In case of an accidental overexposure to ionizing radiation where the dose received by the victim is over 5 Gy, the conventional biological indicator of dose, the dicentric assay, does not provide an accurate enough dose measurement. A more appropriate technique is to measure ring chromosomes in stimulated lymphocytes. Dose-effect relationships were obtained by plotting the frequencies of Premature Chromosome Condensation (PCC)-rings in PCC lymphocytes obtained by chemical induction with Calyculin A in vitro, irradiated with doses between 5 to 25 Gy. Cells were exposed either to neutron or to gamma rays and the corresponding dose effect curves are presented in this paper for the first time in literature. For the elaboration of these curves, 9 675 PCC cells in G1 G2 and M/A stages were analysed. The results were fitted to a lineal model in gamma irradiation up to 25 Gy. For neutron irradiation the data was fitted to a lineal model up to 10 Gy, and then dose saturation was observed. In conclusion, with this technique it is possible to set up dose effect curves up to 25 or 10 Gy according to the gamma or neutron radiation.
To study the genetic effects of low-doses and low-dose-rate ionizing radiation (IR), human lymphoblastoid TK6 cells were exposed to 30 mGy of γ-rays at a dose-rate of 1.2 mGy/hr. The frequency of early mutations (EMs) in the thymidine kinase (TK) gene locus was determined to be 1.7 × 10-6, or 1.9-fold higher than the level seen in unirradated controls. These mutations were analyzed with a loss of heterozygosity (LOH) detection system, a methodology which has been shown to be sensitive to the effects of radiation. Among the 15 EMs observed after IR exposure, 8 were small interstitial-deletion events restricted to the TK gene locus. However, this specific type of event was not found in unirradiated controls. Although these results were observed under the limited conditions, they strongly suggest that the LOH detection system can be used for estimating the genetic effects of a low-dose IR exposure delivered at a low-dose-rate.
Although radiation-induced gene expression has been extensively studied, most of the studies to date have focused on that after single-dose irradiation. As split-dose irradiation, rather than single-dose irradiation, is usual in clinical situations, we investigated the effects of split-dose irradiation on nuclear factor κB (NF-κB) in the human rectum carcinoma cell line, LS174T. After either single- or split-dose irradiation with a different interval, nuclear localization of NF-κB was examined by Western blot and immunofluorescence and its DNA-binding activity was measured by ELISA-based assay. Irradiation-induced NF-κB nuclear accumulation and DNA binding activity increased in a dose-dependent manner. The peak of NF-κB nuclear accumulation and DNA binding activity was seen 2 to 6 hours after a single dose of 4 Gy irradiation and returned to control levels after 12 hours. In split-dose irradiation, NF-κB activity was similar after the first and second doses of 4 Gy irradiation separated by 12 hours. In addition, NF-κB activity was decreased by lengthening the interval between irradiation. The cell survival, which was assessed by colony formation assay, showed inverse correlation to this: the surviving fraction was higher after split-dose irradiation than after single-dose irradiation of the same total dose and it increased as the interval between irradiation was lengthened. Thus the present results showed a correlation between NF-κB activation and the repair of sublethal damage in split-dose irradiation.
The effects of solar radiation on the ESR signals in human tooth enamel were investigated. Enamel samples were exposed to the sunlight in Hiroshima, Japan, on sunny days for a total of about 228 hours over one year. The intensity of the illuminating sunlight was measured with a light meter, which then was converted to the intensity of the solar radiation. Three types of signals caused by the solar radiation were identified. The signal named S1 is identical to the gamma ray radiation-induced axially symmetrical signal and contains two signals S1⊥ and S1// corresponding to the perpendicular and parallel components of the g-tensor characterized by g⊥ = 2.0018 and g// = 1.9975 respectively. In addition, two other peaks named S2 and S3 are induced by the solar radiation. S2 is very near the inherent signal with g = 2.0052, possibly created by the same paramagnetic centers as the natural signal, and S3 is a weaker signal with g = 2.010. On increasing the amount of solar radiation S1 increases linearly, but S2 and S3 reach saturation. The average effect of the solar radiation on the S1⊥ signal is estimated by dose equivalent gamma ray irradiation as 7.8 ± 0.5 mGy (MJ m-2)-1, which corresponds to 19.6 ± 1.3 mGy h-1 at the latitude of Hiroshima. Signals S2 and S3 may be used to recognize the effect of solar radiation on the enamel.
When cell lines are held in a quiescent state after irradiation, survival rates are greater than those from cells that are stimulated to grow immediately after irradiation. These differences in survival rates correspond to rates of potentially lethal damage repair. The effects of confluent holding recovery after γ-irradiation were investigated using normal human fibroblasts (AG1522) and ataxia telangiectasia fibroblasts (GM02052). Calyculin-A-induced premature chromosome condensation and fluorescent in situ hybridization were applied to study G2/M chromosomal aberrations. Survival results indicated normal capacity for PLDR in AG1522 cells but that PLDR was extremely compromised in GM02052 cells. The chromosomal aberration frequency decreased when AG1522 cells were allowed to repair for 24-h, whereas 24-hour incubation had little effect on the aberration frequency in GM02052 cells. Since the main mechanism for dsbs repair during G0/G1 phases of the cells cycle involve the non-homologous end-joining (NHEJ) process, our study indicates that for AG1522 cells the NHEJ repair process is more likely to induce accurate chromosome repair under quiescent G0 conditions than proliferating G1 phase, while in GM02052 cells the fidelity of NHEJ is similarly defective at either cell cycle phase. Reduced fidelity of NHEJ may be responsible for PLDR defect and its hyper-radiosensitivity in A-T cells.
Lipocalin 2 (Lcn2, NGAL) is a member of the lipocalin superfamily with diverse functions such as the transport of fatty acids and the induction of apoptosis. Previous reports indicated that expression of Lcn2 is induced under harmful conditions. However, the mechanisms of the induction of Lcn2 expression remain to be elucidated. In this report, we intended to identify the factor or factors that induce Lcn2 expression. Up-regulation of Lcn2 expression after X-ray exposure was detected in the heart, the kidney and especially in the liver. Primary culture of liver component cells revealed that this up-regulation in the liver was induced in hepatocytes. Up-regulation of Lcn2 expression was also detected in HepG2 cells after the administration of X-rays or H2O2. Interestingly, up-regulation of Lcn2 expression after H2O2 treatment was canceled by the addition of the anti-oxidants, dimethylsulfoxide or cysteamine. These results strongly suggest that Lcn2 expression is induced by reactive oxygen species. Therefore, Lcn2 could be a useful biomarker to identify oxidative stress both in vitro and in vivo.
Apigenin, a common dietary flavonoid present in many fruits and vegetables, is a nonmutagenic chemopreventive agent. In the present study, we investigated the effect of apigenin on the radiosensitivity of SQ-5 cells, which are derived from a human lung carcinoma. Actively growing cells were incubated for 16 h at 37°C in medium containing 40 μM apigenin. The cells were then irradiated with X-rays and incubated with apigenin for a further 8 h. Radiosensitivity was assessed using a clonogenic assay. Apoptosis and necrosis were assessed using acridine orange/ethidium bromide double staining. Cells incubated with apigenin exhibited significantly greater radiosensitivity and apoptosis levels than cells not incubated with apigenin. Protein levels were measured by Western blotting. Incubation with apigenin increased protein expression of WAF1/p21 and decreased protein expression of Bcl-2. Furthermore, apigenin sensitized SQ-5 spheroids (cell aggregates growing in a three-dimensional structure that simulate the growth and microenvironmental conditions of in vivo tumors) to radiation. Thus, apigenin appears to be a promising radiosensitizing agent for use against human carcinomas.
Low-doses of irradiation have been reported to have beneficial effects, particularly anti-tumor effects. In this paper, we show the effects of the low-dose irradiation on T cell activation induced by dendritic cells (DCs). DCs, which had been pre-irradiated at 0.02-1.0 Gy from a 137Cs source, were cultured with allogeneic T cells, and the proliferation of T cells was then examined. The 0.05Gy-pre-irradiated DCs showed the highest proliferation capacity of T cells. The 0.05Gy-irradiation does not augment the expression of major histocompatibility complexes (MHCs) or costimulatory molecules on DCs, as with non-irradiated DCs or 1Gy-irradiated DCs, but does augment the production of IL-2, IL-12 and IFN-γ DCs. These results suggest that the low-dose irradiation augments T cell-activation capacity through cytokine production by DCs, which might shift naïve helper T cells to Th1 cells.
Mammals can barely survive total-body ionizing irradiation greater than 10 Gy. To date, there are few drugs available for radioprotective therapy under such circumstances. Inosine, a natural derivative of adenosine, has been known to provide powerful protection for many kinds of cells and tissues against various insults both in vitro and in vivo. In the present study, we examined whether inosine was also beneficial for mammals subjected to an absolutely lethal total-body ionizing irradiation. Immediately after adult Balb/c mice were exposed to 60Co γ-rays at a single dose of 12 Gy, a moiety of them were administered daily with inosine or adenosine, either at doses of 375 or 750 micromol/kg up to death, and their body weight and survival time were recorded. Some irradiated mice were administered inosine or adenosine daily at doses of 750 micromol/kg and assessed for spatial memory abilities using the Morris water maze. The results demonstrated that, although inosine could not prevent body weight loss in irradiated mice, it was able to significantly prolong their survival time at doses of 750 micromol/kg. Moreover, inosine but not adenosine could suppress spatial memory deficit in irradiated mice. The data suggested that inosine had protective effects on mammals suffering from total-body ionizing irradiation at a single lethal dose.
The radioprotective effect of hawthorn (Crataegus microphylla) fruit extract against genotoxicity induced by gamma irradiation has been investigated in mouse bone marrow cells. A single intraperitoneal (ip) administration of hawthorn extract at doses of 25, 50, 100 and 200 mg/kg 1h prior to gamma irradiation (2 Gy) reduced the frequencies of micronucleated polychromatic erythrocytes (MnPCEs). All four doses of hawthorn extract significantly reduced the frequencies of MnPCEs and increased the PCE/PCE+NCE ratio (polychromatic erythrocyte/ polychromatic erythrocyte + normochromatic erythrocyte) in mice bone marrow compared with the non drug-treated irradiated control (p < 0.02-0.00001). The maximum reduction in MnPCEs was observed in mice treated with extract at a dose of 200 mg/kg. Administration of amifostine at dose 100 mg/kg and hawthorn at dose 200 mg/kg reduced the frequency of MnPCE almost 4.8 and 5.7 fold; respectively, after being exposed to 2 Gy of gamma rays, compare with the irradiated control group. Crataegus extract exhibited concentration-dependent activity on 1,1-diphenyl 2-picrylhydrazyl free radical showing that Crataegus contained high amounts of phenolic compounds and the HPLC analysis determined that it contained chlorogenic acid, epicatechin and hyperoside. It appeared that hawthorn extract with antioxidant activity reduced the genotoxicity induced by gamma irradiation in bone marrow cells.
Present day use of mobile phones is ubiquitous. This causes some concern for human health due to exposure to high-frequency electromagnetic fields (HFEMF) from mobile phones. Consequently, we have examined the effects of 2.45 GHz electromagnetic fields on bacterial mutations and the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations. Using the Ames test, bacteria were exposed to HFEMF for 30 min at specific absorption rates (SARs) from 5 to 200 W/kg. In all strains, there was no significant difference in the frequency of revertant colonies between sham exposure and HFEMF-exposed groups. In examination of mutations of the HPRT gene, Chinese hamster ovary (CHO)-K1 cells were exposed to HFEMF for 2 h at SARs from 5 to 200 W/kg. We detected a combination effect of simultaneous exposure to HFEMF and bleomycin at the respective SARs. A statistically significant difference was observed between the cells exposed to HFEMF at the SAR of 200 W/kg. Cells treated with the combination of HFEMF at SARs from 50 to 200 W/kg and bleomycin exhibited increased HPRT mutations. As the exposure to HFEMF induced an increase in temperature, these increases of mutation frequency may be a result of activation of bleomycin by heat. We consider that the increase of mutation frequency may be due to a thermal effect.
To understand the role of proteins involved in DSB repair modulating SLD recovery, chicken B lymphoma (DT 40) cell lines either proficient or deficient in RAD52, XRCC2, XRCC3, RAD51C and RAD51D were subjected to fractionated irradiation and their survival curves charted. Survival curves of both WT DT40 and RAD52-/-cells had a big shoulder while all the other cells exhibited small shoulders. However, at the higher doses of radiation, RAD51C-/-cells displayed hypersensitivity comparable to the data obtained for the homologous recombination deficient RAD54-/-cells. Repair of SLD was measured as an increase in survival after a split dose irradiation with an interval of incubation between the radiation doses. All the cell lines (parental DT40 and genetic knockout cell lines viz., RAD52-/-, XRCC2-/-, XRCC3-/- RAD51C-/- and RAD51D-/-) used in this study demonstrated a typical split-dose recovery capacity with a specific peak, which varied depending on the cell type. The maximum survival of WT DT40 and RAD52-/-was reached at about 1-2 hours after the first dose of radiation and then decreased to a minimum thereafter (5h). The increase in the survival peaked once again by about 8 hours. The survival trends observed in XRCC2-/-, XRCC3-/-, RAD51C-/- and RAD51D-/-knockout cells were also similar, except for the difference in the initial delay of a peak survival for RAD51D-/-and lower survival ratios. The second phase of increase in the survival in these cell lines was much slower in XRCC2-/-, XRCC3-/-, RAD51C-/-and RAD51D-/-and further delayed when compared with that of RAD52-/-and parental DT40 cells suggesting a dependence on their cell cycle kinetics. This study demonstrates that the participation of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in the DSB repair via homologous recombination is of less importance in comparison to RAD54, as RAD54 deficient cells demonstrated complete absence of SLD recovery.