ENVIRONMENTAL HYDRAZINES AND METHYLATION OF DNA GUANINE

拟议研究的目标是提供严格的生化 细胞毒性和改变的维持甲基化的证据 (复制后不久DNA中5-甲基胞嘧啶的形成) 在啮齿动物肝脏中是机械相关的，并导致 由环境因素引起的癌症。结果将是 适用于下列癌症生物测定的解释 肿瘤发病率的增加只出现在暴露水平上 对靶组织造成细胞毒性。假说 建议用于实验研究的是：细胞毒性具有 改变维持状态在肝癌发生中的致病作用 甲基化模式和/或水平，从而损害 代偿期新生子代细胞的分化 再生。实验将使用两种分析工具 化学和分子生物学测量定量 新的5-甲基胞嘧啶水平和模式的程度 合成的DNA是否被改变以及是否有特定的 与以下因素相关的维护甲基化改变的模式 细胞毒性。细胞毒性的剂量-反应关系 以及联氨引起的维持甲基化改变和 其他致癌物质将被确定。长期接触 仓鼠饮用水中的联氨提供了一种模型 其中肝细胞癌的出现显然是 与DNA加合物的积累和 发展为混合性坏死和增生；这一模型将 用于调查维护变更的关联 甲基化与细胞毒性和致癌作用。具体的 拟议研究的目的是：1)确定剂量- 超甲基化的响应关系(超过5- 基因组肝DNA的甲基胞嘧啶)和 由肼和其他致癌物引起的肝脏DNA；2)发展成 肝DNA含量变化的分析方法 5-甲基胞嘧啶的杂交定位 带有特定探针的DNA，用于检查特定定义的DNA 序列；3)确定化学诱导的持久性 肝脏DNA(5-甲基胞嘧啶)高甲基化；4)测定 形成的关系和事件的先后顺序 甲基鸟嘌呤，肝脏5-甲基胞嘧啶模式的改变 DNA与金黄地鼠肝坏死的发生发展 两年来每天饮用水中肼的剂量水平， 5)确定DNA加合物的结构 苯肼和肼。

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.