome. Using the anti-Guo-8-AAF antiserum, positive results were obtained 6 days after a single AAF dose of 0.5-10 mg/kg, corresponding to a detection limit of less than or equal to 0.4 mumol dGuo-8-(A)AF/mol DNA-P. Staining was rather homogenously distributed over the liver lobules. Persistency of the AAF-DNA interaction products was investigated both after 10 and 2 mg/kg AAF. Increased nuclear staining could be observed up to 8 weeks after 10 mg/kg and 4 weeks after 2 mg/kg.
用兔制备的抗O6-乙基鸟苷（O6-EtGuo）和N-（鸟苷-8-基）-N-乙酰-2-氨基芴（Guo-8-AAF）牛血清白蛋白偶联物的抗体，已用于双过氧化物酶-抗过氧化物酶染色试验，以观察用二乙基亚硝胺（DEN）、乙基亚硝基脲（ENU）或AAF分别处理的大鼠肝切片中DNA-O6-乙氧基脱氧鸟苷（O6-EtdGuo）的定位以及N-乙酰-2-氨基芴（AAF）与DNA鸟嘌呤的一些相互作用产物。注射DEN（12 - 50 mg/kg）或ENU（140 mg/kg）后，可在实质细胞核中检测到O6-EtdGuo。观察到明显的时间和剂量依赖性。注射后5小时，免疫组化可检测到O6-EtdGuo的DEN最低剂量为12 mg/kg；6 mg/kg注射后5小时，未观察到处理组和未处理组大鼠之间有一致的差异。DEN注射后观察到染色模式存在显著异质性：小叶中心区域的染色比周边区域深得多。单次注射50 mg/kg DEN后7天，仍可在中央静脉周围观察到小的明显染色的肝细胞边缘。ENU注射后2小时未观察到染色模式的异质性。与DEN不同，ENU还导致非实质细胞显著染色。ENU注射后24小时，未检测到肝细胞的显著染色，但胆管细胞、血管内皮细胞和窦状细胞仍呈阳性染色。结果表明检测水平约为5 μmol O6-EtdGuo/mol DNA-P，即每个二倍体基因组有5×10⁴个O6-EtdGuo残基。使用抗Guo-8-AAF抗血清，单次给予0.5 - 10 mg/kg AAF后6天获得阳性结果，对应检测限小于或等于0.4 μmol dGuo-8-(A)AF/mol DNA-P。染色在肝小叶中分布相当均匀。研究了10 mg/kg和2 mg/kg AAF后AAF-DNA相互作用产物的持续性。10 mg/kg AAF后长达8周以及2 mg/kg AAF后4周可观察到核染色增加。
