Oxidative Stress and Radiation
What Is Oxidative Stress?
Scientists at PNNL are studying the signal transduction pathways involved in radiation and oxidative stress. Using retroviral techniques, live human mammary epithelial cells (HMEC) have been tagged with a green fluorescent probe for the protein, mitogen-activated protein kinase (MAPK). Using fluorescent microscopy, the movement of tagged proteins in response to stimuli, such as radiation, can be tracked in real time. Cell nuclei are shown in red.
Oxidative stress describes cell damage caused by an overabundance of oxidants, including reactive oxygen species (ROS, e.g., oxygen ions, free radicals, and peroxide). ROS are harmful in excess, but some level of them is necessary for important cellular functions. Some cells produce ROS to kill invading microbes, and ROS are involved in cell signaling (1). Also, oxidative stress plays a role in cellular processes, such as aging and apoptosis.
In a balanced cell state, ROS are produced as a byproduct of metabolic processes and the level of ROS can be controlled with antioxidants, such as small molecular weight dietary supplements, including vitamin E and vitamin C; small molecular weight peptides and cofactors, including glutathione and pyruvate; and enzymes, including superoxide dismutase and catalase (2). In a state of cellular imbalance, in which the levels of oxidants outweigh the levels of antioxidants, damage is caused to nuclear and mitochondrial DNA, proteins, and lipids. If this damage is irreparable, then injury, mutagenesis, carcinogenesis, accelerated senescence, and cell death can occur (2). Oxidative stress has been linked to diseases, including some allergic and inflammatory skin diseases (3), Alzheimers (4), atherosclerosis in diabetes patients (5), and Lou Gehrig disease (6).
How Are Oxidative Stress and Radiation Related?
A state of oxidative stress can be induced by a number of factors, including chemical agents and radiation. Radiation-induced damage and oxidative stress are closely tied. Irradiated cells produce damaging ROS. Treatment with antioxidants can dampen the detrimental effects of radiation exposure (2). Cytoplasmic irradiation can result in damage to nuclear DNA, and experiments with free radical scavengers have shown this DNA damage is dependent on ROS generation (2, 7).
Evidence suggests that a cell’s oxidative state not only plays a role at the time of radiation exposure, but also has effects long after exposure. As the result of irradiation, cells can produce ROS for several minutes or even hours after being exposed (2). In addition to ROS production, cells are stimulated to increase their expression of antioxidants (2). This abundance of antioxidant defenses may play a role in the radioadaptive response (2), defined as the triggering of cellular effects upon radiation exposure that protect a cell when exposed to a subsequent radiation challenge.
In response to ionizing radiation exposure, several signal transduction pathways (e.g., ERK1/2, JNK, p38, and ATM) and transcription factors (e.g., AP1, NF?B, GADD153, and p53) are activated (2). Many of these signaling and gene expression pathways are sensitive to changes in intracellular oxidation/reduction reactions (2). Perhaps it is the case that as the result of radiation exposure, changes are induced in intracellular metabolic redox reactions that initiate the activation of signal transduction, transcription factors, and gene expression (2).
Oxidative Stress and Radiation Research at PNNL
At Pacific Northwest National Laboratory (PNNL), we are studying radiation and oxidative stress in RAW 264.7 macrophages and human mammary epithelial cells (HMEC) with emphases on the calcium-binding protein, calmodulin (CaM), and on low dose radiation. RAW 264.7 and HMEC display similar calcium-dependent stress responses, and are expected to permit the generalization of cellular stress responses. CaM is a central point in calcium signaling for both immediate and long-term responses to calcium transients for the regulation of energy metabolism and gene transcription. We are investigating the hypothesis that cells use and interpret subtle changes in intracellular calcium as a cue for changes in their extracellular environment, and that this information is integrated through competing calmodulin (CaM) protein complexes that modulate signal transduction. With regard to radiation, it would follow from this hypothesis that CaM and associated signaling complexes are sensors of radiation, including low dose radiation, that activate changes in energy metabolism and gene expression in response to exposure. The overall goals of our oxidative and radiation stress research are:
- to define better the signal transduction pathways involved in radiation and oxidative stress
- to identify the molecular mechanisms of the radioadaptive response
- to gain insight into the cellular repair system for radiation-induced DNA damage and DNA damage produced by endogenous oxidative stress.
Proteomics of Membrane Proteins: Relating Calcium Signaling and Oxidative Stress – At PNNL, we are developing technical capabilities for proteomic analyses of membrane protein complexes and post-translational modifications. Our current research is focused on the proteomics of oxidative modifications of myocyte proteins.The following projects are supported by the U.S. Department of Energy's (DOE's) Low Dose Radiation Research Program, which is managed by the Life Sciences Division of DOE's Office of Biological and Environmental Research (OBER).
Molecular Mechanisms Underlying Cellular Adaptive Response to Low Dose Radiation Project - Researchers at PNNL are studying the molecular mechanisms by which cells adapt to low dose radiation exposure with an emphasis on calmodulin (CaM), a protein sensitive to oxidative stress.
Mechanisms of Three-Dimensional Intercellular Signaling in Mammary Epithelial Cells in Response to Low Dose, Low Linear Energy Transfer (LET) Radiation: Implications for the Radiation-Induced Bystander Effect - Using a high-speed confocal microscope that can simultaneously acquire two-color images, PNNL researchers are visualizing radiation-induced bystander signaling in model, mammary epithelial, three-dimensional cell cultures in situ. As part of this project, we are discerning the mechanism of low linear energy transfer radiation-induced bystander effects. Also, different types or levels of cell signaling may be identified than in traditional, two-dimensional cell cultures.
Molecular Energetics of Clustered Damage Sites - Using state-of-the-art computational chemistry models, scientists at PNNL are studying the qualitative and quantitative similarities and differences in the DNA repair of clustered damage sites caused by ionizing radiation and singly damaged sites produced by endogenous oxidative stress. Through this work we will gain insights into the way biological responses to ionizing radiation differ from responses to endogenous damage.
Effects Of Low Doses of Radiation on DNA Repair - Researchers at PNNL are studying the repair of different types of DNA damage caused by low dose radiation as well as by the normal internal operation of the cell. As part of this project, we are also determining if unique forms of DNA repair system damage are induced by low doses of cosmic radiation exposure present during space missions.
Cell Transforming Bystander Signals - Researchers at PNNL are defining the role of annexin A2 and osteopontin, paracrine signals produced by irradiated cells that are firmly linked to human carcinogenesis, in the transformation response induced in bystander cells by low dose radiation.
Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation – We are studying the mechanisms of non-targeted radiation damage using a GM10115 cell model. Through this project, we have the opportunity to apply state-of-the-art proteomic and bioinformatic tools to study the mechanisms of radiation-induced genomic instability.
Using a Low LET Electron Microbeam to Investigate Non-Targeted Effects of Low Dose Radiation – In collaboration with the University of Maryland in Baltimore, researchers at PNNL are using a low LET electron microbeam to examine non-targeted effects associated with low dose radiation exposure, including induced genomic instability and bystander effects.
The following projects are supported by the National Institutes of Health.
Regulation of Calcium Transport in Cardiac Muscle – This research team is identifying the physical mechanisms that regulate calcium resequestration by the Ca-ATPase in cardiac sarcoplasmic reticulum (SR) membranes. Identifying the structural mechanisms underlying the regulation of Ca-ATPase function will permit the design of effective therapies to alleviate the loss of cardiac function in the failing heart.
Structural Basis – Altered Calcium Homeostatis of Aging – Researchers at PNNL are working to identify the molecular mechanisms that result in the age-dependent loss of critical cellular functions, which correlate with an increased sensitivity to stress and diminished capabilities of the elderly.
3-Nitrotyrosine in Aging of Skeletal Muscle and Heart - The long-term goal of this research team is to understand the origin of the prolonged intracellular calcium transients that accompany aging of skeletal and cardiac muscle.
Magnesium NMR of DNA Repair Proteins - The research team at PNNL is applying a novel method using low temperature (10K) solid-state 25Mg NMR to study DNA repair proteins and their model systems.
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(7) Wu LJ, G Randers-Pehrson, A Xu, CA Waldren, CR Geard, Z Yu, and TK Hei. 1999.“Targeted Cytoplasmic Irradiation with Alpha Particles Induces Mutations in Mammalian Cells.” Proceedings of the National Academy of Sciences. 96(9):4959-4964.