REGULATION OF MICROSOMAL HEMOPROTEINS

描述(摘自申请者摘要)：一个尚未解决的主要问题 毒理学具有广泛的医学、经济、法律和政治后果的毒理学 是人类对测试结果的外推的不确定性 给实验动物服用高剂量的外源物质。我们的拨款方案 将利用分子和细胞生物学的进展来帮助定义 可能影响寄主的遗传和环境因素 对某一特定个体健康不良影响风险的抵抗力。我们 关注的是CYP3A基因亚家族的细胞色素P450，一组 人体肝脏中突出的微粒体血色素蛋白在 糖皮质激素和外源物质等的“应激”条件 苯巴比妥和多卤代芳香族环境化学品。细胞色素P3A 参与临床上重要药物的代谢，如 环孢素和硝苯地平等致癌物的生物活性 黄曲霉毒素和苯并[a]芘。利用新定义的系统 大鼠和人肝细胞在Matrigel上的原代培养 基底膜，并允许保留分化状态在 在体外，我们将进行近距离的比较 控制细胞色素P3A结构基因及其产物的DNA片段 在严格控制下的动物和人类细胞之间 条件。分子分离和鉴定技术将是 用于指定调控细胞色素P3A基因表达的核心DNA元件 肝脏与配体依赖的调节转录蛋白因子 与这些DNA反应元件相互作用。我们还将使用反转 基因分析，以研究以前未被识别的 受相同“压力”控制的基因超出了 肝脏协调行动以适应环境应激源。促进 这项工作是一个细胞培养和组织库，为动物和 这些调查所需的人力材料。通过一个深思熟虑的 结合这些方法，我们希望能够在数量上 描述细胞表达变化背后的分子事件 药物、环境化学物质、内分泌控制所致的细胞色素P3A基因 以及其他因素。我们将调查这些影响是如何 可能因人而异。我们完全期待在这方面的改进 了解基因结构、基因表达和疾病结果可以 通过建议的研究计划实现。

DESCRIPTION (from Applicant's Abstract): A major unsolved problem in toxicology having broad medical, economic, legal, and political consequences is the uncertainty of extrapolation to humans of the results of tests of high doses of xenobiotics given to laboratory animals. Our grant proposal will utilize advances in molecular and cellular biology to help define the genetic and environmental factors that may contribute to the host's resistance to risks for an adverse health effect to a given individual. We are focusing on the cytochromes P450 of the CYP3A gene subfamily, a group of microsomal hemoproteins prominent in human liver which are induced under conditions of "stress" by glucocorticoids and also by such xenobiotics as phenobarbital, and polyhalogenated aromatic environmental chemicals. CYP3A are involved in the metabolism of clinically important drugs such as Cyclosporin and nifedipine and the bioactivation of such carcinogens as aflatoxins and benzo[a]pyrene. Taking advantage of a newly defined system primary culture of rat and human hepatocytes on Matrigel, a reconstituted basement membrane and permits retention of the differentiated state in vitro, we will make proximate comparisons of the regulation of the controlling DNA segments of the CYP3A structural genes and their products between animal and human cells under defined, rigorously controlled conditions. Molecular isolation and characterization techniques will be used to specify the core DNA elements that regulate CYP3A gene expression in the liver and the igand dependent regulatory transcription protein factors that interact with these DNA response elements. We will also use reverse genetic analysis to investigate a previously unrecognized assortment of genes regulated under the same "stress" controls that extends beyond the liver acting in concert to adapt to environmental stressors. Facilitating this work is a Cell Culture and Tissue Bank which provides the animal and human material needed for these investigations. Through a thoughtful combination of these approaches, we expect to be able to quantitatively describe the molecular events that underlie changes in cellular expression of the CYP3A genes due to drugs, environmental chemicals, endocrine controls and other factors. We will investigate the question of how these effects may differ among individuals. We fully expect that refinements in understanding gene structure, gene expression, and disease outcome can be achieved by the proposed research program.