Collaborative Projects In Environmental Health Sciences

工作摘要：该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； 4) 制定生物学重要化合物分析策略。该项目不包括在严格的服务基础上进行的许多分析。我们继续与 LPP D. Zeldin 合作，通过负离子化学电离 (NICI) 质谱下的选择离子监测 (SIM) 来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 (EET) 和羟基二十碳三烯酸 (HETE) 表现出显着的生物效应，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非常重要。此外，与花生四烯酸代谢相关的酶对环境挑战的反应可能是环境引起的疾病的重要因素/指标。除了花生四烯酸代谢物之外，我们还对雄性和雌性 CYP2J5 敲除小鼠以及对照野生型小鼠的尿液中的异前列烷进行定量。雌性基因敲除小鼠患有高血压，我们正在测定异前列腺素的水平，作为高血压引起的氧化应激的标志物。 D. Zeldin 的另一项研究旨在检查 CYP2J2 在培养的牛主动脉内皮细胞 (BAEC) 缺氧-复氧诱导的损伤中的作用。与用不相关的绿色荧光蛋白（GFP）cDNA转染的细胞相比，用CYP2J2 cDNA转染BAEC导致CYP2J2表达和花生四烯酸环氧化酶活性增加。 HR 对 GFP 转染的 BAEC 造成显着损伤；然而，在 CYP2J2 转染细胞中，HR 诱导的损伤明显减弱 (p < 0.01)。在 GFP 转染的 BAEC 中，HR 增加细胞 8-异前列腺素 F-2 (P < 0.05)，并降低 eNOS 表达、L-精氨酸摄取和转化以及亚硝酸盐产生 (p < 0.01)。 CYP2J2 转染减弱了 HR 诱导的 8-异前列腺素 F-2 α 增加 (P < 0.05)，并减少了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量 (p < 0.05)，但没有显着影响 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。

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.