DNA DAMAGE FOLLOWING EXPOSURE TO GENOTOXIN

在这个过程中，已经开发了几种免疫学方法。 之前为检测致癌物质修饰的DNA成分提供的拨款。 这些研究表明，免疫分析是非常迅速的。 灵敏而特异的探头。0(6)-乙基郭加合物和胸腺嘧啶核苷二聚体 在0(6)-EtdGuo/dGuo摩尔比为2.2×10到7和 在极小的体内处理的DNA中紫外线剂量小于1J/m2 样本。我们现在提议研制出抗病毒的单抗 0(6)-EtdGuo，0(6)-EtdThy和0(2)-EtdCyt表现出更高的亲和力 单一或单独致癌物修饰的改变 双链DNA。这些抗原更接近于 致癌物-DNA加合物将在体内和体内设计和使用 体外免疫方案。具有适当特异性的抗体 将通过筛选杂交瘤克隆来对抗 抗原。敏感的分析，如酶联免疫吸附试验、USERIA和示踪剂RIA将是 用于筛选允许定量检测的单特异性抗体 直接在未水解的DNA样本中发生个别变化。检测 器官和细胞类型的特定碱基改变将在 通过使用免疫荧光的间接检测来损伤特定抗体， 免疫染色、流式细胞术和放射自显影。低温恒温器 在不同时间取出的人类皮肤活组织切片 紫外光照射后将用于免疫学定量 与环境有关的嘧啶二聚体的初始和持续水平 作为细胞类型的函数。不同修饰碱基在不同条件下的去向 大鼠乳腺、脑和肝脏的DNA(敏感和耐药器官 到ENU诱导的肿瘤)将利用这些免疫技术进行研究 探讨细胞和细胞因子诱导肿瘤的机制 器官类型。

Several immunological methods have been developed during the course of the previous grant for the detection of carcinogen-modified DNA components. These studies have indicated that immunoassays are rapid, extremely sensitive and specific probes. 0(6)-EtdGuo adducts and Thymidine dimers in DNA were measured at 0(6)-EtdGuo/dGuo molar ratios of 2.2 x 10 to the 7 and UV doses of less than 1 J/m2 in extremely small in vivo treated DNA samples. We now propose to develop monoclonal antibodies against 0(6)-EtdGuo, 0(6)-EtdThy and 0(2)-EtdCyt exhibiting higher affinity for individual carcinogen modified alterations within single- or double-stranded DNA. Antigens that more closely resemble the carcinogen-DNA adduct will be designed and used in modivied in vivo and in vitro immunization protocols. The antibodies of appropriate specificity will be chosen by screening the hybridoma clones against a combination of antigens. Sensitive assays like ELISA, USERIA and tracer RIA will be employed to screen the monospecific antibodies allowing quantitation of individual alterations directly in unhydrolysed DNA samples. Detection of specific base alterations in organ and cell types will be performed with damage specific antibodies by indirect assays using immunofluorescent, immunostaining, flow cytometry and autoradiographic procedures. Cryostat sections of the biopsies of human skin removed at various times post-UV-irradiation will be used for the immunologic quantitation of the initial and persistent levels of environmentally relevant pyrimidine dimers as a function of cell type. Fate of various modified bases induced in the DNA of rat mammary gland, brain and liver (organs sensitive and resistant to ENU induced tumors) will be studied utilizing these immuno-techniques to investigate the mechanisms of tumor induction as a function of cell and organ type.