Radioprotective action of WR-1065 on radiation-induced DNA strand breaks in cultured Chinese hamster ovary cells

We have examined the radioprotective effect of WR-1065 on cultured Chinese hamster ovary cells. The effects of the drug on the induction and rejoining of gamma-ray-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) were measured using alkaline (pH 12.1) and neutral (pH 7.0) elution, respectively. Molecular protection factors (PFs) calculated from these data allowed us to determine whether the degree of modification of strand breakage accurately predicted the PFs measured using the biological end point of cell survival. The drug did protect against the induction of both SSBs and DSBs, although to an extent that did not appear to fully account for the degree of radioprotection in terms of cell killing measured under identical conditions. It is therefore unlikely that radioprotection by WR-1065 occurs simply as a consequence of a general lowering of all types of gamma-ray-induced DNA lesions, and it is possible that the drug could differentially protect against the induction of subsets of these DNA lesions. The rate of SSB rejoining was retarded following preirradiation treatment of cells with WR-1065, but there was no effect on DSB rejoining. Postirradiation treatment with WR-1065 also appeared to retard SSB rejoining but without an accompanying effect on either DSB rejoining or cell survival; however, this effect was largely reversed by the addition of catalase and was therefore probably a result of H2O2 generated by autoxidation of the drug. Based on these observations, it would appear that the molecular actions of aminothiol radioprotective compounds that lead to reduced cell killing are much more complex than previously thought.     

Effect of thiols on micronucleus frequency in gamma-irradiated mammalian cells

The effects of the thiols cysteamine, WR-1065, and WR-255591 on radiation-induced micronucleus (MN) frequency and cell killing were compared in cultured Chinese hamster ovary cells. MN were measured using the cytochalasin B assay of Fenech and Morley (1985), which minimizes the effect of cytokinetic perturbations on MN expression. The dose-response curves for MN induction were curvilinear both for control cells at doses between 1.5 and 4.5 Gy and for thiol-treated cells at doses between 3 and 9 Gy. Protection against MN induction by each thiol was independent of radiation dose. Furthermore, there was a close correlation between the degree of modification of MN induction and cell survival by each thiol, i.e., the MN frequency closely predicted the survival level regardless of the presence of absence of the thiols. A similar predictive relationship has also been reported by us for cell survival and DNA double-strand break (DSB) induction in this cell line following treatment with these same thiols. Collectively, these data support the hypothesis that, for DNA-repair-proficient mammalian cells treated with radiomodifying agents that do not alter DNA-repair processes, MN and DSB induction are predictive of the level of radiation lethality and of each other.     

Protection by WR-3689 against gamma-ray-induced intestinal damage: comparative effect on clonogenic cell survival, mouse survival, and DNA damage

The aminophosphorothioate WR-3689 was characterized for its ability to protect mouse jejunal cells in vivo from single doses of X or gamma radiation. First, the effect of the drug on the survival of jejunal stem cells was examined using a clonogenic end point, the crypt microcolony assay. When WR-3689 was administered 30 min prior to whole-body irradiation, the number of surviving crypt cells was markedly increased at all doses of the drug, although protection began to level out at doses larger than 600 mg/kg. Protection was maximal when the drug was given 30 min before whole-body irradiation and declined rapidly with both shorter and longer intervals. Protection factors (PFs) were obtained by measuring survival curves for clonogenic crypt cells as a function of radiation dose; WR-3689 given 30 min before whole-body irradiation protected jejunum in the microcolony assay with a PF of 1.26 +/- 0.02, 1.50 +/- 0.10, and 1.65 +/- 0.10 at doses of 200, 400, and 800 mg/kg, respectively. Next, the effect of WR-3689 on the survival of jejunal stem cells was determined by assaying the survival of mice given X-ray doses to the whole abdomen in the range leading to death from the gastrointestinal syndrome. The PFs based on the LD50 values for 11-day survival were 1.31 +/- 0.05 (200 mg/kg) and 1.48 +/- 0.05 (400 mg/kg). Crypt-cell survival and animal survival were thus modified to a similar extent by this agent. Finally, the effect of WR-3689 on the induction of DNA single-strand breaks (SSBs) in jejunal cells was measured using an adaptation of the alkaline elution methodology. In mice treated with WR-3689 (400 or 800 mg/kg) 30 min prior to whole-body irradiation with 10 Gy there was no significant reduction in the number of DNA SSBs induced either in samples of the jejunum or in the cycling crypt cells, providing further evidence that there is no simple relationship between the modification of DNA SSBs and the survival of jejunal stem cells.     

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.     

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.     

DNA double-strand break misrejoining after exposure of primary human fibroblasts to CK characteristic X rays, 29 kVp X rays and 60Co gamma rays

The efficiency of ionizing photon radiation for inducing mutations, chromosome aberrations, neoplastic cell transformation, and cell killing depends on the photon energy. We investigated the induction and rejoining of DNA double-strand breaks (DSBs) as possible contributors for the varying efficiencies of different photon energies. A specialized pulsed-field gel electrophoresis assay based on Southern hybridization of single Mbp genomic restriction fragments was employed to assess DSB induction and rejoining by quantifying the restriction fragment band. Unrejoined and misrejoined DSBs were determined in dose fractionation protocols using doses per fraction of 2.2 and 4.4 Gy for CK characteristic X rays, 4 and 8 Gy for 29 kVp X rays, and 5, 10 and 20 Gy for 60Co gamma rays. DSB induction by CK characteristic X rays was about twofold higher than for 60Co gamma rays, whereas 29 kVp X rays showed only marginally elevated levels of induced DSBs compared with 60Co gamma rays (a factor of 1.15). Compared with these modest variations in DSB induction, the variations in the levels of unrejoined and misrejoined DSBs were more significant. Our results suggest that differences in the fidelity of DSB rejoining together with the different efficiencies for induction of DSBs can explain the varying biological effectiveness of different photon energies.     

Cellular responses to DNA double-strand breaks after low-dose γ-irradiation

DNA double-strand breaks (DSBs) are a serious threat to genome stability and cell viability. Although biological effects of low levels of radiation are not clear, the risks of low-dose radiation are of societal importance. Here, we directly monitored induction and repair of single DSBs and quantitatively analyzed the dynamics of interaction of DNA repair proteins at individual DSB sites in living cells using 53BP1 fused to yellow fluorescent protein (YFP-53BP1) as a surrogate marker. The number of DSBs formed was linear with dose from 5 mGy to 1 Gy. The DSBs induced by very low radiation doses (5 mGy) were repaired with efficiency similar to repair of DSBs induced at higher doses. The YFP-53BP1 foci are dynamic structures: 53BP1 rapidly and reversibly interacted at these DSB sites. The time frame of recruitment and affinity of 53BP1 for DSB sites were indistinguishable between low and high doses, providing mechanistic evidence for the similar DSB repair after low- and high-dose radiation. These findings have important implications for estimating the risk associated with low-dose radiation exposure on human health.     

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.     

Radiation-induced DNA single-strand breaks in the intestinal mucosal cells of mice treated with the radioprotectors WR-2721 or 16-16 dimethyl prostaglandin E2

Both S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) and 16-16 dimethyl prostaglandin E2 (dm PGE2) protected the intestinal clonogenic cells to some degree from the effects of 137Cs gamma-irradiation. The D0 was increased from 1.1 +/- 0.12 Gy in controls to 1.55 +/- 0.48 Gy in 16-16 dm PGE2 treated and 2.12 +/- 0.20 Gy in WR-2721 treated mice. Both agents also increased the shoulder of the clonogenic-cell survival curve. Studies were done to measure the effects of these two different radioprotectors on radiation-induction of DNA single-strand breaks in cells comprising the murine intestinal mucosa. The number of DNA single-strand breaks increased with increasing doses of gamma-rays in animals killed immediately following exposure. WR-2721 reduced the number of initial radiation-induced DNA single-strand breaks when given one-half hour before exposure; the time of maximum protection. In contrast, 16-16 dm PGE2 given 1 hour before irradiation (the time required to afford maximum protection from radiation cytotoxicity) did not reduce the number of initial DNA breaks. Both agents impeded the rate of rejoining of DNA breaks with increasing time after irradiation. However, the relationship between these effects on the rate of strand rejoining and cell survival is unknown. These results suggest that either both agents are similarly distributed within the cells but the mechanisms of radioprotection are different, or the mechanisms by which these agents protect are similar, but the two agents affect different subcellular targets, the protection of which contributes to increased cell survival.     

Two-lesion kinetic model of double-strand break rejoining and cell killing

Radiobiological models, such as the lethal and potentially lethal (LPL) model and the repair-misrepair (RMR) model, have been reasonably successful at explaining the cell killing effects of radiation. However, the models have been less successful at relating cell killing to the formation, repair and misrepair of double-strand breaks (DSBs), which are widely accepted as the main type of DNA damage responsible for radiation-induced cell killing. A fully satisfactory model should be capable of predicting cell killing for a wide range of exposure conditions using a single set of model parameters. Moreover, these same parameters should give realistic estimates for the initial DSB yield, the DSB rejoining rate, and the residual number of unrepaired DSBs after all repair is complete. To better link biochemical processing of the DSB to cell killing, a two-lesion kinetic (TLK) model is proposed. In the TLK model, the family of all possible DSBs is subdivided into simple and complex DSBs, and each kind of DSB may have its own repair characteristics. A unique aspect of the TLK model is that break ends associated with both kinds of DSBs are allowed to interact in pairwise fashion to form irreversible lethal and nonlethal damages. To test the performance of the TLK model, nonlinear optimization methods are used to calibrate the model based on data for the survival of CHO cells for an extensive set of single-dose and split-dose exposure conditions. Then some of the postulated mechanisms of action are tested by comparing measured and predicted estimates of the initial DSB yield and the rate of DSB rejoining. The predictions of the TLK model for CHO cell survival and the initial DSB yield and rejoining rate are all shown to be in good agreement with the measured data. Studies suggest a yield of about 25 DSBs Gy(-1) cell(-1). About 20 DSBs Gy(-1) cell(-1) are rejoined quickly (15-min repair half-time), and 4 to 6 DSBs Gy(-1) cell(-1) are rejoined very slowly (10- to 15-h repair half-time). Both the slowly and fast-rejoining DSBs make substantial contributions to the killing of CHO cells by radiation. Although the TLK model provides a much more satisfactory formalism to relate biochemical processing of DSBs to cell killing than did the earlier kinetic models, some small differences among the measured and predicted CHO cell survival and DSB rejoining data suggest that additional factors and processes not considered in the present work may affect biochemical processing of DSBs and hence cell killing.     

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.     

Variation in radiation-induced formation of DNA double-strand breaks as a function of chromatin structure.

The influence of chromatin structure on induction of DNA double-strand breaks (DSBs) by X radiation was studied in DNA from CHO cells. Whole cells, nuclei with condensed or relaxed chromatin, and deproteinized DNA in agarose plugs were irradiated and DSB formation was measured as a decrease in the length of DNA by nondenaturing, pulsed-field, agarose gel electrophoresis. The yield of DSBs in deproteinized DNA (2.3 x 10(-10) DSBs Da-1 Gy-1) was observed to be 70 times greater than the yield of DSBs (3.1 x 10(-12) DSBs Da-1 Gy-1) observed in DNA in the intact cell nucleus. Organization of DNA into the basic nucleosome repeat structure and condensation of the chromatin fiber into higher-order structure protected DNA from DSB induction by factors of 8.3 and 4.5, respectively. An additional twofold protection of DNA in fully condensed chromatin occurred in the intact cell nucleus. Since this protection did not appear to involve chromatin structure, we speculate that this additional protection may result from the association of soluble protein and nonprotein sulfhydryls with DNA in the intact cell nucleus. The results are consistent with the organization of nuclear DNA into both basic nucleosome repeat structure and higher-order chromatin structure providing significant protection against DSB induction.     

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.     

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.     

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 and PARP

Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t(1/2) = 7-8 min) and slow (t(1/2) = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.     

A comparison of radioprotection from three neutron sources and 60Co by WR-2721 and WR-151327

Two thiophosphoroate radiation protectors (WR-2721 and WR-151327) were assessed for their ability to modify the effects of neutron or gamma irradiation on the gastrointestinal tract. Three neutron sources (DOSAR, JANUS, and FERMILAB) were compared to the response obtained after 60Co irradiation. The end points studied were intestinal stem cell survival and LD50(6). DOSAR and JANUS, both fission-spectrum neutrons, showed somewhat different gut sensitivities [LD50(6)] of about 240 and 400 cGy respectively. The intestinal LD50 obtained with FERMILAB neutrons (25 meV) was closer (875 cGy) to that obtained after 60Co (1068 cGy) irradiation. WR-151327 protected against the lethal effects of fission neutron (DOSAR and JANUS) to a greater degree (DMF = 2.2) than with lower LET sources such as FERMILAB neutrons (DMF = 1.7) or 60Co (DMF = 1.7). The results did not correlate with the intestinal stem cell assays where WR-2721 when compared to WR-151327 showed either similar (DOSAR; fission spectrum neutrons) or somewhat better (60Co and FERMILAB neutrons) protection. Possible explanations for the differing results are discussed.     

Evidence from nondenaturing filter elution that induction of double-strand breaks in the DNA of Chinese hamster V79 cells by gamma radiation is proportional to the square of dose

We have used nondenaturing filter elution performed at both pH 7.2 and pH 9.6 to measure the induction of double-strand breaks (DSBs) in the DNA of Chinese hamster V79 cells by 60Co gamma-radiation doses between 10 and 120 Gy. The absolute DSB yields as measured by this assay were determined by using our recent calibration of the assay based upon disintegrations of 125I incorporated into the DNA. An analysis of the dose-response relationship for the induction of DSBs by 60Co gamma rays showed that the number of DSBs induced per dalton of DNA was proportional to the square of the applied dose throughout the dose range used. The contribution made by the dose to the first power was small at pH 9.6 and negligible at pH 7.2. These results suggest that DSB induction in cells by gamma rays may be entirely a two-hit event.     

Induction and processing of oxidative clustered DNA lesions in 56Fe-ion-irradiated human monocytes

Space and cosmic radiation is characterized by energetic heavy ions of high linear energy transfer (LET). Although both low- and high-LET radiations can create oxidative clustered DNA lesions and double-strand breaks (DSBs), the local complexity of oxidative clustered DNA lesions tends to increase with increasing LET. We irradiated 28SC human monocytes with doses from 0-10 Gy of (56)Fe ions (1.046 GeV/ nucleon, LET = 148 keV/microm) and determined the induction and processing of prompt DSBs and oxidative clustered DNA lesions using pulsed-field gel electrophoresis (PFGE) and Number Average Length Analysis (NALA). The (56)Fe ions produced decreased yields of DSBs (10.9 DSB Gy(-1) Gbp(-1)) and clusters (1 DSB: approximately 0.8 Fpg clusters: approximately 0.7 Endo III clusters: approximately 0.5 Endo IV clusters) compared to previous results with (137)Cs gamma rays. The difference in the relative biological effectiveness (RBE) of the measured and predicted DSB yields may be due to the formation of spatially correlated DSBs (regionally multiply damaged sites) which result in small DNA fragments that are difficult to detect with the PFGE assay. The processing data suggest enhanced difficulty compared with gamma rays in the processing of DSBs but not clusters. At the same time, apoptosis is increased compared to that seen with gamma rays. The enhanced levels of apoptosis observed after exposure to (56)Fe ions may be due to the elimination of cells carrying high levels of persistent DNA clusters that are removed only by cell death and/or "splitting" during DNA replication.     

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.     

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.