Collaborative Projects In Environmental Health Sciences

工作概述：本项目包括质谱工作组与研究所内外其他小组合作解决共同关心的问题的努力。这些项目的主要重点是：1)未知化合物的结构测定；2)生物学途径的识别和/或确认；3)定量;4)开发生物重要化合物的分析策略。本项目不包括在严格的服务基础上执行的许多分析。我们继续与dr . Zeldin， LPP合作，在负离子化学电离（NICI）质谱下选择离子监测（SIM）鉴定和定量生理样品中的花生四烯酸代谢物。酶促花生四烯酸氧化的产物环氧二十碳三烯酸（EETs）和羟基二十碳三烯酸（HETEs）表现出显著的生物效应，既有有害的，也有有益的。在氧化过程中所涉及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性疾病的重要因素/指标。除了花生四烯酸代谢物外，我们还定量测定了雄性和雌性CYP2J5基因敲除小鼠和对照野生型小鼠尿液中的异前列腺素。雌性敲除小鼠患有高血压，我们正在测定异前列腺素水平，作为高血压引起的氧化应激的标志。

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.