Collaborative Projects In Environmental Health Sciences

工作总结：该项目包括质谱分析师与研究所内外其他团体合作解决共同感兴趣的问题的努力。这些项目的主要重点是：1）未知化合物的结构测定; 2）生物途径的鉴定和/或确认; 3）定量;和4）开发用于分析生物重要化合物的策略。本项目中不包括在严格的服务基础上进行的许多分析。我们将继续与D。Zeldin，LPP，在负离子化学电离（NICI）质谱下通过选择离子监测（SIM）鉴定和定量生理样品中的花生四烯酸代谢物。花生四烯酸的酶促氧化产物环氧二十碳三烯酸和羟基二十碳三烯酸具有重要的生物学效应，既有有害的，也有有益的。参与氧化过程的酶的表征和酶的位置对于理解酶的功能意义是重要的。此外，参与花生四烯酸代谢的酶对环境挑战的反应可能是环境诱导的疾病的重要因素/指标。除花生四烯酸代谢产物外，我们还定量测定了雄性和雌性CYP 2 J5敲除小鼠和对照野生型小鼠尿液中的异前列烷。雌性基因敲除小鼠患有高血压，我们正在测定异前列烷的水平，作为高血压引起的氧化应激的标志物。 另一项研究与D。Zeldin旨在研究CYP 2 J2在缺氧-复氧诱导的牛主动脉内皮细胞（BAECs）损伤中的作用。与转染不相关的绿色荧光蛋白（GFP）cDNA的细胞相比，转染CYP 2 J2 cDNA的BAEC的CYP 2 J2表达和花生四烯酸环氧合酶活性增加。HR对GFP转染的BAEC有明显的损伤作用，而对CYP 2 J2转染的BAEC损伤作用明显减弱（p < 0.01）。HR增加了GFP转染BAECs细胞8-iso-PGF-2的表达（P < 0.05），降低了eNOS的表达、L-精氨酸的摄取和转化以及亚硝酸盐的产生（P < 0.01）。CYP 2 J2转染减弱了HR诱导的8-异前列腺素F-2 α的增加（P < 0.05），并减少了在常氧条件下通过细胞色素c还原检测到的细胞外超氧化物的量（p < 0.05），但对HR诱导的eNOS表达、L-精氨酸摄取和转化以及亚硝酸盐产生的减少没有显著影响。

Summary of Work: This project includes those endeavors in which the mass spectrometry workgroup collaborates with other groups, both inside and outside the Institute to solve problems of mutual interest. The major focuses of these projects are: 1) structure determination of unknown compounds; 2) identification and/or confirmation of biological pathways; 3) quantitation; and 4) development of strategies for the analysis of biologically important compounds. Not included in this project are the many analyses performed on a strictly service basis. We continue to collaborate with D. Zeldin, LPP, on the identification and quantitation of arachidonic acid metabolites in physiological samples by selected ion monitoring (SIM) under negative ion chemical ionization (NICI) mass spectrometry. The products of enzymatically induced arachidonic acid oxidation, epoxyeicosatrienoic acids (EETs) and hydroxyeicosatrienoic acids (HETEs), exhibit significant biological effects, both harmful and beneficial. The characterization of the enzymes involved in the oxidation process and location of the enzymes is important in understanding the functional significance of the enzymes. Additionally, the response of the enzymes involved with arachidonic acid metabolism to environmental challenges may be a significant factor/indicator of environmentally induced disorders. In addition to the arachidonic acid metablolites, we are quantitating isoprostanes in the urine of male and female CYP2J5 knockout mice and control wild-type mice. The female knockout mice are hypertensive and we are determining the levels of isoprostane as a marker of oxidative stress resulting from hypertension. Another study with D. Zeldin was designed to examine the role of CYP2J2 in hypoxia-reoxygenation-induced injury in cultured bovine aortic endothelial cells (BAECs). Transfection of BAECs with the CYP2J2 cDNA resulted in increased CYP2J2 expression and arachidonic acid epoxygenase activity, compared with cells transfected with an irrelevant green fluorescent protein (GFP) cDNA. HR induced significant injury in GFP-transfected BAECs; however, the HR-induced injury was markedly attenuated in CYP2J2-transfected cells (p < 0.01). HR increased cellular 8-iso-prostaglandin F-2 (P < 0.05), and decreased eNOS expression, L-arginine uptake and conversion, and nitrite production (p < 0.01) in GFP-transfected BAECs. CYP2J2 transfection attenuated the HR-induced increase in 8-iso-prostaglandin F-2 alpha (P < 0.05) and decreased the amount of extracellular superoxide detected by cytochrome c reduction under normoxic conditions (p < 0.05) but did not significantly affect HR-induced decreases in eNOS expression, L-arginine uptake and conversion, and nitrite production.