Papanikolaou A, Shank R C, Delker D A, Povey A, Cooper D P, Rosenberg D W
Department of Pharmaceutical Sciences, University of Connecticut, Storrs 06269-2092, USA.
Toxicol Appl Pharmacol. 1998 May;150(1):196-203. doi: 10.1006/taap.1998.8393.
Inbred mice vary in susceptibility to colon carcinogens such as 1,2-dimethylhydrazine (DMH). Differential susceptibility may depend, in part, on formation of promutagenic DNA methyl adducts within target colonic mucosa. The present study was undertaken to evaluate the extent of DNA adduct formation in susceptible (SWR) and resistant (AKR) mice acutely exposed to the colon carcinogen azoxymethane (AOM), a direct metabolite of DMH. In the first experiment, 8-week-old SWR and AKR mice were treated i.p. with 20 mg/kg AOM and sacrificed 6 h later. DNA was isolated from distal colon and liver, and O6-methylguanine (O6-MeGua) adduct levels were assessed by immunoslot blot (ISB) analysis, using a monospecific antibody raised against O6-methyldeoxyguanosine. HPLC-fluorescence detection was also used to quantitate 06-MeGua and 7-methylguanine (7-MeGua), and to generate standard curves. At 6 h, both O6-MeGua and 7-MeGua were significantly higher (2- to 3-fold, p < 0.05) in AKR colon, while an opposite pattern was found in liver. In Experiment 2, mice were injected with AOM (20 mg/kg) and euthanized 12 and 48 h later. At 12 h, O6-MeGua levels were higher in colons (1.4-fold) of SWR mice. Forty-eight hours after treatment, however, adduct levels in colon were markedly (5-fold) reduced in SWR but were unchanged from 12 h in AKR. To further compare activation of AOM in both strains, colon microsomes were incubated with AOM and calf thymus DNA. Comparable levels of O6-MeGua were detected by ISB, demonstrating equivalent metabolic capacity in both SWR and AKR mice. These studies suggest that differential susceptibility to AOM-induced colon carcinogenesis is not based on initial target tissue DNA alkylation and unlikely to depend on differential metabolic capacity.
近交系小鼠对1,2 - 二甲基肼（DMH）等结肠致癌物的易感性存在差异。易感性差异可能部分取决于靶结肠黏膜内促诱变DNA甲基加合物的形成。本研究旨在评估急性暴露于结肠致癌物偶氮甲烷（AOM，DMH的直接代谢产物）的易感（SWR）和抗性（AKR）小鼠中DNA加合物形成的程度。在第一个实验中，8周龄的SWR和AKR小鼠经腹腔注射20 mg/kg AOM，并在6小时后处死。从远端结肠和肝脏中分离DNA，使用针对O6 - 甲基脱氧鸟苷产生的单特异性抗体，通过免疫印迹（ISB）分析评估O6 - 甲基鸟嘌呤（O6 - MeGua）加合物水平。高效液相色谱 - 荧光检测也用于定量O6 - MeGua和7 - 甲基鸟嘌呤（7 - MeGua），并生成标准曲线。在6小时时，AKR结肠中的O6 - MeGua和7 - MeGua均显著升高（2至3倍，p < 0.05），而在肝脏中发现相反的模式。在实验2中，给小鼠注射AOM（20 mg/kg），并在12小时和48小时后实施安乐死。在12小时时，SWR小鼠结肠中的O6 - MeGua水平较高（1.4倍）。然而，在治疗48小时后，SWR小鼠结肠中的加合物水平显著降低（5倍），而AKR小鼠结肠中的加合物水平与12小时时相比没有变化。为了进一步比较两种品系中AOM的活化情况，将结肠微粒体与AOM和小牛胸腺DNA一起孵育。通过ISB检测到相当水平的O6 - MeGua，表明SWR和AKR小鼠具有同等的代谢能力。这些研究表明，对AOM诱导的结肠癌发生的易感性差异并非基于初始靶组织DNA烷基化，也不太可能取决于代谢能力的差异。
