Ex vivo determination of the effect of whole-body exposure to fast neutrons on murine spleen cell viability and apoptosis

The effects of high-linear energy transfer (LET) radiations on lymphoid tissues and lymphocytes are not well understood. As a first approach to delineate these effects, the present work was conducted to assess the effects of high-LET radiations on murine spleen cells ex vivo and in vitro. BALB/c mice were irradiated whole-body with 65 MeV neutrons or 15 MV X rays at doses ranging from 0.2 to 3 Gy. Spleens were removed 1 day postirradiation and weighed, and single cell suspensions were prepared and cultured for several days. Apoptosis occurring in vitro was determined at different times by flow cytometry analysis of cells labeled with propidium iodide. It was found that irradiation with fast neutrons reduced spleen weight and cellularity to a greater extent than photons. Considering the spleen cellularity as end point, the relative biological effectiveness (RBE) of fast neutrons was 2. However, for both modes of irradiation, apoptosis of recovered spleen cells in vitro increased as a function of dose and the duration of culture. The level of apoptosis occurring at various times postirradiation was found to be identical for high- and low-LET radiations. Taken together, these results suggest that external as well as cellular factors might differentially modulate the sensitivity of lymphocytes to fast neutrons and photons.     

Laminin 332 deposition is diminished in irradiated skin in an animal model of combined radiation and wound skin injury

Skin exposure to ionizing radiation affects the normal wound healing process and greatly impacts the prognosis of affected individuals. We investigated the effect of ionizing radiation on wound healing in a rat model of combined radiation and wound skin injury. Using a soft X-ray beam, a single dose of ionizing radiation (10-40 Gy) was delivered to the skin without significant exposure to internal organs. At 1 h postirradiation, two skin wounds were made on the back of each rat. Control and experimental animals were euthanized at 3, 7, 14, 21 and 30 days postirradiation. The wound areas were measured, and tissue samples were evaluated for laminin 332 and matrix metalloproteinase (MMP) 2 expression. Our results clearly demonstrate that radiation exposure significantly delayed wound healing in a dose-related manner. Evaluation of irradiated and wounded skin showed decreased deposition of laminin 332 protein in the epidermal basement membrane together with an elevated expression of all three laminin 332 genes within 3 days postirradiation. The elevated laminin 332 gene expression was paralleled by an elevated gene and protein expression of MMP2, suggesting that the reduced amount of laminin 332 in irradiated skin is due to an imbalance between laminin 332 secretion and its accelerated processing by elevated tissue metalloproteinases. Western blot analysis of cultured rat keratinocytes showed decreased laminin 332 deposition by irradiated cells, and incubation of irradiated keratinocytes with MMP inhibitor significantly increased the amount of deposited laminin 332. Furthermore, irradiated keratinocytes exhibited a longer time to close an artificial wound, and this delay was partially corrected by seeding keratinocytes on laminin 332-coated plates. These data strongly suggest that laminin 332 deposition is inhibited by ionizing radiation and, in combination with slower keratinocyte migration, can contribute to the delayed wound healing of irradiated skin.     

Regional blood-to-tissue transport in an irradiated rat glioma model

To assess vascular permeability in intracerebral grafts of the 36B-10, F-344 rat glioma following 20 Gy 137Cs whole brain irradiation, the blood-to-tissue transport constant, K, of [14C]-alpha-aminoisobutyric acid (AIB) was measured with quantitative autoradiography. Mean, 90th percentile, and 95th percentile values of K were determined in individual tumors and in treatment groups. In 15-day-old unirradiated control tumors, mean, 90th percentile, and 95th percentile values of K were, respectively, 11.3, 18.4, and 20.8 ml kg-1 min-1. In 15-day-old tumors irradiated on Day 14 (Day 1 postirradiation tumors) the K values were 5.9, 9.4, and 10.4, all of which were significantly less than the respective control values (P less than 0.01). In 16-day-old tumors irradiated on Day 14 (Day 2 postirradiation tumors), the K values were 10.8, 15.0, and 16.0, respectively, none of which was significantly different from control tumors. Mean K values for Day 2 vs Day 1 postirradiation tumors (10.8 vs 5.9) yielded P less than 0.05, but the 90th percentile and 95th percentile values for Day 2 vs Day 1 yielded 0.05 less than P less than 0.10. Separate experiments measured AIB and 86RbCl uptake in 36B-10 cells in vitro 1 and 2 days following 20 Gy irradiation to assess whether this radiation dose reduced the capacity of tumor cells to trap AIB or Rb+. Irradiation did not reduce the accumulation of either tracer, but rather was associated with an increased accumulation of AIB. Therefore, the AIB transport data suggest that vascular permeability and/or surface area decreases significantly in the day following 20 Gy irradiation and that this decrease reverses by the second day following irradiation.     

Ionizing radiation alters myofilament calcium sensitivity in vascular smooth muscle: potential role of protein kinase C

Radiation exposure increases vascular responsiveness, and this change involves endothelial damage, as well as direct effects on vascular smooth muscle. In this study, we tested the hypothesis that myofilament Ca(2+) sensitivity in vascular smooth muscle is increased from single whole body gamma irradiation (6 Gy). We measured contractile responses from intact and permeabilized rat thoracic aortic rings combined with cytosolic Ca(2+) ([Ca(2+)](i)) measurements. The sensitivity to KCl and phenylephrine increased significantly in tissues from animals on the 9th and 30th days postirradiation compared with control. Irradiation also significantly increased Ca(2+) sensitivity in beta-escin permeabilized smooth muscle on the 9th and 30th days postirradiation. Inhibitors of protein kinase C, chelerythrine, and staurosporine, had no effect on the pCa-tension curves in control permeabilized tissues but significantly decreased Ca(2+) sensitivity in permeabilized tissues on the 9th and 30th days postirradiation. Phorbol dibutyrate (PDBu, 10(-7) M) increased Ca(2+) sensitivity in control skinned smooth muscle but was without effect in irradiated vascular rings. Simultaneous measurement of contractile force and [Ca(2+)](i) showed that myofilament Ca(2+) sensitivity defined as the ratio of force change to [Ca(2+)](i) significantly increased following gamma-irradiation. PDBu (10(-6) M) stimulation of intact aorta produced a sustained contraction, while the increase in [Ca(2+)](i) was transient. In irradiated tissues, PDBu-induced contractions were greater than those seen in control tissues but there was no elevation in [Ca(2+)](i). Taken together, these data strongly support the hypothesis that irradiation increases the sensitivity of vascular smooth muscle myofilaments to Ca(2+) and this effect is dependent on activation of protein kinase C.     

Initiation of apoptosis in cells exposed to medium from the progeny of irradiated cells: a possible mechanism for bystander-induced genomic instability?

Genomic instability and bystander effects have recently been linked experimentally both in vivo and in vitro. The aim of the present study was to determine if medium from irradiated cells several passages distant from the original exposure could initiate apoptosis in unirradiated cells. Human keratinocytes (from the HPV-G cell line) were irradiated with 0.5 Gy or 5 Gy gamma rays. Medium was harvested at each passage up to the 7th passage (approximately 35 population doublings) postirradiation and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential, and the level of reactive oxygen species were all monitored for 24 h after medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential, and increases in reactive oxygen species (from 6 h after medium transfer) were observed in the recipient cells. There was no significant difference between medium conditioned by cells irradiated with 0.5 or 5 Gy. The effect of medium from progeny was the same as the initial effect reported previously and did not diminish with increasing passage number. The data suggest that initiating events in the cascade that leads to apoptosis are induced in unirradiated cells by a signal produced by irradiated cells and that this signal can still be produced by the progeny of irradiated cells for several generations.     

The relative biological effectiveness of low doses of 14 MeV neutrons in steady-state murine spermatogenesis as determined by flow cytometry

The relative biological effectiveness of 14 MeV neutrons in the low-dose range < or =1 Gy has been determined in differentiating and differentiated spermatogonia. Male NMRI mice were exposed to single doses of 2 cGy to 3 Gy of (60)Co gamma rays or neutrons. The ratios of testicular S-phase cells, 4c primary spermatocytes, and elongated spermatids were quantified by DNA flow cytometry 2 to 70 days after irradiation and were found to decrease. Histological samples and testis weight were analyzed in parallel. Doses of 2-5 cGy neutrons and 10-50 cGy gamma rays significantly (P<0.05) decreased the proportions of S-phase cells, spermatocytes and elongated spermatids at 4, 14 and 28 days postirradiation. For S-phase cells, the biphasic shape of the cell survival curves was described with a D(50) of 5 cGy neutrons. The D(50) for (60)Co gamma rays and the relative biological effectiveness could not be determined. The relative biological effectiveness of neutrons at 50% reductions of testis weight, primary spermatocytes, and elongated spermatids were 2.5, 10.0 and 6.1, respectively. This in vivo assay is interesting because of its sensitivity at dose ranges that are relevant for exposures in the environment, the workplace and radiotherapy.     

Erythrocyte Stiffness during Morphological Remodeling Induced by Carbon Ion Radiation

The adverse effect induced by carbon ion radiation (CIR) is still an unavoidable hazard to the treatment object. Thus, evaluation of its adverse effects on the body is a critical problem with respect to radiation therapy. We aimed to investigate the change between the configuration and mechanical properties of erythrocytes induced by radiation and found differences in both the configuration and the mechanical properties with involving in morphological remodeling process. Syrian hamsters were subjected to whole-body irradiation with carbon ion beams (1, 2, 4, and 6 Gy) or X-rays (2, 4, 6, and 12 Gy) for 3, 14 and 28 days. Erythrocytes in peripheral blood and bone marrow were collected for cytomorphological analysis. The mechanical properties of the erythrocytes were determined using atomic force microscopy, and the expression of the cytoskeletal protein spectrin-α1 was analyzed via western blotting. The results showed that dynamic changes were evident in erythrocytes exposed to different doses of carbon ion beams compared with X-rays and the control (0 Gy). The magnitude of impairment of the cell number and cellular morphology manifested the subtle variation according to the irradiation dose. In particular, the differences in the size, shape and mechanical properties of the erythrocytes were well exhibited. Furthermore, immunoblot data showed that the expression of the cytoskeletal protein spectrin-α1 was changed after irradiation, and there was a common pattern among its substantive characteristics in the irradiated group. Based on these findings, the present study concluded that CIR could induce a change in mechanical properties during morphological remodeling of erythrocytes. According to the unique characteristics of the biomechanical categories, we deduce that changes in cytomorphology and mechanical properties can be measured to evaluate the adverse effects generated by tumor radiotherapy. Additionally, for the first time, the current study provides a new strategy for enhancing the assessment of the curative effects and safety of clinical radiotherapy, as well as reducing adverse effects.     

Downscaling Fourier transform infrared spectroscopy to the micrometer and nanogram scale: secondary structure of serotonin and acetylcholine receptors

High signal-to-noise Fourier transform infrared (FTIR) spectra of the 5-hydroxytryptamine (serotonin) receptor (5-HT(3)R) and the nicotinic acetylcholine receptor (nAChR) were obtained by microscope FTIR spectroscopy using micrometer-sized, fully hydrated protein films. Because this novel procedure requires only nanogram quantities of membrane proteins, which is 4-5 orders of magnitude less than the amount of protein typically used for conventional FTIR spectroscopy, it opens the possibility to access the structure and dynamics of many important mammalian receptor proteins. The secondary structure of detergent-solubilized 5-HT(3)R determined by curve fitting of the amide I band yielded 36% alpha-helix, 33% beta-strand, 15% beta-turn, and 16% nonregular structures, which remained unchanged upon reconstitution in lipid membranes. From hydrogen-deuterium exchange, the secondary structure of the water-accessible part of 5-HT(3)R was determined as 14% alpha-helix, 16% beta-strand, 26% beta-turn, and 14% nonregular structures. Interestingly, we found that both the overall and the water-accessible nAChR secondary structures were nearly identical to those of 5-HT(3)R, in agreement with predicted structures of this class of receptors. This is the first time that structural investigations were obtained for two closely related ligand-gated ion channels under strictly identical experimental conditions.     

Late ROS accumulation and radiosensitivity in SOD1-overexpressing human glioma cells

This study investigates the hypothesis that CuZn superoxide dismutase (SOD1) overexpression confers radioresistance to human glioma cells by regulating the late accumulation of reactive oxygen species (ROS) and the G(2)/M-checkpoint pathway. U118-9 human glioma cells (wild type, neo vector control, and stably overexpressing SOD1) were irradiated (0-10 Gy) and assayed for cell survival, cellular ROS levels, cell-cycle-phase distributions, and cyclin B1 expression. SOD1-overexpressing cells were radioresistant compared to wild-type (wt) and neo vector control (neo) cells. Irradiated wt and neo cells showed a significant increase (approximately twofold) in DHE fluorescence beginning at 2 days postirradiation, which remained elevated at 8 days postirradiation. Interestingly, the late accumulation of ROS was suppressed in irradiated SOD1-overexpressing cells. The increase in ROS levels was followed by a decrease in cell growth and viability and an increase in the percentage of cells with sub-G(1) DNA content. SOD1 overexpression enhanced radiation-induced G(2) accumulation within 24 h postirradiation, which was accompanied by a decrease in cyclin B1 mRNA and protein levels. These results support the hypothesis that long after radiation exposure a "metabolic redox response" regulates radiosensitivity of human glioma cells.     

Prolonged increase in T-cell receptor (TCR) variant fractions of spleen T lymphocytes in pregnant mice after gamma irradiation

To investigate the relationship between the radiation-induced increase of T-cell receptor (TCR) defective variant fractions and physiological status such as pregnancy, C57BL/ 6N mice were irradiated with 3 Gy of gamma rays at various days of gestation, just before and just after pregnancy. While the highest level of variant fractions in spleen T lymphocytes appeared at 9 days postirradiation and resolved fairly rapidly for nonpregnant mice, the increased variant fractions for pregnant mice irradiated at 16.5 days of gestation reached a plateau at 14 days postirradiation and remained at high levels until 28 days after irradiation. Therefore, variant fractions 28 days postirradiation were measured to determine the overall effect of radiation on the kinetics of TCR variant fractions during gestation. There was no significant difference in the baseline TCR variant fraction between unirradiated nonpregnant and pregnant mice. TCR variant fractions after irradiation were about twofold higher in pregnant mice (from 10.5 days of gestation until delivery) than those in nonpregnant mice. Both gamma radiation and pregnancy caused a decrease in the proportion of na?ve T-cell subsets and an increase in TCR variant fractions of na?ve T cells. In addition, the prolonged postirradiation increase in the TCR variant fractions of pregnant mice was associated with an increase in serum progesterone level. Differences between pregnant and nonpregnant mice in the kinetics of postirradiation restoration of T-cell systems may be involved in producing the differences in residual TCR variant fractions of these mice.     

Quantitative magnetic resonance spectroscopy reveals a potential relationship between radiation-induced changes in rat brain metabolites and cognitive impairment

To test the efficacy of magnetic resonance spectroscopy (MRS) in identifying radiation-induced brain injury, adult male Fischer 344 rats received fractionated whole-brain irradiation (40 or 45 Gy given in 5-Gy fractions twice a week for 4 or 4.5 weeks, respectively); control rats received sham irradiation. Twelve and 52 weeks after whole-brain irradiation, rats were subjected to high-resolution MRI and proton MRS. No apparent lesions or changes in T(1)- or T(2)-weighted images were noted at either time. This is in agreement with no gross changes being found in histological sections from rats 50 weeks postirradiation. Analysis of the MR spectra obtained 12 weeks after fractionated whole-brain irradiation also failed to show any significant differences (P > 0.1) in the concentration of brain metabolites between the whole-brain-irradiated and sham-irradiated rats. In contrast, analysis of the MR spectra obtained 52 weeks postirradiation revealed significant differences between the irradiated and sham-irradiated rats in the concentrations of several brain metabolites, including increases in the NAA/tCr (P < 0.005) and Glx/tCr (P < 0.001) ratios and a decrease in the mI/tCr ratio (P < 0.01). Although the cognitive function of these rats measured by the object recognition test was not significantly different (P > 0.1) between the irradiated and sham-irradiated rats at 14 weeks postirradiation, it was significantly different (P < 0.02) at 54 weeks postirradiation. These findings suggest that MRS may be a sensitive, noninvasive tool to detect changes in radiation-induced brain metabolites that may be associated with the radiation-induced cognitive impairments observed after prolonged fractionated whole-brain irradiation.     

Meiotic non-disjunction induced by fission neutrons relative to X-rays observed in mouse secondary spermatocytes. II. Dose-effect relationships after treatment of pachytene cells

(C57B1/Cne X C3H/Cne)F1 male mice were irradiated with single acute doses of 0.4 MeV neutrons (from 0.11 to 0.72 Gy) or 250 kV X-rays (from 0.25 to 3 Gy) and sacrificed 5 days later. Chromosome preparations of secondary spermatocytes, irradiated at the stage of pachytene, were analysed and the incidence of hyper-haploidies and chromosome fragments was recorded. Data on numerical aberrations were fitted by highly significant linear relationships for both types of radiation. A relative biological effectiveness (RBE) value of 5.65 was estimated by the ratio between the slopes of the two regression lines. The same linear fitting was applied to frequencies of cells with fragments, even if in this case other types of functions could not be excluded. An RBE value was estimated in the same way as for numerical aberrations and yielded a comparable figure of 5.23. A significant correlation was also found between the incidence of numerical and structural aberrations, which points to the chromosome itself as the prevalent target for radiation-induced non-disjunction (ND). In addition, the highly significant linearity of the dose-effect relationship observed for the induction of aneuploidies suggests, as the simplest hypothesis, a single-hit mechanism of radiation action, possibly through pre-non-disjunctional damage to the centromeric region, rather than an indirect induction of segregational difficulties after primarily induced chromatid interchanges.     

In situ assessment of erythrocyte membrane properties during cold storage

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.     

Effect of neutron and gamma radiation on physical endurance of rats in the swimming test

Three groups of adult male Wistar rats were exposed to neutrons (a single dose of 2 Gy) and gamma radiation (a continuous dose of 6 Gy delivered within 10.5 days) and a combination of both types of radiation. The physical fitness of irradiated and control rats was measured using a forced swimming test in the course and after the termination of the irradiation. A marked decrease in the physical fitness of all irradiated animals was followed by its normalization 3 weeks after irradiation.     

Detection of large deletions of mitochondrial DNA in tissues of mice exposed to X-rays

Large mtDNA deletions in mouse brain and spleen cells, induced by X-radiation at doses of 2 and 5 Gy were studied within four weeks after the exposure of animals to X-rays. Variations in the content of extracellular (deletion) mtDNA were examined in the blood plasma of mice irradiated with 5 Gy in the same postirradiation times. Ionizing radiation was shown to effectively induce large mtDNA deletions at the doses chosen. The level of deletion mtDNA was dependent on dose and postirradiation time.     

Dose-incidence relationships for exencephalia, anophthalmia and prenatal mortality in mouse embryos irradiated with fission neutrons or 250 kV X-rays

Groups of pregnant mice were irradiated at selected times between 10.00 hours on gestation day 7 and 16.00 hours on day 8. Each group received 0.39 Gy of neutrons or 1.60 Gy of X-rays, or was sham irradiated. We identified a period of high susceptibility of the embryos to radiation-induced exencephalia, anophthalmia and prenatal mortality early in gestation day 8. Dose-incidence relationships in this period were investigated with 0.19-0.48 Gy of neutrons and with 0.40-2.00 Gy of X-rays. With increasing neutron dose, incidence of exencephalia in live embryos rose and then declined. This response suggests that embryos with neutron injury of the type that leads to exencephalia are at a greater risk of dying in utero than are similarly irradiated embryos not so injured, and that this risk increases with dose. A model is proposed that accounts for the shape of the neutron dose-incidence curve. X-ray-induced exencephalia showed only an increase with dose. In X-irradiated litters, almost invariably, the incidence of anophthalmia was higher in exencephalic than in nonexencephalic embryos and the ratio of these incidences (relative risk) decreased toward 1 with increasing dose. A model is proposed that accounts for these observations. The incidence of bilateral anophthalmia in X-irradiated embryos was higher than would be expected if the bilateral form resulted solely from independent injury at each of two equally susceptible sites.     

振动光谱与蛋白质二级结构

红外光谱和拉曼光谱(统称分子振动光谱)应用于蛋白质构象研究经历了定性阶段和半定量阶段.近5年来,去卷积和二阶导教等提高分辨率方法以及曲线拟合方法的应用,使得振动光谱在定量计算蛋白质二级结构和监测蛋白质构象变化等方面取得突破性的进展,进入了定量化的阶段.     

Effect of gamma irradiation on insulin receptor interaction in rat erythrocytes

An insulin receptor interaction has been studied in rat erythrocytes after whole-body gamma irradiation (1 Gy). Specific binding of insulin was found to increase 30 days following irradiation against the background of a decreased immunoreactive insulin concentration in the blood. A change in the postirradiation activity of insulin receptors is considered as a manifestation of the homeostatic mechanism of "up" regulation in exposed animals.     

Oncogenic transformation by fractionated doses of neutrons

Oncogenic transformation was assayed after C3H 10T1/2 cells were irradiated with monoenergetic neutrons; cells were exposed to 0.23-, 0.35-, 0.45-, 5.9-, and 13.7-MeV neutrons given singly or in five equal fractions over 8 h. At the biologically effective neutron energy of 0.45 MeV, enhancement of transformation was evident with some small fractionated doses (below 1 Gy). When transformation was examined as a function of neutron energy at 0.5 Gy, enhancement was seen for cells exposed to three of the five energies (0.35, 0.45, and 5.9 MeV). Enhancement was greatest for cells irradiated with 5.9-MeV neutrons. Of the neutron energies examined, 5.9-MeV neutrons had the lowest dose-averaged lineal energy and linear energy transfer. This suggests that enhancement of transformation by fractionated low doses of neutrons may be radiation-quality dependent.