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

铁

• 批准号: 6258562
• 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/6258562
• 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病患者易发生心力衰竭、肝功能衰竭和II型糖尿病。 尽管（极有可能）推测铁介导的组织损伤涉及细胞氧化和还原当量的共谋，但病理生理事件尚未完全阐明。 目前的调查代表了一种尝试，以确定铁对细胞内细胞器，特别是线粒体和溶酶体的毒性作用。 处于危险中的组织-心脏，肝脏和胰腺β细胞-都具有高度活性的线粒体，这些线粒体偶然产生能够与细胞内铁产生协同毒性的活性氧物质。我们的研究围绕三个具体的假设：（1）铁对具有活性线粒体的细胞更具毒性。 (2)线粒体基因组优先被铁介导的氧化反应损伤，突变事件的积累导致线粒体功能障碍。 (3)“松散的”细胞内铁也会导致溶酶体的氧化不稳定，导致消化酶泄漏到细胞质中，并最终导致细胞凋亡或坏死性细胞死亡。 这些假设将通过以下方式进行检验：（1）确定培养的具有活性线粒体的骨髓细胞和成肌细胞是否比线粒体功能耗尽的细胞更容易受到铁负荷的损伤（通过在溴化乙锭中长期培养或用选定的抑制剂处理）。 (2)使用全长定量聚合酶链反应，我们将确定是否在铁负载培养成肌细胞的线粒体基因组积累的修改，防止通读的聚合酶相比，核基因组内的基因长度相似。 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)还将对先天性或后天性铁超载的小鼠、表达50%正常锰超氧化物歧化酶（线粒体形式的酶）、镰状血红蛋白和地中海贫血的小鼠进行线粒体与核DNA损伤和溶酶体完整性的类似研究。 由此产生的信息将被用来帮助探测目前可用的和实验性螯合剂的效率，不仅在促进铁的排泄，而且在预防确定类型的细胞损伤。