Radioprotection of cultured mammalian cells by the aminothiols WR-1065 and WR-255591: correlation between protection against DNA double-strand breaks and cell killing after gamma radiation

We compared the effects of the radioprotective aminothiols WR-1065 and WR-255591 on the induction of DNA double-strand breaks (DSBs) and on the survival of aerated Chinese hamster ovary cells exposed to 60Co gamma radiation. DSBs were measured using the pH 9.6 neutral elution method. In agreement with earlier studies, protection factors for both drugs measured using the end point of clonogenic cell survival were significantly greater than the protection factors for DSB induction when DSBs were measured after gamma-ray doses ranging from 20 to 90 Gy. However, when DSBs and cell survival measurements were made on the same cell populations after low radiation doses (between 3 and 30 Gy) using the replicate plating method, there appeared to be a close correlation between the modification of DSB induction and the modification of cell survival produced by both drugs. The major influence accounting for the differences between these and previously obtained results appears to be the range of radiation doses used, suggesting that protection against DSB induction is radiation-dose dependent.     

Protective effect of RH-3 with special reference to radiation induced micronuclei in mouse bone marrow

Effect of pre-irradiation administration of different doses of RH-3, the herbal preparation of an Indian medicinal plant Hippophae rhamnoides, 30 min before 10 Gy whole body gamma irradiation was studied. Doses between 25 to 35 mg/kg body wt. were found to render > 80 % survival in mice. In order to investigate whether RH-3 protected against radiation induced genotoxicity, mice were administered different doses of RH-3, 30 min before 2 Gy dose and compared with untreated, RH-3 treated and irradiated controls. The bone marrow cells were collected at different time intervals following various treatments and processed for scoring micronuclei (MN). Administration of RH-3 alone did not enhance the MN frequency as compared to the control, and radiation dose of 2 Gy significantly enhanced the MN frequency (3.1 %, P < 0.01). Pre-irradiation treatment with RH-3, however, reduced the radiation induced MN frequency in a drug dose dependent manner suggesting its radioprotective efficacy. The protective effect of RH-3 on radiation induced perturbations in cell cycle progression was studied flowcytometrically in mouse bone marrow cells. RH-3 treatment (30 mg/kg body wt.) enhanced DNA synthesis (S-phase) in unirradiated controls and also countered radiation induced depression of S-phase to facilitate replenishment of cells lost due to radiation injury.     

ADPRT抑制剂对不同辐射敏感性人肿瘤细胞株的作用比较

从细胞的克隆形成能力和细胞DNA双链断裂及修复几方面分析了两个人卵巢癌细胞株HOC8和A2780对电离辐射的敏感性并探讨了ADP-核糖基转移酶（ADPR）的特异性抑制剂3-氨基苯甲酰胺（3－AB）对二者的辐射增敏效应,结果表明A2780细胞的辐射敏感性大大高于HOC8细胞,其D0值分别为0．9和2．5Gy;γ射线所致两株细胞的初始DNA双链断裂水平没有显著差异,但A2780细胞对DNA双链断裂的修复能力比HOC8细胞低．3AB能降低受照细胞的克隆形成能力及细胞对双链断裂的修复能力,其中对HOC8细胞的作用更为明显．     

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.     

Relationship between phosphorylated histone H2AX formation and cell survival in human microvascular endothelial cells (HMEC) as a function of ionizing radiation exposure in the presence or absence of thiol-containing drugs

Human microvascular endothelial cells (HMEC) were exposed to ionizing radiation at doses ranging from 0 to 16 Gy in either the presence or absence of the active thiol forms of amifostine (WR1065), phosphonol (WR255591), N-acetyl-l-cysteine (NAC), captopril or mesna. Each of these clinically relevant thiols, administered to HMEC at a dose of 4 mM for 30 min prior to irradiation, is known to exhibit antioxidant properties. The purpose of this investigation was to determine the relationship(s), if any, between the frequency of radiation-induced histone H2AX phosphorylation at serine 139 (gamma-H2AX) in cells and subsequent survival, as assessed by colony-forming ability, in exposed cell populations as a function of the presence or absence of each of the five thiol compounds during irradiation. gamma-H2AX formation in irradiated cells, as a function of relative DNA content, was quantified by bivariant flow cytometry analysis with FITC-conjugated gamma-H2AX antibody and nuclear DAPI staining. gamma-H2AX formation in cells was measured as the relative fold increase as a function of the treatment conditions. The frequency of gamma-H2AX-positive cells increased with increasing dose of radiation followed by a dose- and time-dependent decay. The most robust response for gamma-H2AX formation occurred 1 h after irradiation with their relative frequencies decreasing as a function of time 4 and 24 h later. To assess the effects of the various thiols on gamma-H2AX formation, all measurements were made 1 h after irradiation. WR1065 was not only effective in protecting HMEC against gamma-H2AX formation across the entire dose range of radiation exposures used, but it was also significantly more cytoprotective than either its prodrug (WR2721) or disulfide (WR33278) analogue. WR1065 had no significant effect on gamma-H2AX formation when administered immediately or up to 30 min after radiation exposure. An inhibitory effect against gamma-H2AX formation induced by 8 Gy of radiation was expressed by each of the thiols tested. NAC, captopril and mesna were equally effective in reducing the frequency of gamma-H2AX formation, with both WR1065 and WR255591 exhibiting a slightly more robust protective effect. Each of the five thiols was effective in reducing the frequency of gamma-H2AX-positive cells across all phases of the cell cycle. In contrast to the relative ability of each of these thiols to inhibit gamma-H2AX formation after irradiation, NAC, captopril and mesna afforded no protection to HMEC as determined using a colony-forming survival assay. Only WR1065 and WR255591 were effective in reducing the frequencies of radiation-induced gamma-H2AX-positive cells as well as protecting against cell death. These results suggest that the use of gamma-H2AX as a biomarker for screening the efficacy of novel antioxidant radioprotective compounds is highly problematic since their formation and disappearance may be linked to processes beyond simply the formation and repair of radiation-induced DSBs.     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

Molecular-biological properties of blood lymphocytes of Hodgkin's lymphoma patients. Plausible possibility of treatment effect prognosis

Hodgkin's lymphoma (HL) patients were investigated before and during chemical and radiation therapy. The properties of peripheral blood lymphocytes of the HL patients before treatment have been compared with healthy donors and the patients during the treatment. The genetic damage--frequency of cells with micronuclei (MN), the level of DNA single- and double-strand breaks (SSB and DSB), DNA-protein cross-links (DPC) have been studied. Biochemical and physiological parameters have been compared as well: the concentration of reactive oxygen species (ROS), the ability to the adaptive response induction. The radiosensitivity of lymphocytes in vitro exposed to the 1 Gy irradiation has also been determined (by MN test). It was shown that in Hodgkin's lymphoma patients' lymphocytes (in comparison with healthy donors) the frequency of cells with MN does not change, the level of SSBs and DSBs increases, the amount of DPC does not change, and ROS concentration (on average) significantly increases because of the part of the population that have high ROS content. The ROS concentration decreases to control level, the frequency of cells with MN increases, the level of DSBs does not change but the level of DPCs (which prevents the determination of DSB) increases in the patients during treatment. It was also discovered that lymphocyte radiosensitivity correlates with the MN cells frequency before treatment and the ROS concentration. These results make it possible to suppose that the high MN frequency and high ROS concentration in Hodgkin's lymphoma patient lymphocytes (before treatment) can serve as prognostic factors for the effectiveness of radio and chemical therapy.     

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.     

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.     

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.     

On the new mechanism of adaptive response

The role of changes in cell composition of population of human blood lymphocytes in the forming of an adaptive response (AR) has been studied. By micronuclei assay and cytokinetic block with cytohalasin B the frequency of mono-, bi- and multinuclear cells with micronuclei (MN) and without MN were determined in the initial population. The same parameters have been studied after exposure of the population to the adaptive (0.05 Gy), challenge (1.0 Gy) doses and to doses 0.05 + 1.0 Gy 5 hours after. 13 from 23 investigated individuals manifested the AR: the decreasing of the ratio of damaged binuclear cells to the all binuclear cells after the adaptive and challenge exposure. It was shown that the ways of an AR forming are different: in 7 of 13 individuals with AR the number of binuclear cells with MN did not decrease but the amount of binuclear undamaged cells increased. The ratio of these parameters enhances but not for the account of cells with MN decreasing. There is the linear correlation between the frequency of cells with MN and the frequency of binuclear cells in population (spontaneous, after irradiation with doses of 0.05, 1.0 and 0.05 + 1.0 Gy) with the coefficient of correlation about -1. These results show the presence of new mechanism of AR forming, which is not connect with the induction of damage repair and rather with the stimulation of cell division. In the another group of individuals the decrease in damaged cells number after irradiation with doses of 0.05 + 1.0 Gy have been observed. Probably the stimulation of repair system occurred to the moment of 1.0 Gy irradiation. Thus, the mechanism of an AR forming depends on the individual properties of organism. The work was suppoted by RFBR grant 03-04-48325a.     

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.     

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.     

Induction of apoptosis in bone marrow cells after treatment of mice with WR-2721 and gamma-rays: relationship to the cell cycle

Apoptosis and cell proliferation are accepted to be responsible for the maintenance of homeostasis in the hematopoietic system. Understanding of the mechanisms of action of the aminothiols and ionizing radiation on normal hematopoietic cells requires determination of the correlation between apoptotic cell death and cell cycle distribution. The effects of WR-2721 ((S)-2-/3-aminopropylamino/ethylphosphorothioic acid; Amifostine) and ⁶⁰Co gamma-rays on apoptosis and cell cycle progression in the mouse bone marrow were determined. Adult male Swiss mice were exposed to 6 Gy gamma-rays only, or pretreated with WR-2721, at a dose of 400 mg/kg body weight, 30 min before gamma-irradiation. The laser scanning cytometry APO-BRDU^TM assay based on simultaneous analysis of cellular DNA content and the in situ detection of DNA strand breaks was used to identify apoptotic cells and to reveal the cell cycle position of apoptotic and nonapoptotic cells. Temporary changes in the frequency of apoptotic cells with fluorescein isothiocyanate (FITC) labeling of DNA strand breaks, and all bone marrow cells including apoptotic and nonapoptotic ones, whose DNA stained with propidium iodide, were observed in the particular phases of the cell cycle throughout the 96-h period after WR-2721 application and gamma-irradiation. The cell cycle phase specificity of WR-2721 and ⁶⁰Co gamma-irradiation was shown in terms of induction of apoptosis in bone marrow cells. The patterns of alterations in the frequency of apoptotic cells and all bone marrow cells with respect to their cell cycle position were dependent on the agent(s) applied and the time interval after treatment of mice with WR-2721 and/or gamma-rays. A modulatory, suppressive action of WR-2721 on apoptosis induction and the cell cycle perturbation caused in normal cells of the mouse bone marrow by gamma-rays was found.     

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.     

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.     

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.     

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.