Association of WR-1065 with CHO AA8 cells, nuclei, and nucleoids

The radioprotector WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) has been shown to be the active moiety involved in protecting mammalian cells from the cytotoxic and mutagenic effects of ionizing radiation after administration of WR-1065 or the phosphorylated form, WR-2721. Initial experiments demonstrated that, in our hands, WR-1065 protects Chinese hamster AA8 cells from killing by (a) mechanism(s) other than induction of hypoxia. AA8 cells were then incubated in the presence of [14C]WR-1065 to determine whether association of WR-1065 in vivo was random or targeted to the nucleus or the nuclear matrix. The kinetics of incorporation of labeled material showed rapid incorporation for the first 30 min and little, if any, additional incorporation over the next 2.5 h. Examination of nuclei and nucleoids generated from the AA8 cells indicated that approximately 10% of the drug was localized in the nucleus and the drug that remained was not dislodged with repeated washes of the filters. Association kinetics of the drug with nuclei and nucleoids indicated that there was little increase in drug association with time, suggesting that there may be a limited number of strong association sites in the nucleus, but these sites are either with DNA or with matrix proteins. Exposure of the AA8 cells to 6 Gy of 60Co gamma rays did not significantly alter the association of the drug with AA8 cells. Incubating AA8 cells in [14C]WR-1065 for 30 min and then incubating in drug-free medium indicated that nearly all of the drug was lost from cells within the first 5 min of incubation in drug-free medium. The low level of tightly bound matrix-associated label may be important in generating alterations in matrix organization that have been observed previously in this laboratory.     

Interaction of cultured mammalian cells with WR-2721 and its thiol, WR-1065: implications for mechanisms of radioprotection

An isothermal microcalorimeter was used to measure changes in heat flow when radioprotective drugs were added to cultured mammalian cells. The heat produced when WR-2721 was added continued for at least 90 min. WR-2721 was dephosphorylated by the cells to thiol (WR-1065) which oxidizes to disulphide. In the microcalorimeter, thiols give an immediate burst of heat due to this oxidation. A biological oxygen monitor revealed that WR-1065 and cysteamine rapidly consumed all the oxygen in culture medium. (10 mM WR-1065 deoxygenated medium in 2 min.) Rapid consumption of oxygen by radioprotective thiols indicates that they will not co-exist with oxygen for long in cells. This has two important implications with respect to mechanisms of radioprotection: (1) oxygen in tissues will be consumed rapidly and could result in local hypoxia; and, (2) at modest doses of protective agents the thiol will be consumed in oxic cells and hence very little will be available for reactions such as hydrogen donation. Our results indicate that anoxia is probably the principal mechanism of protection by aminothiols in mammals and aerated cells. This has major implications for clinical applications of radioprotectors and these are discussed.     

The nuclear matrix is a thermolabile cellular structure

Heat shock sensitizes cells to ionizing radiation, cells heated in S phase have increased chromosomal aberrations, and both Hsp27 and Hsp70 translocate to the nucleus following heat shock, suggesting that the nucleus is a site of thermal damage. We show that the nuclear matrix is the most thermolabile nuclear component. The thermal denaturation profile of the nuclear matrix of Chinese hamster lung V79 cells, determined by differential scanning calorimetry (DSC), has at least 2 transitions at Tm = 48 degrees C and 55 degrees C with an onset temperature of approximately 40 degrees C. The heat absorbed during these transitions is 1.5 cal/g protein, which is in the range of enthalpies for protein denaturation. There is a sharp increase in 1-anilinonapthalene-8-sulfonic acid (ANS) fluorescence with Tm = 48 degrees C, indicating increased exposure of hydrophobic residues at this transition. The Tm = 48 degrees C transition has a similar Tm to those predicted for the critical targets for heat-induced clonogenic killing (Tm = 46 degrees C) and thermal radiosensitization (Tm = 47 degrees C), suggesting that denaturation of nuclear matrix proteins with Tm = 48 degrees C contribute to these forms of nuclear damage. Following heating at 43 degrees C for 2 hours, Hsc70 binds to isolated nuclear matrices and isolated nuclei, probably because of the increased exposure of hydrophobic domains. In addition, approximately 25% of exogenous citrate synthase also binds, indicating a general increase in aggregation of proteins onto the nuclear matrix. We propose that this is the mechanism for increased association of nuclear proteins with the nuclear matrix observed in nuclei Isolated from heat-shocked cells and is a form of indirect thermal damage.     

Accumulation of CHO cells in G2 phase following exposure to WR-1065

The radioprotector WR-1065 (2-[(aminopropyl)amino]ethanethiol) is known to protect mammalian cells from the cytotoxic and mutagenic effects of radio- and chemotherapeutic agents, but the exact mechanisms involved in this protection are not fully known. To help determine the effects of WR-1065 alone on cells, we examined its effect on a variety of cellular processes. Incubation of AA8 cells in 4 mM WR-1065 did not significantly affect the rate of DNA synthesis. Autoradiographic analysis of heavily labeled (S-phase population) nuclei of AA8 cells showed no significant difference in the S-phase population of WR-1065-treated versus control cells for up to 3 h. An examination of the effect of WR-1065 on repair synthesis, as measured by unscheduled DNA synthesis (UDS) in cells exposed to 15 Gy, showed no difference between treated and sham-treated cells for up to 2 h exposure. A significant reduction in the amount of UDS was seen in cells treated with the protector for 2.5 and 3 h. Incubation of cells in WR-1065 did alter the cell cycle distributions. An increase in the G2-phase population with a corresponding decrease in the G1-phase population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065. After the removal of WR-1065 at 3 h, a redistribution of the cells throughout the cell cycle occurred as has been observed in cells treated with other synchronization agents. These data suggest that perturbations in cell cycle progression, rather than direct effects on the rate of DNA synthesis, could play a role in the increased survival and reduced mutation frequencies observed in the presence of WR-1065.     

N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) protects thymocytes from programmed cell death.

Gamma-irradiation, glucocorticoid hormones, and calcium ionophores stimulate a suicide process in thymocytes, known as apoptosis or programmed cell death, that involves internucleosomal DNA fragmentation by a Ca(2+)- and Mg(2+)-dependent nuclear endonuclease. In this study we report that N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) blocked DNA fragmentation and cell death in thymocytes exposed to gamma-radiation, dexamethasone, or calcium ionophore A23187. WR-1065 protected the thymocytes from radiation-induced apoptosis when incubated with cells after irradiation but not before and/or during irradiation. WR-1065 inhibited Ca(2+)- and Mg(2+)-dependent DNA fragmentation in isolated thymocyte nuclei. Our results suggest that WR-1065 protects thymocytes from apoptosis by inhibiting Ca(2+)- and Mg(2+)-dependent nuclear endonuclease action.     

Factors influencing the oxidation of the radioprotector WR-1065

N-(2-Mercaptoethyl)-1,3-diaminopropane (WR-1065) is the free thiol form of the radio- and chemoprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Interest currently exists in the clinical use of WR-2721 and WR-1065 as radio- and chemoprotectors of normal tissues. However, measurement of plasma levels of WR-1065 has proven difficult, due to rapid drug oxidation. Therefore, we studied factors influencing the oxidation of WR-1065, in Hepes-buffered saline as well as in tissue culture media containing 10% fetal bovine serum. The rate of oxygen consumption by WR-1065, as determined using the Clark oxygen electrode system, was faster in medium plus serum than in Hepes-buffered saline. That this effect is largely due to the presence of trace metal ions in tissue culture media and serum was indicated by the observation that addition of Cu2+ or Fe3+ to buffer stimulated oxygen consumption. Addition of KCN inhibited the reaction of WR-1065 with oxygen, and this effect was dependent on KCN concentration. That KCN blocked WR-1065 oxidation to the disulfide was verified using Ellman's reagent to quantitate the free thiol form. The rate of oxygen consumption was shown to be affected by temperature as well as concentration of WR-1065. Catalase reduced the rate of oxygen consumption of WR-1065, indicating that peroxide is formed in this system. Superoxide dismutase had a stimulatory effect. WR-1065 was found to stimulate the hexose monophosphate shunt in A549 cells. Since this stimulation was prevented by the presence of catalase, it appeared to be due to the response of the cells to peroxide, formed as a result of WR-1065 autooxidation.     

Alkaline phosphatase promotes radioprotection and accumulation of WR-1065 in V79-171 cells incubated in medium containing WR-2721

Addition of alkaline phosphatase and WR-2721 to culture medium containing V79-171 cells leads to production of WR-1065 and its disulphide forms in the medium, to cellular accumulation of WR-1065, and to radioprotection which correlates with cellular WR-1065 level.     

Manganese superoxide dismutase (SOD2)-mediated delayed radioprotection induced by the free thiol form of amifostine and tumor necrosis factor alpha

RKO36 cells, a subclone of RKO colorectal carcinoma cells that have been stably transfected with the pCMV-EGFP2Xho vector, were grown to confluence and then exposed to either the radioprotector WR-1065, i.e. the active thiol form of amifostine, for 30 min at doses of 40 microM and 4 mM or the cytokine tumor necrosis factor alpha (TNFalpha, TNFA) for 30 min at a concentration of 10 ng/ml and then washed. Total protein was isolated as a function of time up to 32 h after these treatments. Both doses of WR-1065 as well as the concentration of TNFalpha used were effective in elevating intracellular levels of the antioxidant protein SOD2 (also known as MnSOD) at least 15-fold over background levels as determined by Western blot analysis, while measured SOD2 activity was elevated between 5.5- and 6.9-fold. SOD2 reached a maximal level 24 h and 20 h after WR-1065 and TNFalpha treatments, respectively. The antioxidant proteins catalase and glutathione peroxidase (GPX) were also monitored over the 32-h period. In contrast to the robust changes observed in intracellular levels of SOD2 as a function of time after exposure of cells to WR-1065, catalase levels were elevated only 2.6-fold over background as determined by Western blot analysis, while GPX activity was unaffected by WR-1065 exposure. GPX protein levels were extremely low in cells, and analysis of GPX activity using a spectrophotometric method based on the consumption of reduced NADPH also revealed no measurable change as a function of WR-1065 or TNFalpha exposure. RKO36 cells either were irradiated with X rays in the presence of either 40 microM or 4 mM WR-1065 or 10 ng/ml TNFalpha or were irradiated 24 or 20 h later, respectively, when SOD2 protein levels were most elevated. The concentrations and exposure conditions used for WR-1065 and TNFalpha were not cytotoxic and had no effect on plating efficiencies or cell survival compared to untreated controls. No protection or sensitization was observed for cells irradiated in the presence of 40 microM WR-1065 or TNFalpha. Survival was elevated 1.90-fold for cells irradiated in the presence of 4 mM WR-1065. When RKO36 cells were irradiated with 2 Gy 24 h after 40 microM or 4 mM WR-1065 and 20 h after TNFalpha treatments when SOD2 levels were the most increased, survival was elevated 1.42-, 1.48- and 1.36-fold, respectively. This increased survival represents a SOD2-mediated delayed radioprotective effect. SOD2 appears to be an important antioxidant gene whose inducible expression is an important element in adaptive cellular responses in general, and the delayed radioprotective effect in particular. It can be induced by a range of agents including cytoprotective nonprotein thiols such as WR-1065 and pleiotropic cytokines such as TNFalpha.     

Protein denaturation in intact hepatocytes and isolated cellular organelles during heat shock

There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98 degrees C. The onset temperature is approximately 40 degrees C, but some transitions may extend as low as 37-38 degrees C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46 degrees C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40 degrees C and contains thermolabile proteins denaturing at the predicted Tm (46 degrees C) for the critical target. The extent of denaturation at any temperature can be approximately by the fractional calorimetric enthalpy. After scanning to 45 degrees C at 1 degree C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7% is found in hepatocytes, V79 cells, and the isolated organelles other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organelles and components, and protein denaturation is widespread and extensive after even mild heat shock.     

Excess protein in nuclei isolated from heat-shocked cells results from a reduced extractability of nuclear proteins

An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0°C, are present in both the nucleus and cytoplasm. © 1996 Wiley-Liss, Inc.     

Effects of growth media on cell cycle progression in CHO cells exposed to the radioprotector WR-1065

Abstract. WR-1065 (2-[(aminopropyl)amino]ethanethiol) reduces cytotoxic and mutagenic effects caused by exposure of cells to radiation and chemotherapeutic drugs, but the mechanisms involved are not fully known. We have observed an accumulation of cells in G, in WR-1065 treated Chinese hamster ovary cells grown in a-minimal essential medium, while others have found no cell cycle effects in WR-1065 treated Chinese hamster ovary cells grown in McCoy's 5A medium. To determine if the two types of media had an effect on cells treated with WR-1065, we examined survival and cell cycle progression. Population doubling times of 12 h were observed for cells grown in both media. Incubation of AA8 cells grown in McCoy's 5A medium with 4 mM WR-1065 30 min prior to and during irradiation with ^13'Cs gamma-rays resulted in a protection factor of 2.2, in close agreement with the value of 2.0 we previously obtained for AA8 cells grown in α-minimal essential medium. Treatment with WR-1065 caused an alteration in the cell cycles of cells grown in both media. An increase in the G₂ population and a decrease in the G₁ population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065, with a redistribution of the cells throughout the cell cycle occurring following removal of the drug. These data suggest that exposure of cells to WR-1065 is the cause of perturbations in cell cycle progression, and is not affected by the type of medium the cells are grown in.     

Protection of human tumor cells of differing radiosensitivity by WR-1065

We examined the ability of WR-1065, the biologically active aminothiol form of the clinically used drug amifostine (WR-2721, Ethyol), to protect cultures of two human glioblastoma cell lines of greatly differing radiosensitivity from the cytotoxic effects of gamma radiation. M059J cells are extremely radiosensitive compared to M059K cells (which were derived from the same tumor) and are defective in the DNA-dependent protein kinase (DNAPK)-mediated pathway for the repair of DSBs. In spite of their marked phenotypic differences, the two glioblastoma lines were protected equivalently ( approximately 1.8-fold) after a 30-min preirradiation treatment with 4 mM WR-1065. These findings are in agreement with earlier studies that showed no relationship between the ability of another aminothiol, cysteamine, to protect human tumor cells with differing abilities to repair DSBs and/or radiosensitivity. Thus it appears that differences in intrinsic radiosensitivity and ability to repair DSBs are not important general factors in the modulation of the radiosensitivity of human cells by aminothiols. Because of a previous report that the radiosensitive mutant rodent xrs5 cell line (which, like M059J, is defective in the DNAPK-mediated pathway for repairing DSBs) is unusually refractory to the radioprotective effects of WR-1065, we re-examined the ability of WR-1065 to protect these cells. In contrast to the earlier studies, both the wild-type and mutant rodent lines were protected extensively by WR-1065. This discrepancy might be related to some unknown factor, such as differences in chromatin organization among xrs5 subclones that arise during their karyotypic evolution, possibly leading to altered DNA-drug associations.     

Equilibrium dialysis studies of the binding of radioprotector compounds to DNA

Radioprotection by WR-2721, S-2-(3-aminopropylamino)ethyl phosphorothioate, is thought to involve its corresponding thiol (WR-1065) or symmetrical disulfide (WR-33278). It has been suggested that these metabolites concentrate close to the DNA target molecule; to test this hypothesis we have measured their in vitro binding to DNA. The binding of WR-33278 (0.05-0.4 mM) to calf thymus DNA (6 mM, with respect to DNA phosphate) was determined at 50, 100, and 150 mM KCl in 1 mM Tris, pH 7, by equilibrium dialysis. The binding of WR-1065 (0.5-8mM) was determined at 25, 50, and 100 mM KCl, under similar conditions, but with 2 mM EDTA and 3 mM dithiothreitol (DTT) added to the dialysis buffer to prevent thiol oxidation. Drug levels were quantitated by HPLC after fluorescent labeling with monobromobimane; disulfide samples were reduced with DTT prior to analysis. Dissociation constants (Kd = [Free Drug] [DNA site]/ [bound drug] ) under these conditions were found to vary with ionic strength, being in the range of 0.02 +/- 0.01 to 0.18 +/- 0.06 mM for WR-33278 and 0.43 +/- 0.24 to 3.5 +2/- 1.5 mM for WR-1065. WR-2721, glutathione, cysteine, and DTT showed no detectable binding to DNA in 25 mM KCl. However, cysteamine and cystamine did bind to DNA, with unbound drug to bound drug ratios of 8 +/- 2 and 0.6 +/- 0.1, respectively, at total drug concentrations of 1 mM. Cystamine and WR-1065 bound to DNA with comparable affinity under similar conditions. These results indicate that binding of WR-33278 and WR-1065 by DNA phosphate are probably significant in the mechanism of radioprotection by WR-2721.     

Properties of selected S-nitrosothiols compared to nitrosylated WR-1065

WR-1065 ([N-mercaptoethyl]-1-3-diaminopropane), the active form of the aminothiol drug Ethyol/Amifostine, protects against toxicity caused by radiation, chemotherapy and endotoxin. Because WR-1065 and other thiols readily bind nitric oxide (NO), injurious conditions or therapies that induce the production or mobilization of NO could alter the effects of WR-1065. S-Nitrosothiols were prepared from various thiols by a standard method to compare properties and stability. Heteromolecular quantum correlation 2D nuclear magnetic resonance was used to characterize nitrosylated glutathione (GSH) and WR-1065; both S- and N-nitrosothiols were observed, depending on the experimental conditions. Three categories of S-nitrosothiol stability were observed: (1) highly stable, with t(1/2) > 8 h, N-acetyl-L-cysteine nitrosothiol (t(1/2) 15 h) > GSH nitrosothiol (t(1/2) 8 h); (2) intermediate stability, t(1/2) approximately 2 h, cysteamine nitrosothiol and WR-1065 nitrosothiol; and (3) low stability, t(1/2) < 1 h, cysteine nitrosothiol and Captopril nitrosothiol. Similar relative rates were observed for Hg(+2)-induced denitrosylation: WR-1065 reacted faster than GSH nitrosothiol, while GSH nitrosothiol reacted faster than N-acetyl-L-cysteine nitrosothiol. Mostly mediated by mixed-NPSH disulfide formation, the activity of the redox-sensitive cysteine protease, cathepsin H, was inhibited by the S-nitrosothiols, with WR-1065 nitrosothiol > cysteine nitrosothiol > N-acetyl-L-cysteine nitrosothiol and GSH nitrosothiol. These observations indicate that, relative to other nitrosylated non-protein thiols, the S-nitrosothiol of WR-1065 is an unstable non-protein S-nitrosothiols with a high reactive potential in the modification of protein thiols.     

Differential antimutagenicity of WR-1065 added after irradiation in L5178Y cell lines

The purpose of this study was to determine the antimutagenicity of WR-1065 added after irradiation of cells of cell lines differing in their ability to rejoin radiation-induced DNA double-strand breaks (DSBs). The postirradiation antimutagenicity of WR-1065 at the thymidine kinase locus was demonstrated for L5178Y (LY)-S1 cells that are deficient in repair of DNA DSBs. Less postirradiation antimutagenicity of WR-1065 was observed in LY-R16 and LY-SR1 cells, which are relatively efficient in DSB repair. Postirradiation treatment with WR-1065 had only a small stimulatory effect on DSB rejoining. A 3-h incubation of irradiated LY cells with WR-1065 caused slight changes in the distribution of cells in the phases of the cell cycle that differed between LY-S1 and LY-SR1 cells. Both LY-S1 and LY-SR1 cells were protected against the cytotoxic and mutagenic effects of radiation when WR-1065 was present 30 min before and during the irradiation. We conclude that the differential postirradiation effects of WR-1065 in the LY-S1 and LY-SR1 cells are not caused by differences in cellular uptake of the radioprotector or in its radical scavenging activity. Possible mechanisms for the postirradiation antimutagenicity of WR-1065 are discussed.     

Studies on the interaction of the human c-myc protein with cell nuclei: p62c-myc as a member of a discrete subset of nuclear proteins

We have analyzed the localization of the human c-myc product (p62c-myc) at steady state in cells by immunoprecipitation and immunoblotting. We show that p62c-myc is extracted from nuclei by mild salt concentrations (below 200 mM), without affecting gross nuclear structure or causing extraction of major chromatin components. We observe no association between p62c-myc and the nuclear matrix. We also demonstrate that p62c-myc is a member of a discrete subset of nuclear proteins that are all rendered irreversibly insoluble in situ by exposure of isolated nuclei to physiological temperatures (37 degrees C). p62c-myc is sequestered into a similar insoluble complex in cells that have been subjected to heat shock. Finally, we show that avian v-myc and v-myb proteins in isolated nuclei also become insoluble after exposure to temperatures above 37 degrees C. We discuss the possible implications of these results.     

WR-1065, the active metabolite of amifostine, mitigates radiation-induced delayed genomic instability

Compounds that can protect cells from the effects of radiation are important for clinical use, in the event of an accidental or terrorist-generated radiation event, and for astronauts traveling in space. One of the major concerns regarding the use of radio-protective agents is that they may protect cells initially, but predispose surviving cells to increased genomic instability later. In this study we used WR-1065, the active metabolite of amifostine, to determine how protection from direct effects of high- and low-LET radiation exposure influences genomic stability. When added 30 min before irradiation and in high concentrations, WR-1065 protected cells from immediate radiation-induced effects as well as from delayed genomic instability. Lower, nontoxic concentrations of WR-1065 did not protect cells from death; however, it was effective in significantly decreasing delayed genomic instability in the progeny of irradiated cells. The observed increase in manganese superoxide dismutase protein levels and activity may provide an explanation for this effect. These results confirm that WR-1065 is protective against both low- and high-LET radiation-induced genomic instability in surviving cells.