In Vivo Detection of Free Radical Generation

这项研究的目的是更好地了解自由基在从分子/细胞生物学到整个动物乃至最终到人类的各个层次的炎症反应机制中的参与，并推进自由基介导的疾病过程的基础和临床研究。总体而言，该项目测试了以下假设：1)自由基是糖尿病和炎症复杂发病机制中的致病分子；2)特定的生化途径参与触发自由基的产生，这些自由基可能作为与人类疾病相关的炎症反应的介质和/或调节器。在这项研究中，内毒素、外毒素、链脲佐菌素、丙酮等引起的体内自由基的产生在炎症、肺炎、糖尿病和酮病等特定疾病状态下被显示出来。 研究成果： I)糖尿病和酮病中的自由基中间体。我们提供了体内自由基形成的ESR和免疫自旋捕捉数据，这是链脲佐菌素诱导的糖尿病发生的一个机制，有助于肝脏和肾脏的脂质过氧化和蛋白质硝化。研究表明，iNOS参与了羟基自由基介导的脂质过氧化反应，因为iNOS的过度表达与所检查器官中自由基产量的增加有关。此外，我们还表明，糖尿病中酮症的模型丙酮也可以通过自由基依赖机制导致蛋白质氧化和脂质过氧化，这种机制主要是由iNOS过度表达驱动的。 Ii)炎症时产生的自由基。已有研究表明，超抗原(SEB)诱导的间质性肺炎(IP)中自由基的产生依赖于黄嘌呤氧化酶、铁和NO的诱导。结论巨噬细胞毒物、黄嘌呤氧化酶抑制剂、铁络合剂或诱导型一氧化氮合酶抑制剂可能是治疗间质性肺炎肺泡炎和纤维化的潜在药物。 三)金属和有毒化学品产生的自由基。阻燃剂的广泛使用会对环境造成危害，因此确定它们的毒性机理是很有意义的。在所有的BFR中，产量最大的是3，3‘，5，5’-四溴双酚A(TBBPA)。过氧化氢通过Fenton反应生成的羟基自由基在体内与DMSO反应生成甲基自由基，并被POBN捕获。这些结果提示，TBBPA对大鼠的肝毒性可能是由于其代谢产物2，6-二溴氢醌与其相应的半醌自由基发生氧化还原反应而在体内产生羟基自由基所致。

The goal of this study is to better understand the involvement of free radicals in the mechanism of inflammatory responses at all levels from molecular/cellular biology to whole animals and, ultimately, to humans, and to advance both basic and clinical research in free radical-mediated disease processes. Overall this project tested the hypotheses that: 1) free radicals are causative molecules in the complex pathogenesis of diabetes mellitus and inflammation; and 2) specific biochemical pathways are involved in triggering generation of free radicals that may act as mediators and/or modulators of inflammatory reactions associated with human disease. In this study, free radical generation in vivo caused by endotoxin, exotoxin, streptozotocin, acetone and others has been shown in specific disease states of inflammation, pneumonia, diabetes, and ketosis. Research Accomplishments: i) Free Radical Intermediates in Diabetes Mellitus and Ketosis. We have provided ESR and immuno-spin trapping data for in vivo free radical formation as a mechanism that occurs in streptozotocin-induced diabetes and contributes to lipid peroxidation and protein nitration in the liver and kidneys. The studies demonstrated involvement of iNOS in hydroxyl radical-mediated lipid peroxidation since iNOS overexpression correlated with increased free radical production in the organs examined. In addition, we have shown that administration of acetone, a model of ketosis in diabetes, can also lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism also driven mainly by iNOS overexpression. ii) Free Radical Production in Inflammation. It has been demonstrated that free radical production in superantigen (SEB)-induced interstitial pneumonia (IP) depends on xanthine oxidase, iron and NO induction. It is concluded that macrophage toxicants, xanthine oxidase inhibitors, iron chelators, or inducible nitric oxide synthase inhibitors may be potential therapeutic agents against alveolitis and fibrosis in interstitial pneumonia iii)Free Radical Generation by Metals and Toxic Chemicals. Wide use of flame retardants can pose an environmental hazard so it is of interest to determine the mechanism of their toxicity. Of all the BFRs, 3,3',5,5'-tetrabromobisphenol A (TBBPA) is produced in the largest volume. The hydroxyl radical generated via the Fenton reaction from hydrogen peroxide reacts in vivo with DMSO to give the methyl radical, which is trapped by POBN. These observations suggest that the hepatotoxicity of TBBPA in rats may be due to the in vivo generation of the hydroxyl radical as a result of redox reactions involving the TBBPA metabolite 2,6-dibromohydroquinone and its corresponding semiquinone radical.