体内碱性彗星试验和酶修饰碱性彗星试验测量DNA链断裂和DNA氧化损伤大鼠肝
Summary
The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.
Abstract
Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.
Introduction
碱性彗星试验在单细胞水平测量DNA链断裂。单个细胞的悬浮液被嵌入在琼脂糖上的显微镜载玻片和细胞裂解而形成的类核，其中包含的DNA超螺旋环路。电泳在pH> 13的结果在超螺旋的在含有链断裂，与DNA的朝向阳极迁移释放链DNA环的损失，创建可以通过荧光显微镜观察彗星状结构。片段化DNA从彗星的“头”到基于所述片段的大小，以及相对于总强度（头部和尾部）彗星尾巴的相对荧光的“尾巴”迁移可以用来量化DNA断裂1 ，2。该法简便，灵敏，多功能，快速，相对便宜1。引起的DNA损伤剂片段化DNA的检测中使用的测定法用于定量细胞中的DNA损伤或分离自细胞核INDIVI具有潜在基因毒性材料（多个）处理的动物的双重组织。由于它的优点， 体内彗星试验被推荐为第二体内遗传毒性试验（ 与体内微核试验配对）在当前国际协调会议（ICH）3和欧洲食品安全局进行产品安全评估（EFSA ）4监管指引。在我们的实验室，我们已采用的测定法中通过食品成分，医药品，和纳米材料5-10诱导的体内 DNA损伤评估。大鼠肝将被用作在本协议的例子，但可以用实验动物的其他组织/器官，只要完整的单细胞可从组织中分离地进行彗星测定。
某些类型的DNA损伤的难以检测的DNA断裂，而无需修改基本碱性彗星试验。在氧化性DNA损伤的情况下，股breaks可以在氧化损伤中的DNA通过用修复酶诸如人8-羟基鸟嘌呤-DNA -N-糖基化酶1（hOGG1基因，它在8-羟基鸟嘌呤（8-oxoGua）和甲基fapy鸟嘌呤11创建场所消化来创建。此外，核酸内切酶III（内Ⅲ）主要产生场所在氧化嘧啶1，因此，在加入的酶消化步骤使得该测定用于测定体内 12氧化性DNA损伤的特定和灵敏的方法。利用这些测定中，我们已经证明在大鼠和小鼠6-8的肝脏和大鼠10的心脏毒物诱导的氧化性DNA损伤。
碱性彗星试验在遗传毒理学和人体生物监测许多应用程序：1）作为后续体内试验通过敏感的体外鉴别基因毒素测试3,13，2）评价的多个组织外源性诱导的DNA损伤的机制14，3）进行调查，如果一个致癌物操作使用毒性或动作的一个非基因毒性模式（MOA）7，4）以评价DNA损伤修复15，5）调查人类疾病和职业暴露16，和6），其为潜在的高通量筛选测定法器官特异性基因毒性17。
Protocol
Representative Results
Discussion
这个协议描述了在单细胞水平在大鼠肝直接和氧化性DNA损伤的同时测量。一般协议，适用于从单个细胞或细胞核可以用最少的处理诱导的DNA损伤被分离的任何组织（ 即 ，诱导不是由测试试剂，而是由动物组织的处理和加工的DNA损伤）。在我们的研究中，我们对细胞从骨髓7,9，胃6，9肾，膀胱9，肺9，10心脏，乳腺5，子宫5，?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by the US Food and Drug Administration. We acknowledge the original publication of the CPA study by Elsevier B.V.: Ding W, Bishop ME, Peace MG, Davis KJ, White GA, Lyn-Cook LE, Manjanatha MG. Sex-specific dose-response analysis of genotoxicity in cyproterone acetate-treated F344 rats. Mutation Research 774: 1-7, 2014 (PMID: 25440904)
Materials
| Coverslips (No. 1, 24 x 50 mm) | Fisher | 12-544-14 | |
| Microscope Slides | Fisher | 12-550-123 | |
| Dimethylsulfoxide (DMSO) | Fisher | 67-68-5 | |
| EDTA, Disodium | Fisher | BP120-1 | |
| Phosphate buffered saline | Fisher | ICN1860454 | |
| 1X Hanks Balanced Salt Solution (HBSS) (Ca++, Mg++ free) | HyClone | SH30588.02 | |
| HEPES | Fisher | BP310-1 | |
| Low Melting Point Agarose (LMP) | Lonza | 50081 | NuSieve GTG Agarose |
| Normal Melting Agarose (NMA) | Fisher | BP1356-100 | |
| pH testing paper strips (pH 7.5-14) | Fisher | M95873 | |
| Potassium Cloride | Fisher | 7447-40-7 | |
| Potassium Hydroxide | Fisher | 1310-58-3 | |
| slide labels, (0.94 x 0.5 in.) | Fisher | NC9822036 | |
| Sodium Chloride (NaCl) | Fisher | 7647-14-5 | |
| Sodium Hydroxide (NaOH) | Fisher | 1310-73-2 | |
| SYBR™ Gold | Invitrogen | S11494 | |
| Triton X-100 | Fisher | 9002-93-1 | |
| Trizma Base | Fisher | 77-86-1 | |
| 2.0 mL microcentrifuge tubes | Fisher | 05-402-6 | |
| Cell strainer (40 µm) | Fisher | 22363547 | |
| Endonuclease III (Nth) | New England Biolabs | M0268S | Dilution 1:1000 |
| hOGG1 | New England Biolabs | M0241S | Dilution 1:1000 |
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