CYTOKINE REGULATION OF P450 IN CULTURED RAT HEPATOCYTES

本研究的目的是阐明其作用和机制。 急性时相反应的细胞因子介体的作用 血色素蛋白家族细胞色素P450(P450)的调节 负责许多药物、其他化学品的生物转化，以及 某些内源性底物。待研究的细胞因子包括 白介素6、白介素1、干扰素与肿瘤 肿瘤坏死因子(TNF)。细胞因子对P450的影响可能解释改变 在感染和炎症过程中发生的生物转化。在……里面 此外，细胞因子用于癌症时对P450的影响 免疫治疗或其他免疫紊乱可能导致药物-药物 相互作用，影响处置和毒性的同时 吸毒。尽管在急性发作期间对P450的影响已经显示出来 时相反应，或在给予细胞因子后，因为 细胞因子之间相互影响的复杂相互作用 体内合成、释放和生物活性尚不清楚 研究哪些细胞因子直接调节P450。该项目将使用 培养大鼠肝细胞以确定哪些细胞因子直接调节 P450，以及P450亚型是否受细胞因子的不同影响。 大鼠肝细胞将在Williams E的Matrigel基质上培养 含有胰岛素和地塞米松的培养液。最近，这样的老鼠 肝细胞培养可被苯巴比妥(PB)诱导 P450IIB 1/2恢复到体内水平。初步结果： 肝细胞培养证实了IL-6的直接作用。 PB诱导P450IIB 1/2。肝细胞培养将用于 进一步阐明浓度反应和时间关系 IL-6改变PB对苯氧基间苯二酚O-脱乙基酶的诱导 活性，P450IIB1/2酶活性的量度。蛋白质印迹 使用P450IIB1/2抗体的分析将用于研究效果 IL-6对P45011B1/2蛋白水平和转换率的影响。解 肝细胞总RNA的杂交将使用 标记P450IIB1和P450IIB2基因的寡核苷酸探针 评价IL-6对mRNA水平和转换率的影响。转录 将使用核径流分析来确定比率。类似 实验将用于评估IL-1、肿瘤坏死因子和干扰素的能力 抑制P45011B1/2的诱导。第三，项目将确定 如果IL-6、IL-1、肿瘤坏死因子和干扰素影响其他P450的诱导 大鼠肝细胞培养中的异构体。细胞因子对血管内皮细胞3-羟色胺的影响 甲基胆蒽诱导P450IA1/2和乙醇诱导P450IA1/2 将检查P450IIE1。 乙氧基间苯二酚O-脱乙基酶活性和对硝基苯酚羟基酶活性 以P450IA1/2和P450IIE为指标进行活性测定 活动分别为。特异性抗体和标记的cDNA P450IA1/2和P450IIE1的寡核苷酸探针将用于 Western印迹分析和溶液杂交分析， 探讨细胞因子对蛋白质和信使核糖核酸水平及周转的影响。 了解细胞因子对P450的影响是至关重要的 为了了解感染或感染期间药代动力学的变化 炎症，以及在癌症免疫治疗期间。

The objective of the research is to elucidate the role and mechanism of action of cytokines mediators of the acute phase response in regulation of cytochrome P450 (P450), a family of hemoproteins responsible for biotransformation of many drugs, other chemicals, and certain endogenous substrates. The cytokines to be studied are interleukin 6 (IL-6), interleukin 1 (IL-1), interferon (INF), and tumor necrosis factor (TNF). Cytokine effects on P450 may explain altered biotransformation occurring during infection and inflammation. In addition, effects on P450 by cytokines when used in cancer immunotherapy or other immune disorders may result in drug-drug interaction, influencing the disposition and toxicity of concurrently used drugs. Although effects on P450 have been shown during the acute phase response, or after the administration of cytokines, because of the complex interplay among the cytokines to affect each others synthesis, release, and biological activity it is unclear from in vivo studies which cytokines directly regulate P450. The project will use cultured rat hepatocytes to determine which cytokines directly regulate P450, and if P450 isoforms are differentially affected by cytokines. Rat hepatocytes will be cultured on Matrigel matrix in Williams E medium containing insulin and dexamethasone. Recently, such rat hepatocyte cultures been shown to be inducible by phenobarbital (PB) for P450IIB 1/2 to levels obtained in vivo. Preliminary results with hepatocyte culture demonstrated a direct effect of IL-6 to inhibit the PB induction of P450IIB 1/2. Hepatocyte cultures will be used to further elucidate the concentration response and temporal relationship for IL-6 to alter PB induction of benzyloxyresorufin O-deethylase activity, a measure of P450IIB1/2 enzymatic activity. Western blot analysis using antibodies to P450IIB1/2 will be used to study effects of IL-6 on the level and turnover of P45011B 1/2 protein. Solution hybridization on total RNA of hepatocytes will be performed using a labelled cDNA oligonucleotide probe for P450IIB1 and P450IIB2 mRNA to evaluate effects of IL-6 on mRNA level and turnover. Transcription rate will be determined using nuclear run-off assay. Similar experiments will be used to evaluate the ability of IL-1, TNF and INF to inhibit P45011B 1/2 induction. Thirdly, the project will determine if IL-6, IL- 1, TNF, and INF affect the induction of other P450 isoforms in rat hepatocyte cultures. Effects of cytokines on 3- methylcholanthrene induction of P450IA1/2 and ethanol induction of P450IIE1 will be examined. Ethoxyresorufin O-deethylase activity, and p-nitrophenol hydroxylase activity will be assayed as indices of P450IA1/2, and P450IIE enzymatic activity, respectively. Specific antibodies and labelled cDNA oligonucleotide probes for P450IA1/2, and P450IIE1 will be used in Western blot analysis and solution hybridization assays, respectively, to assess cytokine effects on level and turnover of protein and mRNA. Knowledge of the effects of cytokines on P450 is of utmost importance for understanding alterations in pharmacokinetics during infection or inflammation, as well as during cancer immunotherapy.