In vivo measurement of dose distribution in patients' lymphocytes: helical tomotherapy versus step-and-shoot IMRT in prostate cancer

In radiotherapy, in vivo measurement of dose distribution within patients'' lymphocytes can be performed by detecting gamma-H2AX foci in lymphocyte nuclei. This method can help in determining the whole-body dose. Options for risk estimations for toxicities in normal tissue and for the incidence of secondary malignancy are still under debate. In this investigation, helical tomotherapy (TOMO) is compared with step-and-shoot IMRT (SSIMRT) of the prostate gland by measuring the dose distribution within patients'' lymphocytes. In this prospective study, blood was taken from 20 patients before and 10 min after their first irradiation fraction for each technique. The isolated leukocytes were fixed 2 h after radiation. DNA double-stranded breaks in lymphocyte nuclei were stained immunocytochemically using anti-gamma-H2AX antibodies. Gamma-H2AX foci distribution in lymphocytes was determined for each patient. Using a calibration line, dose distributions in patients'' lymphocytes were determined by studying the gamma-H2AX foci distribution, and these data were used to generate a cumulative dose–lymphocyte histogram (DLH). Measured in vivo (DLH), significantly fewer lymphocytes indicated low-dose exposure (<40% of the applied dose) during TOMO compared with SSIMRT. The dose exposure range, between 45 and 100%, was equal with both radiation techniques. The mean number of gamma-H2AX foci per lymphocyte was significantly lower in the TOMO group compared with the SSIMRT group. In radiotherapy of the prostate gland, TOMO generates a smaller fraction of patients'' lymphocytes with low-dose exposure relative to the whole body compared with SSIMRT. Differences in the constructional buildup of the different linear accelerator systems, e.g. the flattening filter, may be the cause thereof. The influence of these methods on the incidence of secondary malignancy should be investigated in further studies.     

γ-H2AX与DNA双链断裂关系的研究进展

H2AX是组蛋白的一个种类,普遍存在于整个基因组中。据报道,DNA双链断裂后可诱导位于丝氨酸-139位C-端保守区域内的H2AX磷酸化形成γ-H2AX,在荧光显微镜下形成可见的γ-H2AX焦点。用特异性抗体的免疫荧光法检测γ-H2AX已成为测定DSB的金标准。目前,多种物理性、化学性和生物性的因素都能诱导形成γ-H2AX焦点。本文综述了不同因素诱导形成γ-H2AX的机制及其与DNA双链断裂之间的关系的研究进展。     

Molecular mechanism of the recruitment of NBS1/hMRE11/hRAD50 complex to DNA double-strand breaks: NBS1 binds to gamma-H2AX through FHA/BRCT domain

DNA double-strand breaks represent the most potentially serious damage to a genome, and hence, many repair proteins are recruited to DNA damage sites by as yet poorly characterized sensor mechanisms. We clarified that NBS1 physically interacts with gamma-H2AX to form nuclear foci at DNA damage sites. The fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT) of NBS1 are essential for this physical interaction and focus formation of NBS1 in response to DNA damage. The inhibition of this interaction by introduction of anti-gamma-H2AX antibody into cells abolishes NBS1 foci formation in response to DNA damage. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for re-localization of the NBS1/hMRE11/hRAD50 complex to the vicinity of DNA damage. Moreover, the foci formation of DNA repair-related proteins containing BRCT domain, such as BRCA1, requires the interaction with gamma-H2AX in response to DNA damage. These findings indicate that the physical interaction between gamma-H2AX and DNA repair-related proteins is indispensable for the recruitment of these proteins. Further, it was recently reported that the NBS1/hMRE11/hRAD50 complex has a crucial role for both the recruitment of ATM to DNA damage sites and the subsequent activation of ATM. Therefore, both gamma-H2AX and the NBS1/hMRE11/hRAD50 complex might function for the initial recognition of DNA damage.     

Effects of GSM 1800 MHz radiofrequency electromagnetic fields on DNA damage in Chinese hamster lung cells

OBJECTIVE: To study the effects of GSM 1800 MHz radiofrequency electromagnetic fields (RF EMF) on DNA damage in Chinese hamster lung (CHL) cells. METHODS: The cells were intermittently exposed or sham-exposed to GSM 1800 MHz RF EMF (5 minutes on/10 minutes off) at a special absorption rate (SAR) of 3.0 W/kg for 1 hour or 24 hours. Meanwhile, cells exposed to 2-acetaminofluorene, a DNA damage agent, at a final concentration of 20 mg/L for 2 hours were used as positive control. After exposure, cells were fixed by using 4% paraformaldehyde and processed for phosphorylated form of H2AX (gammaH2AX) immunofluorescence measurement. The primary antibody used for immunofluorescence was mouse monoclonal antibody against gammaH2AX and the secondary antibody was fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). The gammaH2AX foci and nuclei were visualized with an Olympus AX70 fluorescent microscope. Image Pro-Plus software was used to count the gammaH2AX foci in each cell. For each exposure condition, at least 50 cells were selected to detect gammaH2AX foci. Cells were classified as positive when more than five foci were detected. The percentage of gammaH2AX foci positive cells was adopted as the index of DNA damage. RESULTS: The percentage of gammaH2AX foci positive cell of 1800 MHz RF EMF exposure for 24 hours (37.9 +/- 8.6)% or 2-acetylaminofluorene exposure (50.9 +/- 9.4)% was significantly higher compared with the sham-exposure (28.0 +/- 8.4)%. However, there was no significant difference between the sham-exposure and RF EMF exposure for 1 hour (31.8 +/- 8.7)%. CONCLUSION: 1800 MHz RF EMF (SAR, 3.0 W/kg) for 24 hours might induce DNA damage in CHL cells.     

Current topics in DNA double-strand break repair

DNA double strand break (DSB) is one of the most critical types of damage which is induced by ionizing radiation. In this review, we summarize current progress in investigations on the function of DSB repair-related proteins. We focused on recent findings in the analysis of the function of proteins such as 53BP1, histone H2AX, Mus81-Eme1, Fanc complex, and UBC13, which are found to be related to homologous recombination repair or to non-homologous end joining. In addition to the function of these proteins in DSB repair, the biological function of nuclear foci formation following DSB induction is discussed.     

An alternative mechanism for radioprotection by dimethyl sulfoxide; possible facilitation of DNA double-strand break repair

The radioprotective effects of dimethyl sulfoxide (DMSO) have been known for many years, and the suppression of hydroxyl (OH) radicals induced by ionizing radiation has been thought to be the main cause of this effect. However, the DMSO concentration used was very high, and might be toxic, in earlier studies. In the present study, we administered a lower, non-toxic concentration (0.5%, i.e., 64 mM) of DMSO before irradiation and examined its radioprotective effects. Colony formation assay and micronucleus assay showed significant radioprotective effects in CHO, but not in xrs5, which is defective in the repair function of DNA double-strand breaks. The levels of phosphorylated H2AX and the formation of 53BP1 foci 15 minutes after irradiation, which might reflect initial DNA double-strand breaks, in DMSO-treated CHO cells were similar to those in non-treated cells, suggesting that the radioprotective effects were not attributable to the suppression of general indirect action in the lower concentration of DMSO. On the other hand, 2 hours after irradiation, the average number of 53BP1 foci, which might reflect residual DNA double-strand breaks, was significantly decreased in DMSO-treated CHO cells compared to non-treated cells. The results indicated that low concentration of DMSO exerts radioprotective effects through the facilitation of DNA double-strand break repair rather than through the suppression of indirect action.     

γ-H2AX的辐射剂量效应关系研究

目的用共聚焦显微镜分别检测在整体和离体条件下电离辐射强度与γ-H2AX的剂量效应关系以及整体与离体之间的一致性关系。方法用137Cs源分7个剂量点(0、0.5、1、2、4、6、8Gy)照射Wister雌性大鼠外周全血,提取淋巴细胞,利用共聚焦显微镜检测电离辐射与γ-H2AX的剂量效应关系。结果在离体和整体条件下均发现电离辐射与γ-H2AX存在剂量反应关系,拟合的剂量反应曲线分别为:Y整=0.096+0.13X;Y离=0.040+0.21X。并且在离体和整体两种条件下的剂量反应关系具有一致性(R=0.98)。结论γ-H2AX与照射剂量之间存在正相关,相关系数R大于0.98。表明γ-H2AX检测有作为快速辐射生物剂量指标的可能性,可以用离体照射实验替代整体照射。     

Biological effects of field emission-type X-rays generated by nanotechnology

Thermionic emission (TE)-type X-ray generators have been exclusively used in medicine, but there are many difficulties in making these X-ray sources compact. A field emission (FE)-type X-ray generator using carbon nanotubes is a newly-developed compact system that can be as small as several cm in length. Considering the compactness of the equipment, the FE-type X-ray generator may become a useful tool for endoscopic, intracavitary or intraoperative radiotherapy in the future. The aim of this study was to investigate the biological effects of X-rays generated by the FE-type X-ray source in comparison with those of conventional TE-type X-rays. Mouse thymic lymphoma 3SB cells were irradiated by an FE-type X-ray generator developed by our group and a conventional TE-type X-ray source under identical conditions. DNA damage after radiation was detected by foci formation of phospho-H2AX (gamma-H2AX). Effect on the cell cycle was analyzed by flow cytometry. Activation of the DNA damage checkpoint was analyzed by immunoblotting. Induction of apoptosis was studied using the TUNEL assay. In terms of induction of DNA damage (DNA double-strand breaks), activation of cell cycle checkpoints (p53 stabilization, p21 induction, Chk1 and Chk2 phosphorylations), and induction of apoptotic cell death, FE-type X-rays were as effective as TE-type X-rays, and FE-type X-rays appeared to be applicable to radiation therapy.     

Interference between mobile phones and pacemakers: a look inside

In this study we analyzed the problem of electromagnetic interference (EMI) between mobile telephones and cardiac pacemakers (PM), by looking at the mechanisms by which the radiated radio frequency (RF) GSM signal may affect the pacemaker function. From a literature review on this topic, we noticed that older pacemakers had a higher rate of being affected by mobile phones when compared to newer ones. This is probably due to the fact that new generation of PM are more protected against electromagnetic field, being equipped with RF feedthrough filters incorporated to the internal PM circuitry. In some experiments conducted by our group, we found that modulated RF signals are somehow demodulated by the PM internal non-linear circuit elements, if no feedthrough assembly is incorporated inside the PM. Such demodulation phenomenon poses a critical problem because digital cellular phones use extremely low-frequency modulation (as low as 2 Hz), that can be mistaken for normal heartbeat. The feedthrough assembly seems instead to prevents the RF signals from accessing the PM enclosure, thus attenuating EMI signals over a broad range of frequencies.     

High linear-energy-transfer radiation can overcome radioresistance of glioma stem-like cells to low linear-energy-transfer radiation

Ionizing radiation is applied as the standard treatment for glioblastoma multiforme (GBM). However, radiotherapy remains merely palliative, not curative, because of the existence of glioma stem cells (GSCs), which are regarded as highly radioresistant to low linear-energy-transfer (LET) photons. Here we analyzed whether or not high-LET particles can overcome the radioresistance of GSCs. Glioma stem-like cells (GSLCs) were induced from the GBM cell line A172 in stem cell culture medium. The phenotypes of GSLCs and wild-type cells were confirmed using stem cell markers. These cells were irradiated with ⁶⁰Co gamma rays or reactor neutron beams. Under neutron-beam irradiation, high-LET proton particles can be produced through elastic scattering or nitrogen capture reaction. Radiosensitivity was assessed by a colony-forming assay, and the DNA double-strand breaks (DSBs) were assessed by a histone gamma-H2AX focus detection assay. In stem cell culture medium, GSLCs could form neurosphere-like cells and express neural stem cell markers (Sox2 and Musashi) abundantly in comparison with their parental cells. GSLCs were significantly more radioresistant to gamma rays than their parental cells, but neutron beams overcame this resistance. There were significantly fewer gamma-H2AX foci in the A172 GSLCs 24 h after irradiation with gamma rays than in their parental cultured cells, while there was no apparent difference following neutron-beam irradiation. High-LET radiation can overcome the radioresistance of GSLCs by producing unrepairable DNA DSBs. High-LET radiation therapy might have the potential to overcome GBM''s resistance to X-rays in a clinical setting.     

Expression and radiation-induced phosphorylation of histone H2AX in mammalian cells

The mouse histone H2AX (H2AX) has unique C-terminal Ser residues, which are phosphorylated in response to DNA double-strand breaks (DSBs) by ionizing radiation, suggesting that it plays a role in the maintenance of genomic stability. Here, we show that the H2AX protein was detected in most cells in various tissues, and was abundant in the S phase of the cell cycle. Following X-ray irradiation, H2AX was phosphorylated (gamma-H2AX) in the thymus, small intestine and testis. However, H2AX in epithelial cells in the villi of the small intestine were not strongly phosphorylated, even after X-irradiation. Thus, H2AX was expressed in almost all cells. However, the cells that expressed H2AX were not always phosphorylated by X-irradiation, suggesting a different mechanism of kination in those cells.     

Persistence and dynamics of DNA damage signal amplification determined by microcolony formation and live-cell imaging

Cell cycle checkpoints are essential cellular process protecting the integrity of the genome from DNA damaging agents. In the present study, we developed a microcolony assay, in which normal human diploid fibroblast-like cells exposed to ionizing radiation, were plated onto coverslips at very low density (3 cells/cm(2)). Cells were grown for up to 3 days, and phosphorylated ATM at Ser1981 and 53BP1 foci were analyzed as the markers for an amplified DNA damage signal. We observed a dose-dependent increase in the fraction of non-dividing cells, whose increase was compromised by knocking down p53 expression. While large persistent foci were predominantly formed in non-dividing cells, we observed some growing colonies that contained cells with large foci. As each microcolony was derived from a single cell, it appeared that some cells could proliferate with large foci. A live-imaging analysis using hTERT-immortalized normal human diploid cells transfected with the EGFP-tagged 53BP1 gene revealed that the formation of persistent large foci was highly dynamic. Delayed appearance and disappearance of large foci were frequently observed in exposed cells visualized 12-72 hours after X-irradiation. Thus, our results indicate that amplified DNA damage signal could be ignored, which may be explained in part by the dynamic nature of the amplification process.     

Histone H2AX phosphorylation independent of ATM after X-irradiation in mouse liver and kidney in situ

Histone H2AX undergoes phosphorylation at Ser-139 (gamma-H2AX) rapidly in response to DNA double-strand breaks (DSBs) induced by ionizing radiation. The post-translational modification of H2AX plays a central role in responses to radiation, including the repair of DSBs. Although ataxia telangiectasia mutated (ATM) kinase phosphorylates Ser-139 of H2AX in vitro, the post-translational modification pattern and the modifier of H2AX in organs in vivo are not yet well understood. In this study, we detected phosphorylation of H2AX at Ser-139 in cells of the mouse ear, liver, and kidney after X-irradiation. Moreover, the phosphorylation of H2AX was regulated depending on not only the cell type, but also the organ type and the localization of a cell type in an organ. Following X-irradiation, H2AX was phosphorylated in the liver and kidney of ATM gene knockout mice, suggesting that ATM kinase is not essential for phosphorylation of H2AX in these organs after X-irradiation in vivo.     

Low-dose ionizing irradiation triggers a 53BP1 response to DNA double strand breaks in mouse spermatogonial stem cells

The present study aims to examine the effect of low-dose ionizing irradiation on DNA double strand breaks (DSB) in mouse spermatogonial stem cells (SSCs) and reveal the underlying pathways for the DNA repair for DSB in SSCs. Eighteen one-month-old mice were divided into 6 groups and sacrificed separately at 45 minutes, 2 hours, 24 hours, 48 hours, and 72 hours after 0.1Gy X-ray irradiation (mice without receiving ionizing irradiation served as control). After perfusion fixation, testes were removed, sectioned, and followed by staining of γH2AX, 53BP1, Caspase 3, and promyelocytic leukemia zinc-finger (PLZF) for analysis among the different groups. The staining was observed by immunofluorescence visualized by confocal laser scanning. After low-dose irradiation, only 53BP1, but not Caspase3 or γH2AX was upregulated in PLZF positive SSCs within 45 minutes. The expression level of 53BP1 gradually decreased 24 hours after irradiation. Moreover, low-dose irradiation had no effect on the cell number and apoptotic status of SSCs. However other spermatogenic cells highly expressed γH2AX shortly after irradiation which was dramatically reduced following the events of DNA repair. It appears that low-dose ionizing irradiation may cause the DNA DSB of mouse spermatogenic cells. 53BP1, but not γH2AX, is involved in the DNA repair for DSB in SSCs. Our data indicates that 53BP1 plays an important role in the pathophysiological repair of DNA DSB in SSCs. This may open a new avenue to understanding the mechanisms of DNA repair of SSCs and male infertility.     

Melatonin reduces oxidative stress induced by chronic exposure of microwave radiation from mobile phones in rat brain

PURPOSE: The aim of the study was to evaluate the intensity of oxidative stress in the brain of animals chronically exposed to mobile phones and potential protective effects of melatonin in reducing oxidative stress and brain injury. MATERIALS AND METHODS: Experiments were performed on Wistar rats exposed to microwave radiation during 20, 40 and 60 days. Four groups were formed: I group (control)- animals treated by saline, intraperitoneally (i.p.) applied daily during follow up, II group (Mel)- rats treated daily with melatonin (2 mg kg(-1) body weight i.p.), III group (MWs)- microwave exposed rats, IV group (MWs + Mel)- MWs exposed rats treated with melatonin (2 mg kg(-1) body weight i.p.). The microwave radiation was produced by a mobile test phone (SAR = 0.043-0.135 W/kg). RESULTS: A significant increase in the brain tissue malondialdehyde (MDA) and carbonyl group concentration was registered during exposure. Decreased activity of catalase (CAT) and increased activity of xanthine oxidase (XO) remained after 40 and 60 days of exposure to mobile phones. Melatonin treatment significantly prevented the increase in the MDA content and XO activity in the brain tissue after 40 days of exposure while it was unable to prevent the decrease of CAT activity and increase of carbonyl group contents. CONCLUSION: We demonstrated two important findings; that mobile phones caused oxidative damage biochemically by increasing the levels of MDA, carbonyl groups, XO activity and decreasing CAT activity; and that treatment with the melatonin significantly prevented oxidative damage in the brain.     

No induction of p53 phosphorylation and few focus formation of phosphorylated H2AX suggest efficient repair of DNA damage during chronic low-dose-rate irradiation in human cells

Human fibroblast cells obtained from a normal individual and immortalized by introduction of the hTERT gene were irradiated with 0 to 5 Gy of acute high-dose-rate radiation (1.8 Gy/min) or chronic low-dose-rate radiation (0.3 mGy/min) in the G0 phase, and p53 activation was studied. After high-dose-rate irradiation, a dose-dependent induction of Ser15 phosphorylation was observed, whereas after low-dose-rate irradiation almost none was observed. Then we analyzed the focus formation of phosphorylated histone H2AX protein, which is closely correlated with the induction of double-strand breaks. High-dose-rate radiation induced a significant number of foci in a dose-dependent manner, whereas, low-dose-rate radiation could induce only a few foci even at the highest dose. These results strongly suggest that DNA damage induced by low-dose-rate radiation such as a double-strand break is efficiently repaired during chronic irradiation.     

Spontaneous and induced sister chromatid exchanges in lymphocytes of healthy persons

Spontaneous and mitomycin C-induced sister chromatid exchanges (SCEs) in lymphocytes were analyzed in 46 healthy persons. Our results suggest that the frequency of spontaneous SCE in lymphocytes is useful for evaluation of the biological effects of environmental mutagens, and that the induced-SCE test with a mutagen and lymphocytes may be used to detect persons with a high sensitivity to the mutagen.     

Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector

The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effect. Single ion traversal through a cellular nucleus produces complex and massive DNA damage at a nanometer level, leading to cell inactivation, mutations and transformation. We present a novel approach that uses a fluorescent nuclear track detector (FNTD) for the simultaneous detection of the geometrical images of ion traversals and DNA damage in single cells using confocal microscopy. HT1080 or HT1080–53BP1-GFP cells were cultured on the surface of a FNTD and exposed to 5.1-MeV/n neon ions. The positions of the ion traversals were obtained as fluorescent images of a FNTD. Localized DNA damage in cells was identified as fluorescent spots of γ-H2AX or 53BP1-GFP. These track images and images of damaged DNA were obtained in a short time using a confocal laser scanning microscope. The geometrical distribution of DNA damage indicated by fluorescent γ-H2AX spots in fixed cells or fluorescent 53BP1-GFP spots in living cells was found to correlate well with the distribution of the ion traversals. This method will be useful for evaluating the number of ion hits on individual cells, not only for micro-beam but also for random-beam experiments.     

Comparison of the effects of continuous and pulsed mobile phone like RF exposure on the human EEG

It is not clear yet whether Global System for Mobiles (GSM) mobile phone radiation has the ability to interfere with normal resting brain function. There have been reports that GSM exposure increases alpha band power, and does so only when the signal is modulated at low frequencies (Huber, R., Treyer, V., Borbely, A. A., Schuderer, J., Gottselig, J. M., Landolt, H.P., Werth, E., Berthold,T., Kuster, N., Buck, A and Achermann, P. Electromagnetic fields, such as those from mobile phones, alter regional cerebral blood flow and sleep and waking EEG. J Sleep Res 11, 289-295, 2002.) However, as that research employed exposure distributions that are not typical of normal GSM handset usage (deep brain areas were overexposed), it remains to be determined whether a similar result patterning would arise from a more representative exposure. In this fully counterbalanced cross-over design, we recruited 12 participants and tried to replicate the modulation linked post exposure alpha band power increase described above, but with an exposure source (dipole antenna) more closely resembling that of a real GSM handset. Exposures lasted for 15 minutes. No changes to alpha power were found for either modulated or unmodulated radiofrequency fields, and thus we failed to replicate the above results. Possible reasons for this failure to replicate are discussed, with the main reason argued to be the lower and more representative exposure distribution employed in the present study. In addition we investigated the possible GSM exposure related effects on the non-linear features of the resting electroencephalogram using the Approximate Entropy (ApEn) method of analysis. Again, no effect was demonstrated for either modulated or unmodulated radiofrequency exposures.     

Human brain wave activity during exposure to radiofrequency field emissions from mobile phones

The aim of this study was to determine whether there is an effect of mobile phone electromagnetic field emissions on the human electroencephalograph (EEG). EEG recordings from ten awake subjects were taken during exposure to radiofrequency (RF) emissions from a mobile phone positioned behind the head. Two experimental trials were conducted. In the first trial, RF exposures were generated by a GSM mobile phone with the speaker disabled and configured to transmit at full-radiated power. During the second trial, exposures were generated by a non-modified GSM mobile phone in active standby mode. For each trial, subjects were exposed in five minute intervals to a randomized, interrupted sequence of five active and five sham exposures. The experiment was conducted under single-blind conditions. The average EEG band power in active exposure recordings was compared to corresponding sham recordings. Statistical tests indicated significant difference in the full-power mode trial within the EEG alpha (8-13 Hz) and beta (13-32 Hz) bands. A subsequent statistical analysis of median spectral power in discrete EEG rhythms revealed significant differences in 7 of the 32 distinct frequencies overall. In conclusion, the results of this study lend support to EEG effects from mobile phones activated in talk-mode.