IRON & OXYGEN BIOLOGY IN SCD, HH & BETA-THAL MICE

铁

• 批准号: 6381947
• 负责人: ELIZABETH C THEIL
• 金额: $ 19.34万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6381947
• 关键词: DNA damage; apoptosis; disease /disorder model; iron disorder; iron metabolism; iron poisoning; iron storage disorder; laboratory mouse; lysosomes; mitochondria; mitochondrial DNA; myoblasts; oxidative stress; polymerase chain reaction; sickle cell anemia; superoxide dismutase; thalassemia; tissue /cell culture

Patients with primary or secondary hemochromatosis are liable to cardiac and hepatic failure, and type II diabetes. Despite the (highly likely) conjecture that iron-mediated tissue damage involves the conspiracy of cellular oxidizing and reducing equivalents, the pathophysiologic events have not been fully elucidated. The present investigations represent an attempt to define the toxic effects of iron on intracellular organelles, in particular, mitochondria and lysosomes. The tissues at risk- heart, liver and pancreatic beta cells - all have highly active mitochondria which incidentally generate activated oxygen species capable of causing synergistic toxicity with intracellular iron. Our investigations center about three specific hypotheses: (1) Iron is more toxic to cells with active mitochondria. (2) The mitochondrial genome is preferentially damaged by iron-mediated oxidative reactions and accumulation of mutational events leads to mitochondrial dysfunction. (3) 'Loose' intracellular iron also causes the oxidative destabilization of lysosomes, causing leak of digestive enzymes into the cell cytoplasm and eventuating in apoptotic or necrotic cell death. These hypotheses will be tested by: (1) Determining whether cultured myeloid cells and myoblasts with active mitochondria are more readily damaged by iron loading than are cells depleted in mitochondrial function (via long-term culture in ethidium bromide or treatment with selected inhibitors). (2) Using full-length quantitative polymerase chain reaction, we will determine whether the mitochondrial genome in iron-loaded cultured myoblasts accumulates modification which prevent read-through by the polymerase compared with similar lengths of genes within the nuclear genome. In these experiments, several techniques will also be used to estimate the state of 'intactness' of cellular lysosomes. (3) Similar investigations of mitochondrial vs. nuclear DNA damage and lysosomal integrity will also be conducted on mice with congenital or acquired iron-overload, mice expressing 50 percent of normal manganese superoxide dismutase (the mitochondrial form of the enzyme), sickle hemoglobin and thalassemia. The resulting information will be used to help probe the efficiency of presently available and experimental chelators, not only in promoting iron excretion but also in the prevention of defined types of cellular damage.

原发性或继发性血色素沉着病的患者容易患心脏和肝衰竭，以及II型糖尿病。 尽管（极有可能）猜想铁介导的组织损伤涉及细胞氧化和还原等效物的阴谋，但尚未完全阐明病理生理事件。 本研究是指定铁对细胞内细胞器的毒性作用的尝试，尤其是线粒体和溶酶体。 风险心脏，肝脏和胰腺β细胞的组织都具有高度活性的线粒体，该线粒体偶然产生活化的氧，能够用细胞内铁引起协同毒性。我们的研究集中在三个特定的假设中：（1）铁对活性线粒体的细胞更具毒性。 （2）线粒体基因组优先损害了铁介导的氧化反应和突变事件的积累会导致线粒体功能障碍。 （3）“松散”的细胞内铁还会引起溶酶体的氧化不稳定，从而导致消化酶渗入细胞质中，并在凋亡或坏死细胞死亡中导致渗透。 这些假设将通过：（1）确定与线粒体功能中耗尽的细胞（通过在溴化乙乙层中的长期培养或使用选定抑制剂处理的长期培养），确定具有活性线粒体的培养的髓样细胞和成肌细胞更容易受到铁的损害。 （2）使用全长定量聚合酶链反应，我们将确定在载有铁的培养的肌细胞中的线粒体基因组是否会累积修饰，从而可以防止聚合酶与核基因组中的基因相比。 在这些实验中，还将使用几种技术来估计细胞溶酶体的“完整性”状态。 （3）对线粒体与核DNA损伤和溶酶体完整性的类似研究也将在先天性或获得的铁超载的小鼠上进行，表达50％的正常锰超氧化物歧化酶（酶的线粒体形式）的小鼠，镰状血红蛋白和thalassemia。 所得的信息将用于帮助探测目前可用和实验性螯合剂的效率，这不仅用于促进铁排泄，而且还用于预防定义的细胞损伤类型。