Analyses of the secondary particle radiation and the DNA damage it causes to human keratinocytes

High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.     

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.     

Effects of protein kinase inhibitors on the accumulation kinetics of p53 protein in normal human embryo cells following X-irradiation.

DNA-damaging agents induce phosphorylation of the p53 protein, resulting in its accumulation in the nucleus. To clarify the signal transduction pathway(s) involved in p53 protein accumulation in normal human embryo cells following X-irradiation, the effects of three protein kinase inhibitors were examined. Quercetin, an inhibitor of heat-shock response, dose dependently suppressed the p53 accumulation induced by X-rays at more than 100 microM. No suppression, however, was observed with calphostin-C, a specific inhibitor of protein kinase C, in the range of 0.05 to 0.25 microM. Wortmannin was the most potent inhibitor of p53 accumulation. Its suppressive effect appears within a few minutes of pretreatment with a dose of 25 microM, but posttreatment was less effective. Our findings suggest that PKC is not involved in X-ray-induced p53 accumulation in normal human embryo cells and that a wortmannin-sensitive pathway acts as a sensor of DNA damage.     

Relative biological effects of neutron mixed-beam irradiation for boron neutron capture therapy on cell survival and DNA double-strand breaks in cultured mammalian cells

Understanding the biological effects of neutron mixed-beam irradiation used for boron neutron capture therapy (BNCT) is important in order to improve the efficacy of the therapy and to reduce side effects. In the present study, cell viability and DNA double-strand breaks (DNA-DSBs) were examined in Chinese hamster ovary cells (CHO-K1) and their radiosensitive mutant cells (xrs5, Ku80-deficient), following neutron mixed-beam irradiation for BNCT. Cell viability was significantly impaired in the neutron irradiation groups compared to the reference gamma-ray irradiation group. The relative biological effectiveness for 10% cell survival was 3.3 and 1.2 for CHO-K1 and xrs5 cells, respectively. There were a similar number of 53BP1 foci, indicators of DNA-DSBs, in the neutron mixed-beam and the gamma-ray groups. In addition, the size of the foci did not differ between groups. However, neutron mixed-beam irradiation resulted in foci with different spatial distributions. The foci were more proximal to each other in the neutron mixed-beam groups than the gamma-ray irradiation groups. These findings suggest that neutron beams may induce another type of DNA damage, such as clustered DNA-DSBs, as has been indicated for other high-LET irradiation.     

Microwaves from GSM mobile telephones affect 53BP1 and gamma-H2AX foci in human lymphocytes from hypersensitive and healthy persons

The data on biologic effects of nonthermal microwaves (MWs) from mobile telephones are diverse, and these effects are presently ignored by safety standards of the International Commission for Non-Ionizing Radiation Protection (ICNIRP). In the present study, we investigated effects of MWs of Global System for Mobile Communication (GSM) at different carrier frequencies on human lymphocytes from healthy persons and from persons reporting hypersensitivity to electromagnetic fields (EMFs). We measured the changes in chromatin conformation, which are indicative of stress response and genotoxic effects, by the method of anomalous viscosity time dependence, and we analyzed tumor suppressor p53-binding protein 1 (53BP1) and phosphorylated histone H2AX (gamma-H2AX), which have been shown to colocalize in distinct foci with DNA double-strand breaks (DSBs), using immunofluorescence confocal laser microscopy. We found that MWs from GSM mobile telephones affect chromatin conformation and 53BP1/gamma-H2AX foci similar to heat shock. For the first time, we report here that effects of MWs from mobile telephones on human lymphocytes are dependent on carrier frequency. On average, the same response was observed in lymphocytes from hypersensitive and healthy subjects.     

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.     

Effect of age on the sensitivity of the rat thyroid gland to ionizing radiation

Exposure to ionizing radiation during childhood is a well-known risk factor for thyroid cancer. Our study evaluated the effect of age on the radiosensitivity of rat thyroid glands. Four-week-old (4W), 7 -week-old (7W), and 8-month-old (8M) male Wistar rats were exposed to 8 Gy of whole-body X-ray irradiation. Thyroids were removed 3–72 h after irradiation, and non-irradiated thyroids served as controls. Ki67-positivity and p53 binding protein 1 (53BP1) focus formation (a DNA damage response) were evaluated via immunohistochemistry. Amounts of proteins involved in DNA damage response (p53, p53 phosphorylated at serine 15, p21), apoptosis (cleaved caspase-3), and autophagy (LC3, p62) were determined via western blotting. mRNA levels of 84 key autophagy-related genes were quantified using polymerase chain reaction arrays. Ki67-positive cells in 4W (with high proliferative activity) and 7W thyroids significantly decreased in number post-irradiation. The number of 53BP1 foci and amount of p53 phosphorylated at serine 15 increased 3 h after irradiation, regardless of age. No increase in apoptosis or in the levels of p53, p21 or cleaved caspase-3 was detected for any ages. Levels of LC3-II and p62 increased in irradiated 4W but not 8M thyroids, whereas expression of several autophagy-related genes was higher in 4W than 8M irradiated thyroids. Irradiation increased the expression of genes encoding pro-apoptotic proteins in both 4W and 8M thyroids. In summary, no apoptosis or p53 accumulation was noted, despite the expression of some pro-apoptotic genes in immature and adult thyroids. Irradiation induced autophagy in immature, but not in adult, rat thyroids.     

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.     

乙基亚硝基脲致大鼠脑星形胶质细胞DNA损伤及对P53和P21蛋白表达的影响

目的研究乙基亚硝基脲(N-ethyl-N-nitrosourea,ENU)诱发原代大鼠脑星形神经胶质细胞DNA损伤及对P53、P21蛋白表达的影响.方法采用机械分离技术,在体外培养大鼠脑神经胶质细胞,通过传代培养的方法纯化大鼠脑星形胶质细胞.给予不同浓度ENU(1.7、3.4、6.8、13.6、27.2 mmol/L)染毒,采用单细胞凝胶电泳(single-cell gel electrophoresis,SCGE)和免疫组织化学的方法观察ENU对星形胶质细胞DNA的损伤作用和抑癌基因p53、p21的产物P53、P21蛋白在细胞中表达.结果不同染毒浓度下,各染毒组对星形胶质细胞均有不同程度DNA损伤作用,各染毒组拖尾细胞百分率与阴性对照组比较,差异均有统计学意义(P＜0.01),且随受试物浓度增加而增加,表现出明显的剂量-效应关系(r=0.916).各染毒组细胞DNA的迁移距离(尾长)与阴性对照组比较,差异均有统计学意义(P＜0.01).在13.6、27.2 mmol/L ENU染毒组,P53有阳性表达,与阴性对照组比较,有统计学意义(P＜0.01),而P21在各实验组均未见阳性表达.结论ENU对大鼠脑星型胶质细胞的毒性主要表现在诱导细胞遗传物质DNA损伤以及抑癌基因p53的表达水平升高.     

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.     

Hypergravity induces phosphorylation of p53 at serine 15, but not an expression of p53-downstream genes

We investigated the p53 signaling pathway induced by hypergravity in the human glioblastoma cell line A172. Hypergravity (20 x g) induced the accumulation of p53 and the phosphorylation of p53 at Ser-15. The phosphorylation of p53 with hypergravity was not inhibited by wortmannin, the PI3-kinase inhibitor. This indicated that the p53 signal pathway induced by hypergravity is different from other p53 signal pathways, such as that of the DNA damage signal. Hypergravity did not induce an expression of the genes Waf-1 or Bax, located downstream from p53. We also examined the expression of genes with hypergravity by using a DNA microarray containing oligo DNA from 30,000 human genes. Hypergravity (20 x g, 6 h) did induce the expression of some genes concerned with the cell signaling pathway and cytoskeleton of the cell, but not any of the p53-downstream genes. DNA microarray revealed the induction of many genes to enable the cells to adapt to the hypergravity environment.     

氨基酚类化合物对人淋巴细胞及小鼠脾脏细胞DNA损伤的比较

目的研究氨基酚类化合物对人外周血淋巴细胞及小鼠脾脏细胞DNA的损伤效应。方法选用人淋巴细胞及小鼠脾脏细胞进行研究。暴露时间为1h。应用单细胞凝胶电泳技术,计算细胞损伤率及专用单位。结果3种氨基酚类化合物均能引起两种细胞不同程度的DNA损伤,高剂量组与阴性对照组相比,差异均有显著性(P<0.05),其中对氨基酚的毒性高于邻氨基酚和间氨基酚,其损伤的程度随剂量的增加而增加。结论与小鼠脾脏细胞相比,人外周血淋巴细胞的敏感性较强,更能直接反映氨基酚类化合物对人群的遗传毒性。     

杂色曲霉素对人胚胃粘膜细胞p53基因致突变研究

为探讨杂色曲霉素（ＳＴ）的致癌作用，运用细胞培养、流式细胞术（ｆｌｏｗｃｙｔｏｍｅｔｒｙ，ＦＣＭ）和银染ＰＣＲ－ＳＳＣＰ等方法研究了不同浓度的ＳＴ（１ｍｇ／Ｌ和３ｍｇ／Ｌ）诱发体外培养的人胚胃粘膜细胞恶性转化情况以及此过程中抑癌基因ｐ５３在蛋白及基因水平上的变化。结果表明，ＳＴ处理４周后处理细胞增殖旺盛，并出现恶性转化灶；ＳＴ处理２４周后处理细胞可在软琼脂上形成细胞集落（ＳＴ１ｍｇ／Ｌ和３ｍｇ／Ｌ组每皿细胞集落数平均分别为１５和１７个）；ＦＣＭ检测结果表明，ＳＴ处理的细胞细胞增殖指数增高，ＤＮＡ含量增高，出现ＤＮＡ异倍体，突变型抑癌基因ｐ５３蛋白表达明显增强。ＰＣＲ－ＳＳＣＰ分析结果显示，ＳＴ处理２２周后，处理细胞ｐ５３第８外显子出现异常泳动带型。本研究进一步证实了ＳＴ对体外培养的人胚胃粘膜细胞的致癌作用     

Micronuclei formation induced by X-ray irradiation does not always result from DNA double-strand breaks

X-ray induced formation of micronuclei is generally thought to result from DNA double-strand breaks (DSBs). However, DNA DSBs inhibit the cell cycle progression that is required for micronucleus formation. In order to reconcile this apparent discrepancy, we investigated whether DNA DSBs induced during the G1 phase could lead to micronucleus formation. We irradiated human embryonic (HE17) cells that had been treated with a radical scavenger, either DMSO or ascorbic acid (AsA), and determined the level of suppression of DNA DSBs or micronuclei. When DNA DSBs were evaluated using 53BP1 foci, treatment with 5 mM AsA did not inhibit the numbers of foci at various intervals after X-ray irradiation; however, treatment with 5 mM or 256 mM DMSO did have a significant inhibitory effect. By contrast, an assay of micronucleus numbers showed that treatment with 5 mM or 256 mM DMSO before X-ray irradiation resulted in almost no inhibition of micronucleus formation, but treatment with 5 mM AsA did have a significant inhibitory effect. These results clearly showed that AsA could suppress micronucleus formation, although it was not effective for suppression of DNA DSBs. Therefore, we conclude that DNA DSBs induced in the G1 phase do not directly lead to micronucleus formation.     

CYP2E1 Ras I基因型对苯酚诱导的酶活性和DNA损伤作用的影响

[目的]研究3种CYP2E1 Ras I基因型对苯酚诱导永生化人淋巴细胞的CYP2E1酶活性及DNA损伤作用的影响。[方法]加入不同浓度的苯酚对CYP2E1 Ras I不同基因型的永生化人淋巴细胞进行24～72 h染毒,采用四甲基偶氮唑蓝法(Methyl Thiazolyl Tetrazolium,MTT)测定细胞毒性并确定苯酚染毒剂量;采用苯胺分光光度法测定CYP2E1酶活性;采用单细胞凝胶电泳(SCGE)法测定DNA损伤作用。[结果]0．05％苯酚24 h染毒对CYP2E1 Ras I野生、杂合及突变基因型细胞均产生生长抑制作用,苯酚对细胞24～72 h染毒的最大无作用浓度为0．01％。各基因型细胞经一定浓度苯酚诱导后酶活性升高(P<0．05),其中野生型细胞经0．005％和0．01％浓度的苯酚染毒可出现酶活性增高的效应,突变型和杂合型细胞经0．01％浓度染毒可出现酶活性增高的效应,且染毒72 h时,在0．01％苯酚染毒剂量下各基因型细胞的CYP2E1酶活力出现明显差异,其中野生基因型细胞的CYP2E1酶活力显著高于突变型、杂合型基因型细胞(P<0．05)。各基因型细胞经一定浓度苯酚染毒后可出现DNA损伤效应,与对照相比,细胞的拖尾率、尾DNA含量及尾长显著升高(P<0．05),且染毒48 h、72 h时,野生基因型细胞的DNA损伤效应与突变型、杂合型细胞相比更为明显(P<0．05)。苯酚诱导后细胞的DNA损伤与CYP2E1酶活性之间呈现明显正相关性。[结论]CYP2E1 Ras I不同基因型明显影响苯酚对人淋巴细胞CYP2E1酶的诱导活性和DNA的损伤程度,其中CYP2E1 Ras I野生基因型细胞的作用最为明显。     

Establishment of immortal normal and ataxia telangiectasia fibroblast cell lines by introduction of the hTERT gene

To establish immortal human cells, we introduced the human catalytic subunit of telomerase (hTERT) gene into skin fibroblast cells obtained from normal and ataxia telangiectasia (AT) individuals of Japanese origin. After hTERT introduction, these cells continue to grow beyond a population doubling number of 200 while maintaining their original radiosensitivity. Inductions of p53, phosphorylation of Ser15 in p53, and induction of p21 by X-ray irradiation in immortal cells derived from normal individual were not affected by the hTERT introduction. Both normal and AT immortal cells exhibited an apparent inhibition of growth as original primary cells when they reached confluence. Karyotype analysis has revealed that they are in a diploid range. These results suggest that cells immortalized by hTERT introduction retain their original characteristics except for immortalization, and that they may be useful for analyzing various effects of radiation on human cells.     

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.     

苯并（a）芘作用下人支气管上皮细胞蛋白表达谱的改变

[目的]观察苯并（a）芘（BaP）作用下人支气管上皮细胞（16HBE）蛋白表达谱的改变。[方法]以0、1、2、4、8、16、32、64um01／L的BaP染毒细胞24h,应用双向凝胶电泳和ImageMaster软件分析BaP处理后细胞内蛋白质表达谱的改变,基质辅助激光解吸附电离-飞行时间质谱（matrix-assisted laser desorption／ionization time of flight mass spectrometry,MALDI-TOF）结合数据库检索鉴定差异表达蛋白质斑点。[结果]各浓度染毒组与对照组相比,成功鉴定了17个差异表达蛋白质斑点,其中表达上调的有9种,下调的有8种。这些蛋白质包括细胞骨架蛋白、与能量代谢有关的蛋白、与DNA损伤修复有关的蛋白、肿瘤标志物、伴侣蛋白、与信号转导有关的蛋白和与氧化应激有关的蛋白。[结论]BaP作用于16HBE后导致与细胞损伤、DNA修复、应激、细胞恶性转化等有关的蛋白质表达发生改变。