Radiation protection of murine intestine by WR-2721, 16,16-dimethyl prostaglandin E2, and the combination of both agents

The survival of murine intestinal clonogenic cells (ICC) and the survival of mice after whole-body exposure to 137Cs irradiation were used to measure radiation protection by ethiophos (WR-2721), 16,16-dimethyl prostaglandin E2, and the combination of the two. Doses from 2 to 12.5 mg/mouse of WR-2721 increased cell survival linearly from 3.2 +/- 0.3 in controls given 15.0 Gy to 93.1 +/- 5.2 per jejunal circumference. In contrast, 16,16-dm PGE2 increased ICC survival at 15.0 Gy rapidly from 1 to 10 micrograms/mouse, followed by a plateau up to 100 micrograms/mouse. Animal survival at 6 days (LD50/6) increased from 16.3 +/- 0.4 Gy (95% confidence limits) in controls to 20.3 +/- 0.6 Gy in the PG-treated animals. WR-2721 increased the LD50/6 to 26.1 +/- 1.4 Gy. The dose modification factors were 1.25 and 1.60, respectively. The combination of agents increased ICC survival above that seen with each agent alone up to 8 mg WR-2721, above which no additional protection was seen. Animals given 10 micrograms PG plus 10 mg WR-2721 survived longer than with either agent given alone. The LD50/6 was 36.3 +/- 1.8 Gy for a dose modification factor (DMF) of 2.23. In addition, the slope of the probit curve was reduced from those of each agent alone. PG-induced changes in villus epithelial cell morphology and survival may account, in part, for these observations. The results suggest that either the mechanisms for these two types of radiation protectors are different or they act on separate subcellular targets which are critical to survival from radiation injury.     

Protection from radiation nephropathy by WR-2721

The efficacy of WR-2721 pretreatment against radiation injury to the growing kidney was evaluated in the weanling mouse. Immediately following unilateral nephrectomy, animals received intraperitoneal injections of saline or WR-2721 (220 mg/kg). Thirty minutes later both nonprotected (saline-treated) control animals and protected (WR-2721-treated) animals received 1000-rad single-fraction radiation to the remaining kidney. Other animals received WR-2721 immediately following unilateral nephrectomy but no radiation. Animals were sacrificed at 3 and 24 weeks. Nonirradiated animals treated with WR-2721 only showed normal compensatory renal growth, body growth, and renal function at 24 weeks. The nonprotected, irradiated animals exhibited renal growth inhibition without body growth inhibition, and renal functional abnormalities including elevation of serum BUN and reduction of glomerular filtration rate. Pretreatment with WR-2721 prior to 1000 rad prevented the renal growth inhibition and functional abnormalities seen in the nonprotected irradiated animals. Within the observation period there were no differences in renal morphology by light and electron microscopy between protected and nonprotected groups; only mild glomerular and tubular abnormalities compatible with radiation injury were seen. WR-2721 can modulate renal radiation injury; however, the growth and functional protection is not well correlated with specific histologic change. The dose reduction factor for WR-2721 renal growth protection is between 1.16 and 1.2. WR-2721 may have future clinical utility by increasing radiation tolerance of the kidney.     

Protection of mice against mixed fission neutron-gamma (n: gamma = 1:1) irradiation by WR-2721, 16,16-dimethyl PGE2, and the combination of both agents

The survival of mice after whole-body exposure to a modified fission neutron-gamma field (n: gamma = 1:1) was used to examine radiation protection by WR-2721, 16,16-dimethyl PGE2(DiPGE2), and the combination of both agents. Administration of WR-2721 (453 mg/kg) increased the LD50/30 from 5.24 to 7.17 Gy (DMF = 1.37), whereas pretreatment with DiPGE2 (1.6 mg/kg) increased the LD50/30 to 5.77 Gy (dose modification factor (DMF) = 1.10). The combination of 453 mg/kg WR-2721 and 0.4 mg/kg DiPGE2 resulted in an LD50/30 of 7.33 Gy, yielding a DMF of 1.39. However, no significant difference in protection was obtained with the combination of the two agents compared to that seen with WR-2721 alone.     

Radioprotective effect of combinations of WR-2721 and mercaptopropionylglycine on mouse bone marrow chromosomes

The radioprotective and toxic effects of low to moderate doses of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721) and its combination with mercaptopropionylglycine (MPG, 20 mg/kg body wt) on the chromosomes of the bone marrow cells of Swiss albino mice were studied at 24 h and 14 days postirradiation. Significant protection against radiation-induced chromosome aberrations was observed with 50 mg/kg WR-2721. The protection increased with the dose of the drug administered, and the degree of protection per unit dose increment was more pronounced at lower than at higher doses. A combination of WR-2721 and MPG given before exposure resulted in a significantly greater number of normal metaphases at 24 h postirradiation compared to the respective single-drug treatment groups. On Day 14 postirradiation, when the presence of WR-2721 resulted in an increase in the frequency of aberrant cells, combination with MPG helped to reduce this value markedly, especially at WR-2721 doses below 200 mg/kg. On the basis of these results it is suggested that 150 mg/kg WR-2721 may be considered an optimum dose for combination with MPG for protection of chromosomes of bone marrow cells when repeated drug administrations are not needed. Changes in the level of glutathione (GSH) in the blood were studied at different times following the administration of 150 mg/kg WR-2721 and its combination with MPG (20 mg/kg) before sham irradiation or exposure to 4.5 Gy 60Co gamma rays. The results showed that WR-2721 elevated blood GSH levels significantly above normal values by the time radiation was delivered, while MPG did not. Glutathione appears to have an important role in the action of WR-2721, while protection by MPG may not be mediated through GSH. Injection of MPG after WR-2721 helps to maintain the higher GSH level for a longer duration compared to treatment with WR-2721 alone. It is possible that MPG delays the metabolism of GSH.     

Opposite effects of WR-2721 and WR-1065 on radiation-induced hypothermia: possible correlation with oxygen uptake

Ionizing radiation induces hypothermia in guinea pigs. While systemic injection of the radioprotectant S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) did not block hyperthermia induced by exposure to 10 Gy of gamma radiation, central administration did attenuate it. The dephosphorylated metabolite of WR-2721, N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065), accentuated radiation-induced hypothermia by both routes of administration. In brain homogenates, oxygen uptake was inhibited by WR-2721 but elevated by WR-1065. These results suggest that the antagonism of radiation-induced hypothermia found only after central administration of WR-2721 is due to its direct actions and not to its dephosphorylated metabolite and that this effect may be correlated with the inhibition by WR-2721 of oxygen uptake.     

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.     

Modification of radiation induced damage in mouse intestine by WR-2721

Intestinal protection in mice against radiation injury by WR-2721 (300 mg/kg body wt, i.p., 30 min before irradiation) was studied after whole body gamma irradiation (0.5, 1.5, 3.0, 4.5, or 6.0 Gy). Crypt survival and induction of apoptosis, and abnormal mitoses in crypt cells in the jejunum were studied on day 1, 3 and 7 after irradiation. Irradiation produced a significant decrease in crypt survival, whereas apoptosis and abnormal mitoses showed a significant increase from sham-treated control animals. Maximum changes in all the parameters were observed on day 1 after irradiation and the effect increased linearly with radiation dose. There was recovery at later intervals, which was inversely related to radiation dose. WR-2721 pre-treatment resulted in a significant increase in the number of surviving crypts, whereas the number of apoptotic cells in the crypts showed a significant decrease from respective irradiated controls on day 1 after exposure. The recovery was also faster in WR-2721 pre- treated animals. It is concluded that WR-2721 protects against gastrointestinal death by reducing radiation induced cell death, thereby maintaining a higher number of stem cells in the proliferating compartment.     

Determination of the radioprotective effects of topical applications of MEA, WR-2721, and N-acetylcysteine on murine skin

Topical applications of MEA (beta-mercaptoethylamine or cysteamine), WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid], and N-acetylcysteine (NAC) were tested for their ability to protect the normal skin of the hind legs of mice against acute and late damage from single doses of 137Cs radiation. No significant protection was observed with either WR-2721 or NAC. MEA was shown to offer significant protection against acute skin damage in both buffered and unbuffered forms, but no significant protection against late contraction. The use of topical MEA on unanesthetized animals breathing carbogen (95% O2, 5% CO2) appears to give an enhanced level of radioprotection over that shown for anesthetized, air-breathing animals.     

Postirradiation glucan administration enhances the radioprotective effects of WR-2721

Based on murine survival studies, endogenous hemopoietic spleen colony formation (E-CFU), and recovery of bone marrow and splenic granulocyte-macrophage colony-forming cells (GM-CFC), it was demonstrated that the postirradiation administration of glucan, an immunomodulator and hemopoietic stimulant, enhances the radioprotective effects of WR-2721. LD50/30 dose reduction factors for mice treated with WR-2721 (200 mg/kg approximately 30 min before irradiation), glucan (250 mg/kg approximately 1 h after irradiation), or both agents were 1.37, 1.08, and 1.52, respectively. Enhanced survival in mice treated with both agents appeared to be due in part to glucan's ability to accelerate hemopoietic regeneration from stem cells initially protected from radiation-induced lethality by WR-2721. Following a 10-Gy radiation exposure, E-CFU numbers in mice treated with saline, WR-2721, glucan, or both WR-2721 and glucan were 0.05 +/- 0.03, 6.70 +/- 1.05, 0.95 +/- 0.24, and 33.90 +/- 2.96, respectively. Similarly, bone marrow and splenic GM-CFC numbers were greater in mice treated with both WR-2721 and glucan than in mice treated with either agent alone. These results demonstrated at least additive radioprotective effects when mice were given WR-2721 prior to irradiation and glucan following irradiation. These effects appeared to depend on the sequential cell protection mediated by WR-2721 and hemopoietic repopulation mediated by glucan.     

Late functional and biochemical changes in mouse lung after irradiation: differential effects of WR-2721

The radioprotective effect of WR-2721 on late damage after whole thorax irradiation has been studied after split doses of radiation using the standard death and breathing rate assays at monthly intervals between 3 and 15 months after irradiation, as well as two biochemical measurements of injury at 15 months, hydroxyproline (HP), an indicator of tissue fibrosis, and DNA content, an indicator of tissue cellularity. A comparison of HP/lung and breaths per minute (BPM) in each dose group in the WR-2721 and non-WR-2721-treated mice 15 months after irradiation showed that the relationship between these two assays of late lung injury was not the same. There were large dose-related increases in breathing rate corresponding to relatively small changes in HP in the lungs of mice given radiation alone. In contrast, the mice given WR-2721 before irradiation showed large dose-related increases in HP/lung, but BPM remained relatively constant independent of dose. These data suggest then that changes in breathing rate and deaths later than 9 months after whole lung irradiation may not be due to collagen accumulation in the lung. WR-2721 did protect better against late lung functional changes (protection factors (PF) = 1.6) and late deaths (PF = 1.51) than against earlier changes in these same assays (PF = 1.4 and 1.28, respectively). Although the earlier-appearing injury after whole thoracic irradiation is most likely related to lung damage with deaths and increases in breathing rate resulting from pneumonitis, the cause of the late-appearing functional injury in the lung after radiation is not clear. Thus protection of late lung damage measured from either lethality or breathing rate is not related to the prevention of lung fibrosis.     

Protection of bone marrow of Swiss albino mouse against whole body gamma irradiation by WR-2721.

Preinjected with a radioprotective drug, WR-2721, the Swiss albino mice were whole body irradiated with 5 Gy of 60Co gamma rays. The animals were sacrificed at different intervals and bone marrow films were prepared for differential counting of lymphocytes, pronormoblasts and normoblasts and granulocytes. The results indicated significant protection of the bone marrow cells by the drug against radiation induced damage. It is therefore concluded that WR-2721 protects all types of cells including as sensitive ones as lymphocytes, pronormoblasts and normoblasts.     

Suppression of delayed-type hypersensitivity to oxazolone in whole-body-irradiated mice and protection by WR-2721

The effect of whole-body irradiation on cellular immunity, as measured in vivo by delayed-type hypersensitivity (DTH) to oxazolone (4- ethoxymethylene -2-phenyl- oxazol -5-one), was determined in CD2F1 mice. DTH, determined by changes in ear swelling after challenge with oxazolone, was significantly depressed in irradiated mice (500-900 rad of 60Co) in a dose-dependent fashion when animals were irradiated after sensitization and before challenge with oxazolone. Administration of WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] 30 min before irradiation (2 days after sensitization) resulted in protection against suppression of DTH, which was dependent on drug and radiation dose. An effective dose of WR-2721 (200 mg/kg body wt) provided an approximate dose-modifying factor of 1.3. The data suggest that WR-2721 interacts with cells involved in that DTH response (lymphocytes and/or macrophages) and that WR-2721 may be useful in protecting against radiation-induced decrements in cell-mediated immunity.     

WR-2721, its derivatives and their radioprotective effects on mammalian cells in culture

Although it is well known that WR-2721 is very efficient in protecting mice against lethal irradiation, we could not find any radioprotective effect of WR-2721 on mouse L cells in culture. But WR-1065 alone (free SH form of WR-2721), and WR-2721 pre-incubated with mouse liver homogenate, showed radioprotective ability. It was found that mouse liver homogenate dephosphorylated WR-2721 to WR-1065. The highest WR-2721 metabolizing activity was found in mouse liver homogenate and Chang liver cell homogenate. Homogenates of human liver and kidney were also shown to possess moderate activity for metabolizing WR-2721. These results suggest to us that WR-2721 must be dephosphorylated before exerting its radioprotective effect and that this dephosphorylating activity varies with tissues. It is demonstrated therefore that mouse L cell in culture is a novel system to assess the extent of dephosphorylation of WR-2721 in various tissues.     

Histopathologic effects of soluble glucan and WR-2721, independently and combined in C3H/HeN mice.

Soluble glucan, an immunomodulator, and Walter Reed (WR)-2721, a radioprotectant, increase postirradiation survival when administered before and after exposure, respectively. Combined, these agents act synergistically through WR-2721's ability to spare hematopoietic stem/progenitor cells from radiation injury and glucan's ability to subsequently stimulate spared cells to proliferate. In this study, the histopathologic effects of WR-2721 (200 mg/kg, ip) and glucan (250 mg/kg, iv), at doses capable of increasing survival in lethally irradiated mice, were evaluated in unirradiated and irradiated female C3H/HeN mice. After treatment, whole body weights and wet organ weights of liver, spleen, and kidney, as well as gross and histologic changes in these and other tissues, were monitored on Days 1, 4, 7, 11, 15, 21, and 28. Morphometric studies of splenic white and red pulps were also performed. Soluble glucan, with or without WR-2721, in unirradiated groups, was associated with splenomegaly, transient morphometrically determined perturbations of white and red pulp areas, and histologic alterations of white pulp. In irradiated mice, splenic weight loss was initially dampened in glucan groups and accompanied by morphologic and histologic changes similar to those seen in unirradiated counterparts. The subsequent rebound of splenic parameters in irradiated mice was limited to WR-2721-treated mice and was associated with hematopoietic reconstitution. Glucan, with or without WR-2721, in unirradiated groups was associated with transient hepatomegaly and associated histologic changes. Similar changes in irradiated animals were seen only in the combined treatment group.     

Selenium Pretreatment Enhances the Radioprotective Effect and Reduces the Lethal Toxicity of Wr-2721

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/ kg IP before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.     

Selenium Pretreatment Enhances the Radioprotective Effect and Reduces the Lethal Toxicity of Wr-2721

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/ kg IP before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.     

Comparison of the protective effects of three phosphorothioate radioprotectors in the RIF-1 tumor

Three aminoalkyl phosphorothioates, WR-2721, WR-3689, and WR-77913, were compared as radioprotectors of RIF-1 tumors irradiated in vivo and assayed for cell survival in vitro. The protector doses were 50% of the acute drug LD50. The radiation dose modifying factors for the three drugs were nearly equal, ranging from 1.5 to 1.7 at surviving fractions of 0.1 and 0.05. Using biodistribution data obtained with 35S labeled drugs, the uptake in tumors was calculated as micromoles drug per gram of tumor. On this basis, tumor levels of WR-77913 were 4.5-fold those of WR-2721, and WR-3689 uptake was 2.7-fold greater than uptake of WR-2721. Thus, on a molar basis, WR-2721 appears to be the most effective protector, but all three phosphorothioates protect this tumor moderately well. In diffusible substance autoradiographs of 3H WR-3689 labeled tumors, label was generally distributed over cells with no evidence of preferential localization over nuclei.     

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.     

Transdermal absorption of radioprotectors using permeation-enhancing vehicles

Radioprotectors are not currently used clinically due to concerns regarding toxicity and uncertainties regarding tumor protection. Topical radioprotection of skin might find clinical applications with protectors such as WR-2721, but laboratory studies in which protectors have been applied in water have not been promising. We have studied the absorption of 14C-WR-2721 and [14C]cysteine dissolved in skin permeation-enhancing vehicles through the skin of hairless mice and compared the absorption to that in water. Skin concentration of WR-2721 was increased most by dimethylformamide (DMF), but only propylene glycol increased absorption as far as the dermis, as measured by plasma concentration. Skin concentration of cysteine was improved by DMF, 2-pyrrolidone (2-P), and methyl-2-pyrrolidone (M-2-P); only dimethylsulfoxide (DMSO) resulted in increased plasma levels of the protector. Pretreating skin with DMSO before application of WR-2721, irrespective of the vehicle, improved its concentration within the skin. Plasma levels were improved (10 and 12 times) only with 2-P and DMF. Therefore, by choosing the appropriate vehicle, it is possible to breach the barrier of the stratum corneum and enhance the presence of the protector in all layers of the skin.     

The radioprotective agent, amifostine, suppresses the reactivity of intralysosomal iron

Amifostine (2-[(3-aminopropyl)amino]ethane-thiol dihydrogen phosphate ester; WR-2721) is a radioprotective agent used clinically to minimize damage from radiation therapy to adjacent normal tissues. This inorganic thiophosphate requires dephosphorylation to produce the active, cell-permeant thiol metabolite, WR-1065. The activation step is presumably catalyzed by membrane-bound alkaline phosphatase, activity of which is substantially higher in the endothelium of normal tissues. This site-specific delivery may explain the preferential protection of normal versus neoplastic tissues. Although it was developed several decades ago, the mechanisms through which this agent exerts its protective effects remain unknown. Because WR-1065 is a weak base (pKa = 9.2), we hypothesized that the drug should preferentially accumulate (via proton trapping) within the acidic environment of intracellular lysosomes. These organelles contain abundant 'loose' iron and represent a likely initial target for oxidant- and radiation-mediated damage. We further hypothesized that, within the lysosomal compartment, the thiol groups of WR-1065 would interact with this iron, thereby minimizing iron-catalyzed lysosomal damage and ensuing cell death. A similar mechanism of protection via intralysosomal iron chelation has been invoked for the hexadentate iron chelator, desferrioxamine (DFO; although DFO enters the lysosomal compartment by endocytosis, not proton trapping). Using cultured J774 cells as a model system, we found substantial accumulation of WR-1065 within intracellular granules as revealed by reaction with the thiol-binding fluorochrome, BODIPY FL L-cystine. These granules are lysosomes as indicated by co-localization of BODIPY staining with LysoTracker Red. Compared to 1 mM DFO, cells pre-treated with 0.4 microM WR-1065 are protected from hydrogen peroxide-mediated lysosomal rupture and ensuing cell death. On a molar basis in this experimental system, WR-1065 is approximately 2500 times more effective than DFO in preventing oxidant-induced lysosomal rupture and cell death. This increased effectiveness is most likely due to the preferential concentration of this weak base within the acidic lysosomal apparatus. By electron spin resonance, we found that the generation of hydroxyl radical, which normally occurs following addition of hydrogen peroxide to J774 cells, is totally blocked by pretreatment with either WR-1065 or DFO. These findings suggest a single and plausible explanation for the radioprotective effects of amifostine and may provide a basis for the design of even more effective radio- and chemoprotective drugs.