ENVIRONMENTAL HYDRAZINES AND METHYLATION OF DNA GUANINE

这项研究的目的是提供严格的生物化学 细胞毒性和改变的维持甲基化 （复制后不久DNA中5-甲基胞嘧啶的形成） 在啮齿类动物的肝脏中， 由环境因素引起的癌症。 其结果将 适用于癌症生物测定的解释，其中 肿瘤发病率的增加仅出现在接触水平 其在靶组织中引起细胞毒性。 的假设 提出的实验研究是：细胞毒性有一个 通过改变维持在肝癌发生中的致病作用 甲基化模式和/或水平，从而损害 代偿期新子细胞的分化 再生 实验将使用分析工具和 化学和分子生物学来测量 5-甲基胞嘧啶水平和模式在新生儿中的程度 合成的DNA被改变，是否有特定的 改变的维持甲基化模式与 细胞毒 细胞毒性的剂量-反应关系 以及肼诱导的维持甲基化改变， 其他致癌物质将被确定。 长期暴露于 仓鼠的联氨在饮用水提供了一个模型， 肝细胞癌的表现 与DNA加合物的积累有关， 混合坏死和增生的发展;该模型将 用于调查生活方式改变与 甲基化与细胞毒性和致癌作用。 具体 研究的目的是：1）确定剂量- 超甲基化的反应关系（过量的5- 甲基胞嘧啶）的基因组肝DNA和定义的序列， 肝脏DNA的肼和其他致癌物质; 2）开发一个 用于分析肝DNA的量的变化的方法 和5-甲基胞嘧啶的位置 带有特定探针的DNA，用于检查特定的DNA 3）确定化学诱导的持久性 肝DNA超甲基化（5-甲基胞嘧啶）; 4）测定 形成的关系和事件的顺序 甲基鸟嘌呤，肝脏中5-甲基胞嘧啶模式的改变 DNA和仓鼠肝坏死的发展， 饮用水中联氨的每日剂量水平，持续两年， 和5）确定由以下形成的DNA加合物的结构： 苯肼和肼苯哒嗪。

The goal of the proposed study is to provide rigorous biochemical evidence that cytotoxicity and altered maintenance methylation (formation of 5-methylcytosine in DNA shortly after replication) in the rodent liver are mechanistically related and causative in cancer induced by environmental agents. The results would be applicable to the interpretation of cancer bioassays in which increased incidences of tumors appear only at exposure levels which cause cytotoxicity in the target tissue. The hypothesis proposed for experimental investigation is: cytotoxicity has a causative role in liver carcinogenesis by altering maintenance methylation patterns and/or levels, thus compromising differentiation of new daughter cells during compensatory regeneration. The experiments will use tools of both analytical chemistry and molecular biology to measure quantitating the extent to which 5-methylcytosine levels and patterns in newly synthesized DNA are altered and whether there are specific patterns of altered maintenance methylation related to cytotoxicity. The dose-response relationships for the cytotoxicity and altered maintenance methylation induced by hydrazine and other carcinogens will be determined. Chronic exposure to hamster of hydrazine in the drinking water provides a model in which the appearance of hepatocellular carcinomas is clearly associated with the accumulation of DNA adducts and the development of mixed necrosis and hyperplasia; this model will be used to investigate the association of altered maintenance methylation with cytotoxicity and carcinogenesis. The specific aims of the proposed research are: 1) determine the dose- response relationship for the hypermethylation (excess of 5- methylcytosine) of genomic liver DNA and of defined sequences in liver DNA by hydrazine and other carcinogens; 2) develop a method for the analysis of liver DNA for changes in the amount and position of 5-methylcytosine using hybridization of treated DNA with specific probes to examine particular defined DNA sequences; 3) determine the persistence of the chemically-induced hypermethylation of liver DNA (5-methylcytosine); 4) determine the relationship and sequence of events for the formation of methylguanines, alteration of 5-methylcytosine patterns in liver DNA and development of liver necrosis in hamsters given three dose levels of hydrazine daily in the drinking water for two years, and 5) determine the structures of the DNA adducts formed by phenylhydrazine and hydralazine.