CDNA MICROARRAY TO DETECT CELLULAR RESPONSES TO MIXTURES

毒理学的大部分工作都集中在描述其影响上。 一种单一的化学物质，通常在高剂量和短时间内 句号。然而，人类更经常地接触到多种化学物质。 持续时间长，剂量比通常使用的要低 试验性的。因此，有必要了解潜在的 暴露于多种化学物质的相互作用 细胞和整个生物体水平。目前的请求提议采取 基因表达分析的最新进展及其优势 高密度微阵列探索使用这项技术来 确定与接触多种化学品有关的变化。 这项工作将建立在最近发现的基础上，表明细胞毒性 在代谢激活的全身性肺损伤中，1- 通过预先暴露在臭氧中，硝基萘的含量大大增加。二 将利用各种方法。编码两个阶段I的基因阵列 和第二相代谢酶，参与合成和 谷胱甘肽、几种热休克蛋白的降解与管家 基因将会被准备好。从对照和处理组中提取的mRNA (硝化萘、臭氧和硝化萘加臭氧)大鼠肺将 以此为模板合成荧光标记的cDNA 标签(CY-3(对照)和CY-5(处理))，这些标签将被杂交 以确定治疗是导致向上还是向下 对可能控制新陈代谢激活或 对硝基萘的解毒作用。平行定量组织病理学 将进行研究以确认#年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 肺库将排列在玻璃幻灯片上，并进行屏蔽 标记的mRNA来自对照组(CY-3)和处理组(CY-5)。克隆人 显示上行或下行规则将被排序以供识别。 这些研究将测试使用DNA阵列快速 筛查肺混合液对基因表达的影响 毒药。剂量和时程反应研究相结合的研究 其中包括通过组织病理学对组织进行详细检查 定义对化学物质作出反应而发生的细胞/分子事件 并有望探索使用DNA阵列的有效性 来筛选潜在的化学相互作用。通过检查库克隆， 这些研究可能识别新的基因，这些基因的调控被 接触化学物质。

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.