University of Iowa Radiation and Free Radical Research Core

The Radiation and Free Radical Research Core focuses on providing state of the art technologies to investigators studying the role of oxidative stress and redox biology. There is growing evidence that oxidative stress and redox biology are critical determinants of cancer biology including the processes of initiation, promotion, and progression to malignancey as well as the prevention and treatment of cancer and degenerative diseases of aging. The RFRRC was established to provide easy access to free radical and radiation biology expertise, reagents, technologies, and analysis for investigators doing basic, pre-clinical, and clinical research. The RFRRC offers the following three basic services to the university community and to researchers and private companies across the country:

1) Ionizing Radiation Services (IRS) and phosphorimaging as well as cell cycle analytical tools critical to undertanding cellular responses to radio-chemo-therapy. These services include: whole body radiation; partial body radiation; dosimetry; autoradiography; chemilumenescent quatification; high dose and low dose rate radiation; and preclinical radiotherapy irradiations for aminal experiments as well as cell cycle analytical tools critical to undertanding cellular responses to radio-chemo-therapy. The x-ray source is capable of delivering filtered or unfiltered x-rays, with maximum x-ray energy of 300 kVp. The gamma ray source is capable of delivering low or high dose rates of monoenergetic (0.667 MeV) gamma radiation, the range being from 10-3200 cGy/minute. Over the past several years the total doses to inanimate or animate objects have ranged from 5 cGy-13,000,000 cGy. The x-ray and gamma-ray facilities have been used for total body or partial body irradiation of tumors in experimental mice, rats, dogs, as well as cancer or normal cell cultures grown in vitro.

2) Electron Paramagnetic Resonance Spectroscopy (EPR) services as well as other detection methologies for measuring free radicals, singlet oxygen, nitric oxide and the array of related oxidants and oxidative damage products are available. The EPR facility assists users in the detection of: free radicals in systems that range from solids, solutions, cells, tissues and whole animals, nitric oxide and related metabolites including peroxynitrite, MDA/TBARS, indicators of lipid peroxidation that are detected with fluorescence spectroscopy, oxidation of fluorescent dyes as indicators of oxidation reactions in living cells, UV-Vis spectroscopy for kinetic studies of PhGPx, Cellular oxygen consumption for studying metabolic oxidations reactions, oxidative stress-indicators using HPLC: including DNA damage products, antioxidants such as vitamins C and E, beta-carotene, precise cell volume measurements.

3) Antioxidant Enzyme Services (AES) to provide easy access to technologies for modifying and measuring molecules responsible for pro-oxidant formation, metabolism of reactive oxygen species, and mediators of redox biology including: anti-oxidant proteins/enzymes, small molecular weight cellular thiols and reductants, as well as redox mediated signaling and gene expression pathways governing growth, differentiation, and cell injury processes. These services include molecular biology reagents to transfect and infect cells with adenovirus and plasmid vectors that cause over expression of anti-oxidant proteins, providing QA on the levels of expression and enzymatic activity of cells, and QA to ensure that the desired stable integration or transient expression of various proteins has occurred as well as RTPCR analysis of SNPs and other genes of interest in free radical biology. Resources available through the AES include antibodies, cDNAs, cell lines, transgenic mice models, primers, and well characterized expression vectors. The AES works closely with other cores in this regard, including the Gene Transfer Vector Core and the DNA Core. Techniques include: measurement of thiols and antioxidant enzyme activity using activity assays, activity gels, spectrophotometric assays and HPLC assays; measurement of immunoreactive protein for antioxidant enzymes using western blotting; measurement of steady-state mRNA levels for antioxidant enzymes using northern and PCR analysis; determination of antioxidant enzyme gene copy number and gross chromosomal changes; transfection and characterization of cell lines expressing antioxidant enzyme sense and antisense cDNAs; maintain, distribute, and provide a repository for reagents used in the study of antioxidant proteins: i.e., antibodies, cDNAs, expression vectors, RNAi reagents and cell lines; provide expertise and equipment for studying a range of O2 tensions from radiobiological hypoxia (<0.1% O2) to physiologically relevant tissue O2 tensions (4-6% O2) in tissue culture experiments measuring clonogenic cell survival.

The following enzyme assays are routinely available: copper-and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), catalase (CAT), glutathione transferases (GSTs), glutamyl transpeptidase (GGT), glutamylcysteine synthetase (GCS), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), thioredoxin reductase (TRR), and glutathione peroxidases (GPx). Assays for the detection of prooxidant production in living cells (superoxide, hydrogen peroxide, lipid peroxidation products, etc.) are also be available. Antibodies, cDNA probes, transplatable human tumor xenograph models, will be provided. Services to measure glutathione/glutathione disulfide, thioredoxin/thioredoxin disulfide, glucose, ATP, lactate, NADP+/NADPH and NAD+/NADH are also available for monitoring oxidative metabolism. AES staff will carry out the proposed work or will advise the user and staff on how to perform and analyze the experiments.