LIVER TOXICITY OF HEMOGLOBIN SOLUTIONS

血红蛋白溶液的肝脏毒性

• 批准号: 2230765
• 负责人: Ruth E Billings
• 金额: $ 21.69万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2230765
• 关键词: Kupffer's cell; amine oxidoreductase; antioxidants; blood /plasma substitute; crosslink; cytokine; enzyme activity; fluorescent dye /probe; free radical oxygen; hemoglobin Ss; hepatotoxin; laboratory rat; oxidative stress; oxygenases; phagocytosis; polymerization; recombinant proteins; reticuloendothelial system; tissue /cell culture; transcription factor

This project seeks to understand the potential of hemoglobin solutions (HbS), which are suitable for use as red blood cell substitutes, to cause liver toxicity. The toxicity of HbS may depend upon differences in preparations (e.g. cross-linking reagent, degree of polymerization) as well as the physiological and pathophysiological state of the patient. The liver may be especially susceptible to toxicity because it degrades hemoglobin metabolites such as iron, which causes oxidative injury and because it contains large numbers of resident macrophages (Kupffer cells). These cells (the reticuloendothelial system, RES) are activated to phagocytize foreign materials in the circulation. Although impure preparations of HbS undoubtedly activate the RES, it is not clear to what extend endotoxin-free, cross-linked preparations cause this activation. This is an important question because activation of RES stimulates Kupffer cells to secrete cytotoxic substances including superoxide anion and tumor necrosis factor-alpha (TNFalpha). Superoxide is a toxic reactive oxygen intermediate (ROI) which may also be produced directly by hemoglobin or its breakdown product, heme and iron. Therefore, the hypothesis to be investigated in this project is that ROI which are generated directly by oxidation of hemoglobin, heme, or iron synergize with ROI due to activation of Kupffer cells and together they may cause oxidative injury in hepatocytes. Coincident with these events, however, the gene expression of stress proteins such as heme oxygenase and nitric oxide synthase is up-regulated by these same potentially toxic intermediates. The toxicity which is manifested by HbS will depend upon the balance between activation of such protective proteins and the formation of ROI. Altered physiological states such as infection and ischemia may tip the balance toward toxicity because ROI generation is increased already by the pathophysiology. To investigate this hypothesis, we will infuse HbS into normal rats, volume depleted rats, rats pre-treated with endotoxin, and rats with ischemic levers. We will measure oxidative stress (as indicated by plasma GSSG levels), liver toxicity, the extent and nature of Kupffer cell activation or priming, the induction of heme oxygenase, nitric oxide synthase (potentially protective proteins) as well as cytokines such as TNFalpha, IL-1, and IL- 6. Activation of transcription factors known to be important in the oxidative stress response in the liver will also be determined. The contribution of Kupffer cells to these events will be evaluated by use of inhibitors of Kupffer cell activation. ROI formation in hepatocytes will be directly measured in primary cultures treated with HbS or Kupffer cell conditioned medium and HbS. Using these primary cultures, the effectiveness of anti-oxidants will be determined. This in vitro system will also be used to compare various preparations of HbS (e.g. chemically cross-linked HbS vs recombinant HbS.)

这个项目试图了解血红蛋白溶液的潜力。 (HBS)，适合用作红细胞替代品，以引起 肝脏毒性。HBS的毒性可能取决于 制剂(如交联剂、聚合度) 以及患者的生理和病理生理状态。 肝脏可能特别容易受到毒性的影响，因为它会降解 血红蛋白代谢物，如铁，会导致氧化损伤和 因为它含有大量的常驻巨噬细胞(Kupffer 单元格)。这些细胞(网状内皮系统，RES)被激活 吞噬血液循环中的异物。虽然不纯洁 HBS的筹备无疑激活了RES，目前还不清楚是什么 延长无内毒素，交联型制剂会引起这种激活。 这是一个重要的问题，因为激活RES会刺激 库普弗细胞分泌包括超氧阴离子在内的细胞毒性物质 和肿瘤坏死因子-α(TNFpha)。超氧化物是一种有毒物质 也可以直接生产的活性氧中间体(ROI) 由血红蛋白或其分解产物、血红素和铁组成。因此， 本项目中要研究的假设是ROI，它们是 由血红蛋白、血红素或铁的协同氧化直接生成 由于Kupffer细胞的激活而导致的ROI，它们共同可能导致 肝细胞的氧化损伤。然而，与这些事件同时发生的是， 血红素加氧酶和一氧化氮等应激蛋白的基因表达 氧化合成酶被这些同样的潜在毒性上调 中间体。HBS表现出的毒性将取决于 这类保护性蛋白的激活与 ROI的形成。改变的生理状态，如感染和 缺血可能会使天平向毒性倾斜，因为ROI的产生 已经被病理生理学增加了。来调查这件事 假设，我们将给正常大鼠，容量耗竭大鼠， 内毒素预处理的大鼠和缺血水平的大鼠。我们会 测量氧化应激(以血浆GSSG水平表示)、肝脏 毒性，库普弗细胞激活或启动的程度和性质， 诱导血红素加氧酶、一氧化氮合酶(潜在地 保护性蛋白)以及细胞因子，如肿瘤坏死因子α、白介素1和白介素1- 6.已知的转录因子的激活在 肝脏中的氧化应激反应也将被确定。这个 库普弗细胞对这些事件的贡献将通过使用 库普弗细胞激活的抑制剂。肝细胞中ROI的形成 将在用HBS或Kupffer处理的原代培养中直接测量 细胞条件培养液和HBS。使用这些主要培养物， 抗氧化剂的有效性将被确定。这个体外系统 还将用于比较HBS的各种制剂(例如，化学上的 (交联型HBS与重组HBS。)