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

患有原发性或继发性血色沉着症的患者容易发生心脏和肝脏衰竭，以及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，但其病理生理事件尚未完全阐明。本研究试图确定铁对细胞内细胞器的毒性效应，特别是线粒体和溶酶体。处于危险中的组织-心脏、肝脏和胰腺β细胞-都具有高度活跃的线粒体，这些线粒体顺便产生能够与细胞内铁产生协同毒性的活性氧物种。我们的研究集中在三个具体的假设上：(1)铁对线粒体活跃的细胞毒性更大。(2)线粒体基因组优先受到铁介导的氧化反应的破坏，突变事件的积累导致线粒体功能障碍。(3)“松散”的细胞内铁还导致溶酶体的氧化不稳定，导致消化酶泄漏到细胞质中，最终导致细胞凋亡或坏死性死亡。这些假说将通过：(1)确定具有活跃线粒体的培养的髓系细胞和成肌细胞是否比线粒体功能衰竭的细胞更容易受到铁负荷的损害(通过长期在溴化乙锭中培养或使用选定的抑制剂处理)。(2)利用全长定量聚合酶链式反应，我们将确定与核基因组中类似长度的基因相比，铁负载培养成肌细胞的线粒体基因组是否积累了阻止聚合酶通读的修饰。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)类似的线粒体与核DNA损伤和溶酶体完整性的研究也将在先天性或获得性铁超载小鼠、表达50%正常锰超氧化物歧化酶(酶的线粒体形式)的小鼠、镰刀状血红蛋白和地中海贫血小鼠身上进行。由此得到的信息将被用来帮助探索目前可用的和实验中的螯合剂的效率，不仅在促进铁排泄方面，而且在防止特定类型的细胞损伤方面。

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.