Oxidative Stress and G6PDH Expression

活性氧（ROS）是在细胞内正常氧化代谢过程中产生的。严格控制这一过程对许多生物事件的发生至关重要，包括酶激活、细胞周期调节和程序性细胞死亡。ROS的异常产生可导致氧化还原稳态失衡，即氧化应激。通常情况下，暴露于环境因素，包括紫外线照射，臭氧，除草剂，农药和金属可以产生额外的活性氧，导致这种不平衡。暴露于这些物质后，ROS水平升高会对蛋白质、核酸和脂质造成损伤，并改变信号转导和基因表达等生理反应。氧化应激的长期后果也与多种毒性和疾病的发病机制有关。在某些情况下，细胞可以利用抗氧化防御系统来应对氧化应激过程中产生的过量ROS。然而，抗氧化防御系统中一些关键成分的工作机制尚不完全清楚。例如，NADPH是一种关键的生物辅助因子，有助于维持细胞的促氧化和抗氧化状态之间的平衡。戊糖磷酸途径通过葡萄糖-6-磷酸脱氢酶（G6PDH）和6-磷酸葡萄糖酸脱氢酶的活性产生细胞所需的大部分NADPH。G6PDH是该途径的关键限速酶，不仅调控碳在该途径中的流动，还调控NADPH的产生。由于NADPH是维持细胞氧化还原平衡所必需的，而G6PDH对其产生很重要，因此似乎有理由得出这样的结论：细胞的氧化还原状态会影响这种重要酶的表达。事实上，该实验室和其他实验室的研究表明，G6PDH基因的表达受细胞氧化状态的调节。然而，这种调节发生的机制尚不清楚。因此，本研究旨在验证环境污染物镉通过降低谷胱甘肽水平影响细胞氧化状态，从而通过氧化还原敏感转录事件调节G6PDH的表达的假设。由于塑料、材料、电池、烟草、电镀、焊接和冶炼等工业生产过程中镉的积累，造成了大量的环境问题。由于镉的蒸气压相对较高，生物体很容易接触到镉，这就解释了镉在水中的高溶解度。该研究将在培养的原代大鼠肝细胞中进行。肝脏是急性无机镉暴露的主要靶器官。此外，由于肝细胞对激素、营养物质或环境因素的代谢反应与体内观察到的代谢反应一致，因此肝细胞作为模型是有吸引力的。这些研究将是此类研究的第一次，并大大增加了我们对细胞氧化状态对主要生化途径（如戊糖磷酸途径）中酶表达的作用的理解。这些研究还将为细胞如何保护自己免受环境因素（如金属）的损害提供有价值的信息。团队学习环境将有助于PI和学生完成工作。

Reactive oxygen species (ROS) are produced inside the cell during normal oxidative metabolism. Tight control of this process is crucial for numerous biological events to occur, including enzyme activation, cell cycle regulation and programmed cell death. Abnormal production of ROS can lead to an imbalance in redox homeostasis known as oxidative stress. Often times exposure to environmental agents, including, UV irradiation, ozone, herbicides, pesticides and metals can produce additional ROS that lead to this imbalance.Upon exposure to these agents, this elevated level of ROS can cause damage to proteins, nucleic acids and lipids, as well as, alter physiological responses such as signal transduction and gene expression. Long term consequences of oxidative stress have also been associated with the pathogenesis of a variety of toxicities and diseases. Under some circumstances, cells can utilize an antioxidant defense system in order to cope with the excess ROS produced during oxidative stress. However, the mechanism by which some key components in the antioxidant defense system work is not completely known. For example, NADPH is a critical biological cofactor that helps to maintain a balance between the prooxidant and antioxidant status of the cell. The pentose phosphate pathway through the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase generates most of the NADPH needed by the cell. G6PDH is the key rate-limiting enzyme in this pathway and thus not only regulates the flow of carbon through this pathway, but also the production of NADPH. Since NADPH is necessary for maintaining the cell's redox balance and G6PDH is important for its production then it seems reasonable to conclude that the redox state of the cell would influence the expression of this important enzyme. Indeed, studies from this laboratory and others have suggested that expression of the gene for G6PDH is regulated by the oxidative status of the cell. However, the mechanism by which this regulation occurs is unknown. Therefore, this research is aimed at testing the hypothesis that the environmental contaminant cadmium, which influences the oxidative state of a cell by decreasing levels of glutathione, regulates the expression of G6PDH through a redox sensitive transcriptional event. Cadmium is generating a great deal of environmental concern due to its accumulation from such industrial practices as production of plastics, materials, batteries, tobacco, electroplating, welding and smelting. Organisms are easily exposed to cadmium due to its relatively high vapor pressure, which accounts for its high solubility in water. The studies will be done in primary rat hepatocytes in culture. The liver is the major target organ for acute inorganic Cd exposure. Additionally, hepatocytes are attractive to use as a model since the metabolic responses to hormones, nutrients or environmental agents consistently mimic those observed in vivo.These studies would represent the first of this kind and add significantly to our understanding of the role of the oxidative state of the cell on the expression of enzymes involved in major biochemical pathways such as the pentose phosphate pathway. These studies would also add valuable information about how a cell protects itself from damage induced from environmental agents such as metals. A team learning environment will facilitate the completion of the work by the PI and students.