CDNA MICROARRAY TO DETECT CELLULAR RESPONSES TO MIXTURES

毒理学的大部分工作都集中在描述 单一化学物质的化学反应通常是在高剂量和短时间内 时期 然而，人类更经常接触多种化学物质， 在较长的时间段内，并以低于通常使用的剂量 实验性的 因此，有必要了解潜在的 接触多种化学物质的相互作用 细胞和整个生物体水平。目前的请求建议采取 基因表达分析的最新发展的优势， 高密度微阵列，以探索使用这种技术， 识别与暴露于多种化学物质相关的改变。 这项工作将建立在最近的研究结果表明，细胞毒性， 代谢活化的全身性肺损伤剂，1- 硝基萘，是大大增强了预暴露于臭氧。 两 方法将被使用。 基因阵列编码的第一阶段， 和II相代谢酶，参与合成的酶， 谷胱甘肽、几种热休克蛋白和管家的降解 基因将被准备好。从对照和处理的mRNA中分离 （硝基萘、臭氧和硝基萘加臭氧）大鼠肺将 可用作合成荧光标记cDNA的模板 标签（CY-3（对照）和CY-5（处理的）），并且这些将被杂交 以确定治疗是否会导致向上或向下 调节可能控制代谢活化的基因，或 硝基萘的解毒 平行定量组织病理学 将进行研究，以确认肺部病变的严重程度， 所有治疗组。 在第二种方法中，来自对照大鼠的克隆 肺库将排列在玻璃载玻片上， 来自对照（CY-3）和处理（CY-5）动物的标记mRNA。 克隆 显示上调或下调将被排序以供识别。 这些研究将测试使用DNA阵列的有效性， 筛选对肺混合物反应的基因表达变化 有毒物质 剂量和时间过程反应研究的组合 包括通过组织病理学对组织进行详细检查， 定义响应化学物质而发生的细胞/分子事件 并有望探索使用DNA阵列的有效性 以筛选潜在的化学相互作用。 通过检查文库克隆， 这些研究可能会发现新的基因， 化学暴露

Much of the work in toxicology has focused on delineating the effects of a single chemical entity often at high doses and over short time periods. However, humans are more often exposed to multiple chemicals over long time periods and at lower doses than generally used experimentally. Thus, there is a need to understand potential interactions of exposure to multiple chemical entities at both the cellular and whole organism level. The current request proposes to take advantage of recent developments in analysis of gene expression with high density microarrays to explore the use of this technology to identify alterations associated with exposure to multiple chemicals. This work will build on recent findings showing that the cytotoxicity of the metabolically activated, systemic pulmonary injurant, 1- nitronaphthalene, is considerably enhanced by preexposure to ozone. Two approaches will be utilized. Arrays of genes coding for both Phase I and Phase II metabolizing enzymes, enzymes involved in the synthesis and degradation of glutathione, several heat shock proteins and housekeeping genes will be prepared. mRNA isolated from control and treated (nitronaphthalene, ozone and nitronaphthalene plus ozone) rat lung will be used as a template for synthesis of cDNA labeled with fluorescent tags (CY-3 (control) and CY-5 (treated)) and these will be hybridized to the arrayed targets to determine whether treatments cause up or down regulation of genes likely to control the metabolic activation or detoxication of nitronaphthalene. Parallel quantitative histopathology studies will be done to confirm the severity of the pulmonary lesion in all treatment groups. In the second approach, clones from a control rat lung library will be arrayed on glass slides and screened against labeled mRNA from control (CY-3) and treated (CY-5) animals. Clones showing up or down regulation will be sequenced for identification. These studies will test the validity of using DNA arrays to rapidly screen changes in gene expression in response to mixtures of lung toxicants. The combination of dose and time course response studies which include detailed examination of tissues by histopathology will define cellular/molecular events that occur in response to chemical exposure and are expected to explore the validity of using DNA arrays to screen potential chemical interactions. By examining library clones, these studies may identify new genes whose regulation is altered by chemical exposure.