Delayed Radioprotection by Thiols

描述（由申请人提供）：本研究的目的是表征细胞暴露于非蛋白硫醇（NPT）后诱导的延迟辐射防护效应。潜在的假设是，细胞暴露于NPT导致氧化还原敏感核转录因子kappaB （NFkappaB）的激活和锰超氧化物歧化酶（MnSOD）基因表达的增强，随后细胞内活性MnSOD蛋白水平升高。MnSOD是一种抗氧化蛋白，可以保护机体免受电离辐射诱导的氧化自由基。NPT在较晚时间诱导MnSOD水平升高的能力导致延迟的辐射防护效果，这将对一般的辐射防护，特别是肿瘤防护产生影响。该研究主要针对四种临床批准的NPT：氨磷汀、mesna、nacetyl半胱氨酸（NAC）和卡托普利。这些核不扩散物质目前正在临床使用，它们在接受癌症治疗的患者的肿瘤中诱导延迟放射防护作用的潜力是一个迄今未被认识到的问题。提出了四个具体目标。Aim 1旨在验证假设，即一定剂量的NPT会增强MnSOD基因表达，导致细胞内活性MnSOD蛋白水平升高，同时在培养的肿瘤和非肿瘤细胞中过氧化氢酶和谷胱甘肽过氧化物酶（GPx）活性增加。目标2将检验MnSOD活性升高将导致增强辐射防护的假设，特别是在2gy辐射下。Aim 3的重点是验证动物单次或多次暴露于NPT都会导致正常组织和肿瘤组织中MnSOD基因表达和蛋白活性升高的假设，后者在动物后腿生长为肺肿瘤结节或实体瘤。Aim 4将通过定量生存试验（即脾脏集落试验、肠道微集落试验、肺集落试验和TCD50试验）验证MnSOD活性升高会导致对选定的正常组织和肿瘤组织延迟放射保护的假设。另外两种假设也将被检验。MnSOD活性升高可导致过氧化氢酶和GPx活性相关的抗氧化失衡，导致过氧化氢积累和辐射致敏。第二，MnSOD水平的升高而不伴随酶活性的增加可能是由过氧亚硝酸盐（ONOO-）对MnSOD的硝化作用引起的。

DESCRIPTION (provided by applicant): The goal of this investigation is the characterization of the delayed radioprotective effect induced following exposure of cells to non-protein thiols (NPT). The underlying hypothesis is that NPT exposure of cells results in activation of the redox sensitive nuclear transcription factor kappaB (NFkappaB) and enhancement in manganese superoxide dismutase (MnSOD) gene expression followed by elevated intracellular levels of active MnSOD protein. MnSOD is an anti-oxidant protein that can confer protection against oxidative radical species induced by ionizing radiation. The ability of NPT to induce at a later time elevated MnSOD levels results in a delayed radioprotective effect that will have implications in radioprotection in general, and tumor protection in particular. This study focuses on four clinically approved NPT, amifostine, mesna, Nacetylcysteine (NAC), and captopril. These NPT are currently in clinical use, and the potential for their induction of a delayed radioprotective effect in tumors of patients undergoing cancer treatment is a here-tofore unrecognized problem. Four specific aims are proposed. Aim 1 seeks to test the hypothesis that NPT over a range of doses will enhance MnSOD gene expression, leading to elevated intracellular levels of active MnSOD protein accompanied by increased catalase and glutathione peroxidase (GPx) activities in cultured tumor and non-tumor cells. Aim 2 will test the hypothesis that elevated MnSOD activity will result in enhanced radioprotection, especially at 2 Gy. Aim 3 is focused on testing the hypothesis that either single or multiple exposures of animals to NPT will result in elevated MnSOD gene expression and protein activity in both normal and tumor tissues, the latter grown as pulmonary tumor nodules or solid tumors in the hind legs of animals. Aim 4 will test the hypothesis that elevated MnSOD activity will result in delayed radioprotection to selected normal and tumor tissues as determined by quantitative survival assays, i.e., spleen colony assay, intestinal microcolony assay, lung colony assay, and the TCD50 assay. Two alternative hypotheses will also be tested. One elevated MnSOD activity can result in anti-oxidant imbalance relating to catalase and GPx activities, resulting in hydrogen peroxide buildup and radiation sensitization. Two, elevation in MnSOD level without a concomitant increase in enzymatic activity can result from nitration of MnSOD by peroxynitrite (ONOO-).