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.     

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.     

Radioprotective effects of the thiols GSH and WR-2721 against X-ray-induction of micronuclei in erythroblasts

The frequency of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow and peripheral blood of adult male Swiss mice treated with reduced glutathione (GSH) and S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721), at a dose of 400 mg/kg body weight, and exposed to 6 Gy X-rays. GSH or WR-2721 was applied alone, or 60 and 30 min, respectively, prior to X-ray-exposure. The number of MNPCEs was determined at 24 h after the thiol treatment and X-irradiation. The radioprotection and toxicity caused in the mouse erythroblasts by GSH and WR-2721, as indicated by the number of MNPCEs were dependent on the thiol applied. The stronger radioprotective effect is obtained following WR-2721 administration than after GSH application. WR-2721 showed greater toxicity than GSH. The combination of GSH and WR-2721 given before X-ray-exposure resulted in the most radioprotective effect as compared to the respective single-drug treatment of mice. Application of the both thiols, without subsequent X-irradiation appeared to be the most toxic, compared with administration of WR-2721 or GSH alone. The effective radioprotection by the combined action of GSH and WR-2721 against genomic instability induced in the mouse erythroblasts by X-rays was shown.     

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.     

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.     

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.     

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.     

Radioprotective thiolamines WR-1065 and WR-33278 selectively denature nonhistone nuclear proteins

Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23 degrees C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 degrees C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR-1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein-WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca(2+)ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.     

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.     

Induction of micronucleated erythrocytes by MEA,AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.     

Protection of mice against fission neutron irradiation by WR-2721 or WR-151327

Two phosphorothioate compounds, WR-2721 and WR-151327, were examined for their radioprotective efficacies against the effects of fission neutron irradiation in male and female mice. Within sex groups no significant difference in lethality at 30 or 100 days postirradiation was found between WR-2721 or WR-151327 pretreatment. The dose modification factors (DMFs) for male mice treated with either compound were 1.29 (LD50/30) and 1.24 (LD50/100), and those for drug-treated female mice were 1.21 (LD50/30) and 1.19 (LD50/100). Both WR-2721 and WR-151327 were found to be equally radioprotective when compared using DMFs as the end point. WR-151327 (500 mg/kg, ip) was found to be significantly more toxic to both male and female B6D2F1 mice than equimolar amounts of WR-2721. Small but significant sex differences in radioprotection were found: the DMFs for female mice pretreated with either compound were lower than those for similarly treated male mice; the incidence of mortality 31-100 days postexposure in male mice pretreated with WR-151327 was greater than for female mice. In addition, sex differences were noted in drug toxicity. Toxic death in female mice given WR-151327 (500 mg/kg, ip) is 2.6 times more probable than in males.     

Radioprotection of mouse jejunum by WR-2721 and WR-1065: effects on DNA strand-break induction and rejoining

WR-2721 and its free-thiol metabolite WR-1065 have been characterized for their ability to protect mouse jejunal cells in vivo from the damaging effects of gamma rays with respect to both cytotoxicity and DNA single-strand break (SSB) induction. SSBs were measured both in the whole jejunal epithelium and in the proliferating crypt cells using an adaptation of the alkaline elution methodology. Protection factors (PFs) were also obtained using the microcolony assay for jejunal crypts. In mice treated with WR-1065 (400 mg/kg) 15 or 30 min prior to irradiation, there was a slight but significant reduction in the initial number of SSBs both in the whole jejunum (PF of between 1.17 and 1.22) and in the proliferating crypt cells (PF of between 1.13 and 1.28). At a dose of 200 mg/kg, the PF for SSBs in the proliferating crypt cells was 1.12 +/- 0.07 while that for crypt-cell survival was approximately 2.0. In mice treated with WR-2721 (400 mg/kg) 15 min prior to irradiation, there was little effect on the initial number of SSBs induced both in the whole jejunum (PF of 1.07 +/- 0.11) and in the proliferating crypt cells (PF of 1.04 +/- 0.07). WR-2721 protected jejunum in the microcolony assay with a much greater PF of 1.8. For each drug the PF for SSBs was therefore always much lower than that indicated by the biological end point under identical conditions. Both drugs also retarded the rate of SSB rejoining in each population of cells. These data suggest that mechanisms such as free-radical scavenging by these drugs may contribute to but not completely explain their protective action. Comparison with data obtained previously with cultured CHO cells supports the idea that the action of these drugs at the DNA lesion level may not be dose-modifying, but may also result in a shift in the spectrum of lesions induced by the radiation.     

Measurement of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for automated analysis of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood has been developed in our laboratory. WR-1065 is the active thiol metabolite of the radio- and chemo-protector drug amifostine (WR-2721). Using WR-1065 quality control levels over the experimental range: 7.0, 45.0 and 85.0 μmol/l spiked into plasma, method validation for total WR-1065 included between-run assessment of imprecision (SD/C.V.%: 1.11/16.7%, 6.58/15.5% and 9.24/11.3%, respectively) and % accuracy (94.7, 106.0 and 97.2%).     

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.     

WR-2721 inhibition of radiation-induced prostaglandin excretion in rats

Pre-irradiation administration of the radioprotectant drug WR-2721 to rats resulted in a significant reduction in radiation-induced increases in excretion rates of prostaglandins (PGE and PGF2 alpha) and thromboxane (TxB2). In animals not irradiated. WR-2721 did not significantly alter these excretion rates. Dramatic reductions in the levels of urinary PGE and TxB2 were observed following exposure to 9.0 Gy of whole-body, unilateral gamma-radiation in WR-2721-treated animals, whereas changes in PGF2 alpha levels were less pronounced. Radiation-induced diuresis was also significantly depressed in animals given WR-2721 before irradiation. Reduced prostaglandin excretion rates may reflect the general radioprotective capacity of the chemoprotector WR-2721 on the release of prostaglandins from radiation-damaged tissue. The decrease in diuresis may be related to the observed prostaglandin decreases.     

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.     

Thiol uptake by Chinese hamster V79 cells and aerobic radioprotection as a function of the net charge on the thiol.

A series of thiols having net charge (Z) varying from -2 to +3 were studied using aerobic suspensions of Chinese hamster V79-171 cells in pH 7.4 medium at 297 K to evaluate the rate of uptake by cells and the extent of radioprotection as a function of thiol concentration in cells. For measurement of cellular levels, cells were separated from medium by centrifugation through silicone oil and tritiated water was employed to determine cell water volume. Estimated half-lives for uptake were: 2-mercaptosuccinate (Z = -2), greater than or equal to 1 h; 3-mercaptopropanoate (MPA, Z = -1), less than 2 min; 2-mercaptoethanol (2ME, Z = 0), less than 2 min; cysteamine (CyA, Z = +1), less than 2 min; N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, Z approximately +2), approximately 40 min; N1-(2-mercaptoethyl)spermidine (WR-35980, Z approximately +3), greater than or equal to 10 h. After equilibration the cellular concentration of MPA was 60 +/- 8% of the medium level; the corresponding values for 2ME and CyA were 95 +/- 3 and 180 +/- 12%, respectively, but equilibrium was not reached for the other thiols studied. Those thiols taken up at significant rates were evaluated in terms of their ability to protect against aerobic gamma-ray-induced lethality. The results, summarized in terms of the cellular concentration of thiol (mmol dm-3) needed to achieve an aerobic radioprotection factor of 1.5, were as follows: MPA, 80 +/- 15; 2ME, 24 +/- 2; CyA, 4.7 +/- 1.3; WR-1065, 3.4 +/- 0.6. These values accorded well with those predicted from hydroxyl radical scavenging and DNA radical repair rates obtained using pBR322 DNA as a model system. This shows that hydroxyl radical scavenging and DNA radical repair are important mechanisms in the protection of cells by thiols and that the net charge on the thiol is a significant factor in its effectiveness. The results indicate that in air hydroxyl radical scavenging is the dominant mode of action by MPA, but that chemical repair of DNA radicals becomes significant for 2ME and is the dominant mechanism of protection for CyA and WR-1065.     

Phosphatidylserine externalization during bone marrow cell death after treatment of mice with WR-2721 and gamma-rays

The cell surface exposure of phosphatidylserine (PS) and the plasma membrane impairment were assessed in the bone marrow of adult male Swiss mice exposed to a single 6 Gy dose of 60 Co gamma-rays, and treated intraperitoneally with the aminothiol WR-2721 (Amifostine, S-2-/3-aminopropylamino/ethyl phosphorothioic acid), at a dose of 400 mg/kg body weight, 30 min prior to gamma-irradiation. The bone marrow cells were stained with a combination of fluoresceinated annexin V (annexin V--FITC) and propidium iodide (PI) at 3 h, 7 h, and 24 h after treatment of mice with WR-2721 and 60Co gamma-irradiation. The number of early apoptotic cells (annexin V--FITC positive/PI negative), and late apoptotic and necrotic cells (annexin V--FITC positive/PI positive), was increased at 3 h after exposure of mice to 60Co gamma-rays and thereafter declined with the frequency of apoptotic and necrotic cells remaining lower in WR-2721 pre-treated mice. Using the annexin V--FITC flow cytometric assay, the radioprotective effect of WR-2721 against induction of apoptosis and necrosis in normal cells of the haematopoietic system was shown.