ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 289, No. 1, August 15, pp. 62-70, 1991 Inhibition of Oxygen-Dependent Radiation-Induced Damage by the Nitroxide Superoxide Dismutase Mimic, Tempo1 James B. Mitchell,’ William DeGraff, Dwight Kaufman, Murali C. Krishna, Amram Samuni,* Eli Finkelsteiqt Min S. Ahn, Stephen M. Hahn, Janet Gamson, and Angelo Russo Radiobiology Section, Radiation Oncology Branch, Clinical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; *Molecular Biology, School of Medicine, Hebrew Uniuersity, Jerusalem, Israel 91010; and TRadiation Oncology Department, Elizabeth General Medical Center, Elizabeth, New Jersey 07202 Received January 31,1991, and in revised form April 18, 1991 Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hy- drogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hy- droxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radia- tion. Treating Chinese hamster cells under aerobic con- ditions with 5, 10,50, and 100 IIIM Tempo1 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempo1 afforded no protection. Tempo1 treatment under hypoxic conditions did not provide radioprotec- tion. Aerobic X-ray protection by Tempo1 could not be attributed to the induction of intracellular hypoxia, in- crease in intracellular glutathione, or induction of intra- cellular SOD mRNA. Tempo1 thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation- induced cellular damage and may have broad applications in protecting against oxidative stress. 0 1991 Academic Press, Inc. The deleterious effects of ionizing radiation include cy- totoxicity, mutagenesis, and carcinogenesis. The discovery i To whom correspondence should be addressed at Radiation Oncology Branch, National Cancer Institute, NIH, Bldg. 10, Room B3-B69, Be- thesda, MD, 20892. ’ Abbreviations used: SOD, superoxide dismutase; DF, desferriox- amine; Tempol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; DTPA, diethylenetriaminepentaacedic acid; BSO, L-buthionine sulfoximine; Tempol-H, Tempol-hydroxylamine; RT, room temperature; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate. 62 in the late 1940s that sulfhydryls (thiols), such as cysteine and cysteamine, provide radiation protection to animals ushered in an era of research aimed at elucidating the mechanism(s) of radiation-induced cytotoxicity and de- velopment of more effective protective agents (1, 2). Ra- diation damages biomolecules, in large part (approxi- mately 80%), through its interaction with water to produce free radicals (H’, OH, e&) and HzOz, or through inter- action with oxygen to produce the superoxide anion (‘0;). Thiols, which function by donation of a hydrogen atom to damaged molecules or by “scavenging” radiation- produced free radicals, afford significant protection to animals against whole body radiation (1); however, their use is limited by toxicity. Despite extensive testing and synthetic efforts, no thiol-based radioprotector has been found to be significantly better than cysteamine. Stable nitroxide radicals are useful as NMR imaging contrast agents, EPR spin labels, and pH or oximetry cellular probes (3-7). As part of our studies investigating the chemical and biochemical characteristics of nitroxide radicals, we recently found that they exhibit superoxide dismutase mimetic activity (89) and are capable of pro- tecting mammalian cells from damage by superoxide gen- erated by hypoxanthine/xanthine oxidase and by hydro- gen peroxide (10). These findings encouraged us to test if stable nitroxide radicals could protect mammalian cells against ionizing radiation, since approximately 80% of the damaging effects of ionizing radiation is thought to occur through indirect damage caused by radiolysis prod- ucts of water or through oxygen reacting with radiation- induced carbon-centered free radicals (11). We have found that Tempo1 does protect against ionizing radiation and is the prototypical radiation protector of this group of chemical compounds and, moreover, may provide insight 0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.