HEPATOTOXIN METABOLISM AND ITS REGULATION WITHIN LIVER

该协议的基本前提是， 肝细胞和非实质细胞对 由外源性物质的生物活化引起的毒性主要是 由于异生素活化的细胞特异性差异， 解毒虽然肝内和小叶内 在异生物质代谢及其诱导过程中存在异质性， 根本问题仍然有待回答，为什么这样的 存在异质性。因此，我们在本项目中的目标是：[a] 更全面地描述肝细胞分化能力的差异 穿过小叶以酶促活化和解毒异生物质; [b]更确切地鉴定出所有能代谢 异生物质;和[c]以了解如何表达 这些细胞中的异生物质代谢酶被正常调节， 通过诱导物和个体发育。具体而言，我们建议：[1] 在不同的分布中定义小叶内异质性 细胞色素P-450的形式，确定哪些异生物质代谢 酶存在于非实质细胞中，并确定 肝细胞和非实质细胞中酶的表达与 在未处理的成年大鼠中，[2]以确定是否 异生物质代谢酶可以在非实质细胞中被诱导， 如果在诱导的动力学方面存在区域内差异， 如果细胞中的全酶和mRNA水平增加， [13][14][15][16][17][18][19] 代谢酶及其小叶内的异质性 分布。为了完成这项工作，半定量 免疫组织化学将用于确定肝内 细胞色素P-450的定位和小叶内分布， NADPH-细胞色素P-450还原酶、环氧化物水解酶、谷胱甘肽S- 转移酶和UDP-葡萄糖醛酸转移酶，原位杂交 将用于研究编码这些的mRNA的表达， 酶和组织化学将用于检查异生物质 单加氧酶活性酶及其mRNA的组织学发现， 和单加氧酶活性将与体外 生物化学和免疫化学测定。使用这种 组织学方法将使我们能够研究基因表达及其 单个细胞内的转录和 蛋白质水平这反过来又将使我们能够获得根本的、新的 深入了解细胞特异性 异生物质代谢的差异及其在肝脏和 将有助于实现我们的长期目标， 导致代谢中细胞特异性差异的机制 肝毒素和其他外源性物质。

The basic premise of this protocol is that the differential susceptibilities of hepatocytes as well as nonparenchymal cells to toxicities resulting from the bioactivation of xenobiotics are primarily due to cell-specific differences in xenobiotic activation and detoxication. Although marked intrahepatic and intralobular heterogeneity occur during xenobiotic metabolism and its induction, many fundamental questions remain to be answered regarding why such heterogeneity exists. Thus, our objectives in this project are: [a] to more completely characterize differences in the abilities to hepatocytes across the lobule to enzymatically activate and detoxicate xenobiotics; [b] to more conclusively identify all liver cells which can metabolize xenobiotics; and [c] to gain insight into how the expression of xenobiotic-metabolizing enzymes in these cells are regulated normally, by inducers, and ontogeny. Specifically, we propose: [1] to further define intralobular heterogeneity in the distributions of different forms of cytochrome P-450, determine which xenobiotic-metabolizing enzymes are present in nonparenchymal cells, and ascertain if the expression of enzymes in hepatocytes and nonparenchymal cells correlate with those of their mRNAs in untreated adult rats; [2] to determine if xenobiotic-metabolizing enzymes can be induced in nonparenchymal cells, if there are intrazonal differences in the kinetics of induction of enzymes, and if increases in holoenzyme and mRNA levels in cells correlate; and [3] to investigate the postnatal ontogeny of xenobiotic- metabolizing enzymes and of heterogeneity in their intralobular distributions. To accomplish this work, semiquantitative immunohistochemistry will be employed for determining the intrahepatic localizations and intralobular distributions of cytochromes P-450, NADPH-cytochrome P-450 reductase, epoxide hydrolase, gluthione S- transferases, and UDP-glucuronosyltransferases, in situ hybridization will be utilized for studying the expressions of mRNAs encoding these enzymes, and histochemistry will be used for examining xenobiotic monooxygenase activities. Histologic findings on enzymes, their mRNAs, and monooxygenase activities will be correlated with in vitro biochemical and immunochemical determinations. The use of this histologic approach will allow us to study gene expression and its regulation within individual cells at both the transcriptional and protein levels. This, in turn, will enable us to gain fundamental, new insight into both the presence of and explanation for cell-specific differences in xenobiotic metabolism and its regulation in liver and will aid in the attainment of our long-term goal to elucidate the mechanism(s) responsible for cell-specific differences in the metabolism of hepatotoxins and other xenobiotics.