测量DNA损伤与修复小鼠脾细胞慢性后<em>在体内</em>暴露于β和γ辐射剂量很低
Summary
A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.
Abstract
低剂量的辐射暴露可能产生多种生物效应是在由高辐射剂量所产生的影响的数量和质量不同。处理适当和有科学依据的方式与环境，职业健康和公共卫生安全问题在很大程度上依赖于准确测量低剂量的污染物，如电离辐射，化学物质的生物效应的能力。 DNA损伤和修复是健康风险最重要的早期指标，由于其潜在的长期后果，例如癌症。在这里，我们描述了一种协议来研究慢性体内暴露于低剂量的γ-和β射线对DNA损伤和修复在小鼠脾细胞中的作用。采用DNA双链断裂的一个普遍接受的标记，磷酸化组蛋白H2AX被称为γH2AX，我们展示它如何被用于评估DNA损伤的不仅是水平，而且还改变在DNA修复能力可能通过体内暴露低剂量制备。流式细胞仪允许在大量样品的免疫荧光标记的γH2AX的快速，准确和可靠的测量。 DNA双链断裂修复可以通过暴露提取脾细胞的2戈瑞一个具有挑战性的剂量以产生足够数量的DNA的破坏触发修理和通过测量诱导（1小时后照射）和残留的DNA损伤进行评估（24小时照射后）。残余DNA损伤将指示不完全修复和长期的基因组不稳定和癌症的风险。结合其它测定法和最终点，可以很容易地在这种被测定体内研究（ 例如 ，染色体畸变，在骨髓的网织红细胞微核频率，基因表达等），这种方法允许的生物效应的准确和上下文评价低水平压力。
Introduction
显著争论低或非常低剂量的电离辐射和公众的恐惧辐射，由广岛和长崎原子弹爆炸和珍稀核电站事故的图像驱动的潜在有害影响（通过大众媒体加剧），导致了很严格的辐射防护监管和有潜在不能科学合理的标准。在过去的三十年中，许多报告已经证明了两者的不足和有害诱导低剂量辐射1-4潜在有益的生物效应的存在。主要辐射的健康危险因素是癌症的概率，根据对原子弹爆炸幸存者接受高或中等剂量的辐射流行病学研究估计。这些数据（所谓的线性无阈值或LNT模型）的线性外推来估算的癌症风险在低剂量。然而，这种方法并没有收到世界通用的科学验收并被大量辩论5。
很明显，更多的研究来澄清这一问题，并可能提高辐射防护标准。这些研究应当包括（最好为外推效果对人类）和这样的最终点作为DNA损伤率，DNA修复和诱变慢性治疗（环境和职业暴露最佳逼近）， 在体内动物模型。已知的是，DNA的是用于破坏性辐射效应和不完整或错误修复可导致诱变和癌发展6的主要目标。
DNA双链断裂（DSB）是最有害类型的DNA损伤之一，并且可能会导致细胞死亡和肿瘤发生7。据，因此，重要的是能够可靠地和精确地测量DSB暴露于低剂量辐射和/或其他压力，如化学污染物后的水平。其中最敏感和特异的标记物的DNA DSB的是磷酸化组蛋白H2AX，称为γH2AX8，但其他标记物和方法已被建议9,10。据估计，数千H2AX分子，在诱导的DSB的附近，是参与γH2AX的形成可进行个体的DSB的通过免疫荧光标记的检测与抗γH2AX抗体和荧光显微镜11。该反应是非常快的，分30至60达到其最大值。有证据表明γH2AX利于修复DNA DSB通过吸引其他修复因子来休息的场所，并通过改变染色质结构来断锚定DNA末端，并提供访问其他修复蛋白（12综述）。在修复DNA DSB的完成，得到γH2AX去磷酸化和/或发生退化，新合成的分子H2AXγH2AX替代染色质10的受灾地区。监测γH2AX的形成和损失，因此，提供的DNA DSB修复动力学的精确估计。这种方法已被用于研究修复DSB在照射高剂量的辐射和其速率和残留的DSB水平的各种人肿瘤细胞系已被证实与放射敏感性13-15。
我们修改了这个实验方法并将其应用到小鼠体内研究，探讨低剂量慢性γ-和β辐射对DNA DSB水平和维修（图1）的影响。首先，我们展示了一个方法来执行的小鼠的长期慢性暴露于β辐射由氚或者发射（氢-3）中氚化水（HTO）的形式或作为有机结合的氚（OBT）溶解在饮用水。这两种形式都预期累积和/或分发不同的机构，因此，产生不同的生物学效应。这两种形式是核工业的潜在危险。这种治疗方法由长期暴露于γ射线在等效剂量率并联以允许正确比较的两个辐射类型，这对于它们的相对生物学有效性的评价是至关重要的。的β-辐射是由电子的，使得它从γ射线，高能光子非常不同。由于这种差异，Β辐射表示主要是内部的健康危害和可能产生较γ射线不同的生物学效应。这种并发症导致曝光过度的监管，β射线通过HTO发出显著争议。因此，HTO在饮用水的公共管理水平各不相同，从100贝可/ L的欧洲至75,000贝可/ L，在澳大利亚。它是，因此，重要的是要比较HTO生物效应等效剂量γ射线的。其次，DNA DSB的速率是使用免疫荧光标记的γH2AX检测完成慢性暴露后测定分离的脾细胞通过流式细胞仪主编。这允许通过在体内暴露造成DNA损伤的程度的评价。然而，这是明智的预期，这些低水平暴露可能不会产生DNA DSB任何检出率;相反，一些隐藏更改/应答可以预期这将影响到细胞的修复DNA损伤的能力。这些变化中，如果发现，可以是刺激（产生有益的影响）或抑制（产生有害的影响）。该协议允许通过用高剂量的辐射，产生一个显著量损坏的挑战所提取的脾细胞揭示这样的变化（ 例如 ，2戈瑞产生每细胞约50的DNA DSB或3 – 5倍，增加总的γH2AX水平） 。接着，形成与γH2AX的损失，这反映了DSB修复的起始和结束时，通过流式细胞术监测。以这种方式，不仅基底和DNA DSB的治疗引起的水平可以被测量，但还对细胞的能力的潜在影响作出回应和修复DNA损伤诱导的高得多的水平应激。
Protocol
Representative Results
Discussion
本文提出的协议是用于进行大规模的老鼠体内研究审查低水平各种化学和物理因素，包括电离辐射的遗传毒性作用是有用的。我们独特的无特定病原体动物设施配备了150 GammaBeam辐射和一个30米长的照射大厅允许进行涉及低或非常低剂量率照射在数百甚至数千老鼠终身学习。较小的辐照设施慢性低剂量照射，可设置在一所大学或医院的环境，放射生物学的研究进行例行和辐射防护指导方针/法…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the contribution of our colleagues Sandrine Roch-Lefevre and Eric Gregoire of the Institute of Radioprotection and Nuclear Safety (Paris, France). This work was supported by the Government of Canada Science and Technology program at Canadian Nuclear Laboratories (Chalk River, Ontario, Canada), the CANDU Owners Group (Toronto, Ontario, Canada), the Canadian Nuclear Safety Commission and by the Institute of Radioprotection and Nuclear Safety (Paris, France).
Materials
| HTO: tritiated water, [3H] | locally obtained from a nuclear reactor | stock activity 3.7 GBq/mL; can be substituted with HTO from Perkin Elmer | |
| OBT: Alanine, L-[3-3H]: organically bound tritium (OBT) | Perkin Elmer | NET348005MC | 1mCi/mL (185 MBq) |
| OBT: Glycine, [2-3H]: organically bound tritium | Perkin Elmer | NET004005MC | 1mCi/mL (185 MBq) |
| OBT: Proline, L-[2,3-3H]: organically bound tritium (OBT) | Perkin Elmer | NET323005MC | 1mCi/mL (185 MBq) |
| tritiated water, [3H] (HTO) | Perkin Elmer | NET001B005MC | substitute for HTO of local origin |
| GammaBeam 150 irradiator | Atomic Energy of Canada Limited | locally manufactured | can be substituted with another g-radiation source of sufficiently low activity |
| tween-20 | Sigma Aldrich | P1379-500ML | |
| RPMI | Fisher Scientific | SH3025501 | Hyclone RPMI 1640 with L-Glutamine and HEPES 500mL |
| fetal bovine serum | Sigma Aldrich | F1051-100ML | |
| anti-gH2AX antibody, clone JBW301 | Millipore | 05-636 | |
| Alexa fluor-488 goat anti-mouse antibody | Life Technologies (formerly Invitrogen) | A21121 | |
| propidium iodine | Sigma Aldrich | P4864-10ML | 1mg/mL |
| ethanol | Commercial Alcohols | P006-EAAN | 500ml bottles Absolute Ethanol |
| 1.5 mL tubes | Fisher Scientific | 2682550 | microcentrifuge tubes |
| 15 mL tubes | Fisher Scientific | 05-539-5 | sterile polypropylene centrifuge tubes |
| liquid nitrogen | Linde | P110403 | |
| 12 x 75 mm mL uncapped glass tubes | Fisher Scientific | K60B1496126 | disposable borosilicate glass tubes with plain end |
| T25 flasks | VWR | CA15708-120 | nunc tisue culture 25ml flask (supplier no. 156340) |
| scissors | Fine Science Tools | 14068-12 | Wagner scissors 12cm sharp/sharp |
| forceps, straight | Fine Science Tools | 11008-13 | Semken forceps 13cm, straight |
| forceps, curved | Fine Science Tools | 11003-12 | Narrow Pattern forceps 12 cm, curved |
| 60 mm petri dishes | VWR | CA25382-100 | BD Falcon tissue culture dish 60x15mm |
| cell strainers, 70 mm | Fisher Scientific | 08-771-2 | Falcon cell strainers 50/case |
| PBS recipe: 1 tablet dissolved in 200mL of deionized water, adjust pH to 7.4 if needed. | Sigma Aldrich | P4417-100TAB | Phosphate Buffered Saline Tablets |
| TBS recipe: to make 10X Stock, | |||
| 30g TRIS HCl | Sigma Aldrich | T3253-1KG | Trizma hydrochloride |
| 88g NaCl | Fisher Scientific | S271-500 | Sodium Chloride |
| 2g KCl | Fisher Scientific | P217-500 | Potassium Chloride |
| Dissolve in 1L of deionized water, adjust pH to 7.4 |
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