HEPATOTOXIN METABOLISM AND ITS REGULATION WITHIN LIVER

该协议的基本前提是区分 肝细胞和非实质细胞易感性的研究 外源生物的生物活化所产生的毒性主要是 由于异种生物激活和细胞特异性的差异 解毒。尽管标记为肝内和小叶内 异质性发生在异种代谢及其诱导过程中，许多 关于为什么会出现这种情况，基本问题仍有待回答 异质性存在。因此，我们在这个项目中的目标是：[a] 更完整地描述了肝细胞能力的差异 穿过小叶，以酶法激活和解毒外源物质； [B]更确凿地确定所有能代谢的肝细胞 外源生物；以及[c]以深入了解 这些细胞中的异种生物代谢酶受到正常调控， 通过诱导者和个体发育。具体来说，我们建议：[1]进一步 在不同的分布中定义小叶内的异质性 细胞色素P-450的形态，决定了哪些异体代谢 酶存在于非实质细胞中，并确定是否 肝细胞和非实质细胞中酶表达的相关性 与未治疗的成年大鼠的mRNAs进行比较；[2]以确定 异种生物代谢酶可以在非实质细胞中诱导， 如果在诱导的动力学上存在区域内差异 酶，以及细胞中全酶和mRNA水平的增加 相关；以及[3]研究异种生物的出生后个体发育- 代谢酶及其在小叶内的异质性 分配。为了完成这项工作，半定量的 将采用免疫组织化学方法来确定肝内 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-葡萄糖醛酸基转移酶，原位杂交 将用于研究编码这些基因的mRNAs的表达 酶和组织化学将用于检测异源生物 单加氧酶活性。关于酶及其mRNAs的组织学发现， 单加氧酶活性将在体外与 生化和免疫化学测定。这个的用法 组织学方法将使我们能够研究基因表达及其 单个细胞内转录和转录水平的调节 蛋白质水平。反过来，这将使我们能够获得根本的、新的 洞察特定细胞的存在和解释 异物在肝脏和肝脏中代谢及其调控的差异 将有助于实现我们的长期目标，即阐明 新陈代谢中细胞特异性差异的机制(S) 肝毒素和其他外源生物。

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.