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)

伦理学声明:涉及动物的程序已经批准的机构动物护理和使用委员会（IACUC）在美国FDA /国家毒理学研究中心。 注:这里所描述的研究设计是基于由日本中心的替代方法验证（JaCVAM）为他们的体内啮齿动物碱性彗星试验18验证开发的协议，并进一步修改根据OECD指南建议TG489 19 。 1.准备<ol><li>幻灯片准备<ol><li>在1％溶解定期熔化琼脂糖（重量/体积）在磷酸盐缓冲液（钙离子 ，镁离子的分类和无酚红）通过在微波炉中加热。 </li><li>放置在55℃的水浴中熔化1％标准琼脂糖溶液并平衡同浴的温度。浸显微镜玻片到琼脂糖溶液，流掉任何excesš琼脂糖和擦拭干净的幻灯片的后面。放置幻灯片直立在平坦表面上，并允许滑动在RT干燥;存放在干燥的地方的幻灯片。 </li></ol></li><li>彗星试验方案的准备<ol><li>制备0.5％的低熔点琼脂糖溶液。琼脂糖溶解低熔点磷酸盐缓冲液（钙离子 ，镁离子 ，和无酚红）通过在微波炉中加热。在保持37-45℃的溶液彗星玻片制备过程中;事后丢弃任何剩余的琼脂糖溶液。 </li><li>准备裂解液。使二钠乙二胺四乙酸（EDTA二钠，先生372.24克/摩尔），2.5M氯化钠（NaCl，先生58.44克/摩尔），和10mM三羟甲基氨基甲烷（Tris碱，先生121.14克/摩尔）的100mM溶液在纯水中，并调节至pH 10用10N氢氧化钠水溶液（NaOH，先生为40g /摩尔）。上使用的当天，添加的Triton X100和二甲基亚砜（DMSO，先生78.13克/摩尔）该溶液中来实现最终concentrat的分别为1％和10％，离子和搅拌在4-10℃下在使用前至少30分钟。 </li><li>就使用当天新鲜的酶缓冲液。制备含40mM的2- [4-（2-羟乙基）哌嗪-1-基]乙磺酸（HEPES，先生238.3克/摩尔），100毫氯化钾（KCl，先生74.55克/摩尔），0.5毫摩尔的溶液EDTA二钠，和在纯水中的0.2％牛血清白蛋白（BSA，先生66463克/摩尔）。将pH调节至8.0与10氢氧化钾溶液（KOH 56.9克/摩尔）。搅拌在RT，并验证在使用之前完全溶解。 </li><li>制备含有300mM NaOH和在纯水中1毫EDTA二钠的碱性缓冲溶液。搅拌在4-10℃下在使用前至少30分钟。测量使用和确保pH为&gt; 13之前的溶液的pH值。 </li><li>制备的0.4M Tris碱在纯水中的溶液，并调整到与盐酸（HCl议员，36.46），PH值7.5。存储在4-10℃的溶液中。这是中和缓冲区。 </li><li>让˚FRESH上使用的当天切碎缓冲区。制备含有20mM EDTA二钠和10％DMSO中Hank氏平衡盐溶液中的溶液（HBSS，钙离子 ，镁离子 ，和无酚红）。存储在冰上直到使用。 </li><li>以制备染色溶液中，在1稀核酸荧光染色原液用Tris-硼酸盐EDTA（TBE缓冲液）:10,000（体积/体积）。保护使用铝箔的光解决方案。保存在2-8℃，并用24小时使用。 </li></ol></li></ol> 2.从大鼠肝单细胞悬浮液的制备<ol><li>安乐死在动物组织安乐死美国兽医指导建议的 CO 2气体以下方法鼠:2013年版20。 <ol><li>在2.75设置的 CO 2流速。把大鼠在安乐死室（ 例如 ，干燥器罐子，大鼠笼排出盖），并通过打开罐阀打开的 CO 2。等待1.5至2.5分钟，直到老鼠变得不省人事。增加的 CO 2流至其最大设置。呼吸停止后，离开大鼠在腔室大约1分钟，以确保安乐死。通过触诊，以确认老鼠已经没有心跳，捏脚趾，以确定老鼠不撤回其肢体验证安乐死。 </li></ol></li><li>消毒头发皮肤用70％乙醇。打开腹腔并用手术剪去除完整的肝脏。取出肝脏后，从手术剪剩下的肝脏取出左外叶。放置在切割板上的左外叶，并用一次性单刃剃刀片切断两个相邻3-4毫米厚的横截面。 </li><li>放置在滤纸上一个横截面（防止部的卷曲），并将部分成10％的中性缓冲的福尔马林（NBF）。确保10％NBF中对组织的比例为10:1或更大。后至少72小时，PROC的注视时间ESS组织病理学（见第7，下同）的肝脏。 </li><li>放置第二横向肝脏部分到5毫升冰冷切碎缓冲液（部分1.2.6）在6cm培养皿中，并冲洗3次，以除去残留的血液。 </li><li>转移肝脏部分以含有1毫升冰冷的切碎缓冲器新的2ml离心管中。讳言有一对细剪刀以释放细胞的良好的漂洗肝脏部分。通过40微米的细胞滤网过滤细胞悬浮液，以除去任何组织肿块。 </li><li>放置在冰上的单细胞悬浮液，并使用1小时内使滑动。通过计数（ 例如 ，用血细胞计数器或电子细胞计数器），以确保它的浓度是约2×10 6细胞/ ml验证细胞的浓度。 </li></ol> 3.彗星幻灯片的制备<ol><li>混合的单细胞悬液中的一个体积与10体积的熔融，37℃，0.5％的低熔点琼脂糖溶液（1.2.1节）。立即吸取100微升到涂有常规低熔点琼脂糖（1.1节）的幻灯片。立即将在细胞琼脂糖混合盖玻片。使至少8玻片每个样品。 注意:所有载玻片应由数字或类似代码被识别及其识别记录，从而使滑动可以以盲方式进行评分（参见6.2.2，下文）。 </li><li>莱幻灯片平，冷却至4℃，30分钟。 </li><li>胶凝固后，轻轻取下盖玻片，沉浸在预冷裂解液（第1.2.2节）的幻灯片。从光保护的幻灯片，并保持在裂解缓冲液的幻灯片在从1小时（最小）4℃至O / N（最大）。 </li></ol> 4.幻灯片彗星酶处理<ol><li>除去从裂解缓冲液各组织样品制备的8幻灯片6和冲洗他们3倍与在RT酶缓冲液（部分1.2.3）每次5分钟。用剩下的两个幻灯片碱性彗星试验W¯¯ithout酶治疗（见5.1，下同）。漂洗后，通过排放并用纸巾吸干载玻片除去过量的液体。 </li><li>稀释用酶缓冲液hOGG1基因和远藤酶III股票以1:1000（V / V）。在冰上放置直至使用。 </li><li>应用200微升以下解决方案，从每个治疗组准备的幻灯片:200微升单独酶缓冲液（2张幻灯片作为参考幻灯片），200微升hOGG1基因溶液（2张幻灯片）或200微升远藤III解决方案（2张幻灯片）的。 </li><li>把滑入与湿润的纸巾内衬培养皿，盖用封口膜的幻灯片，并在37°C孵育45分钟的EndoⅢ-处理并参考滑梯和hOGG1基因处理的载玻片30分钟。 </li><li>孵化后，用蒸馏水冲洗幻灯片和低于5.2说明，继续处理。 </li></ol>放料和电泳<ol><li>对于未用酶处理过的载玻片，从裂解缓冲液取出幻灯片，漏的幻灯片，并用冷的中和缓冲液（部分1.2.5）冲洗一次5分钟以去除残留的清洁剂和盐。 </li><li>从步骤4.5和在水平凝胶电泳槽5.1随机发生滑动时，避免了任何空格。装满新鲜的pH值&gt; 13电泳缓冲液（1.2.4节），坦克，直到液面刚好盖住幻灯片（避免在琼脂糖泡沫，并确保坦克水平）。记录在DNA解旋的起始缓冲液的pH和温度。 </li><li>让载玻片保持在碱性缓冲液中20分钟，以允许的碱容易损坏的DNA和表达的退绕。 </li><li>期间展开，程序电源，以在电泳槽（伏/厘米=伏特的正和负电极之间以cm除以距离）0.7伏/厘米。 </li><li>平仓后，按下电源运行按钮。如果需要的话，通过提高或降低的缓冲水平调节电流300毫安。保持日Ë缓冲水平只是幻灯片的表面之上。在4℃下进行电泳20分钟。记录在开始和电泳的端滑动安置，缓冲器的温度和电流（安培）。 注:太多缓冲器将导致较低的电压，可以用DNA迁移干扰。 </li><li>电泳后，通过从罐5分钟轻轻提起的幻灯片，并在中和缓冲液中浸渍（部分1.2.5）中和过量的碱。漏的幻灯片，并重复两次以上。 </li><li>通过在冷100％乙醇中浸渍5分钟，固定在滑动。允许的幻灯片空气干燥并置于贮存干燥的地方。 </li></ol> 6. DNA染色，彗星可视化和分析<ol><li> DNA染色<ol><li>放置在纸板或金属托盘的幻灯片，并添加200μl的核酸荧光染色工作溶液（部分1.2.7）的对每个幻灯片，用玻璃盖玻片覆盖。保护幻灯片光允许滑动到得分之前与染色溶液保持接触至少30分钟。染色在20批次的幻灯片在一个时间。 </li><li>看完每张幻灯片之前，轻轻吸干多余的染色液，小心不要去打扰玻璃盖。 </li></ol></li><li>彗星可视化和评分<ol><li>使用连接到荧光显微镜一台摄像机到电脑显示器上显示的彗星幻灯片的实时图像。 </li><li>随机选择含有细胞在显微镜领域。使用鼠标，选择一个单元格。点击"彗头"，用鼠标的左键的中心。 注:计算机软件计算用于小区中的所有测量参数和数据添加到数据文件。 </li><li>分数在当前场中（见步骤6.2.4）中的所有单元格，然后用显微镜的阶段控制和屏幕上的视频，了解另一组细胞。得分这些细胞如6.2.2所述。 </li><li>分数幻灯片˚FROM从右到左，下到上。随机选取约10场。在此过程中，出现在视野的任何合适的彗星细胞应不带偏见进行评分。 注意:不要得分，彗星如有以下情况是真实的:1）它位于任何幻灯片的边缘; 2）两个或更多个彗星重叠; 3）有一个在尾碎片; 4）软件不能在头部和尾部之间区分;或5）的核和/或尾部的染色从幻灯片上其他彗星大不相同。 注:在尾部几乎所有的DNA荧光彗星通常称为"刺猬"彗星。刺猬彗星不应该被打进，但滑动得分期间检测刺猬的数目应记录并报告为总彗星的百分比。 </li><li>分析每张幻灯片至少75颗彗星，每读器官/组织两个幻灯片获得至少150彗星细胞/总样本。从幻灯片所需数量的细胞后有可恩得分，结果被保存为后期数据分析的电子表格文件。 </li></ol></li></ol> 7.组织病理学分析<ol><li>在10％NBF最少72小时的固定，接着组织修整21后，常规处理和嵌入样品的左侧肝叶中基于介质22石蜡蜡。部分样品在大约5微米，并收集在载玻片上。 </li><li>染色用苏木＆曙红（H＆E）的部分: <ol><li>加热与石蜡的组织切片的载玻片在60℃的烘箱中之前染色过程2小时。 </li><li>使用二甲苯deparaffinize两侧。以补充水分梯度乙醇（乙醇）到正在运行的自来水冲洗的幻灯片。 </li><li>放置在450毫升苏木的玻片2分钟和40秒。用酸漂洗载玻片（2毫升冰醋酸至450毫升去离子水），持续4秒通过5分钟流动的自来水冲洗以下。 </li><li>放置下滑ES在含有1ml氢氧化铵进行2分钟，随后流动的自来水冲洗2分钟，30秒和1分钟70％的乙醇漂洗450毫升70％乙醇。 </li><li>孵育含有4毫升冰醋酸3分钟450毫升曙红Y溶液的幻灯片。 </li><li>脱水用梯度乙醇的幻灯片。清除与二甲苯的幻灯片（3次，每次1分钟），并保持在二甲苯滑动，直到盖玻片放置在载玻片上。 </li></ol></li><li>审查通过光学显微镜，记录的调查结果，并为强度等级非肿瘤性病变的幻灯片为1（最小），2（轻度），3（中度），或4（标记）。 注:施加于一个特定的病变的严重性等级是基于肝脏与病变的相对比例（最小= &lt;1-15％;轻度= 16-35％;中等= 36-60％;及标记= 61- 100％）。 </li></ol> 8.数据分析<ol><li>动物被认为是统计评估试点单位。 DNA损伤表示为％的DNA有尾。 </li><li>氧化性DNA损伤的计算方法如下:参考幻灯片（无酶处理）提供背景的DNA链断裂（SB）的估计值。 注:酶处理载玻片提供链断裂的量度和氧化碱（SB + OX）。假设为％的DNA在尾巴DNA损伤的功能的线性剂量反应，从SB + OX SB的减法给出了从氧化嘧啶/改变嘌呤23的DNA链断裂的估计。 </li><li>分析在尾％DNA作为通过单向ANOVA的剂量函数，随后Dunnett检验对治疗动物的车辆控制的响应的成对比较。 注:所有的测试都是单尾和小于0.05的p值被设定为标准进行统计意义。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+oxidative+DNA+damage+and+its+repair+using+the+comet+assay.">Investigating oxidative DNA damage and its repair using the comet assay.</a> Mutat Res. 681, 24-32 (2009).</li><li>Collins, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comet+assay+in+human+biomonitoring+studies:+reliability,+validation,+and+applications.">Comet assay in human biomonitoring studies: reliability, validation, and applications.</a> Environ Mol Mutagen. 30 (2), 139-146 (1997).</li><li>Gutzkow, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+comet+assay+using+96+minigels.">High-throughput comet assay using 96 minigels.</a> Mutagenesis. 28 (3), 333-340 (2013).</li><li>AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. AVMA Panel on Euthanasia Available from: <a target="_blank" href="https://www.avma.org/KB/Policies/Documents/euthanasia.pdf">https://www.avma.org/KB/Policies/Documents/euthanasia.pdf</a> (2013)</li><li>Ruehl-Fehlert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1.">Revised guides for organ sampling and trimming in rats and mice--part 1.</a> Exp Toxicol Pathol. 55 (2-3), 91-106 (2003).</li><li>Carson, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Self-Instrumental+Text.">A Self-Instrumental Text.</a> Histotechnology. , 25-37 (1996).</li><li>Dusinska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=THE+COMET+ASSAY,+modified+for+detection+of+oxidised+bases+with+the+use+of+bacterial+repair+endonucleases.">THE COMET ASSAY, modified for detection of oxidised bases with the use of bacterial repair endonucleases.</a> Dusinska Protocol [Internet]. , (2000).</li><li>Schuppler, J., Gunzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+tumors+and+steroid+hormones+in+rats+and+mice.">Liver tumors and steroid hormones in rats and mice.</a> Arch Toxicol Suppl. 2, 181-195 (1979).</li><li>Hobkirk, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+sulfotransferases+and+steroid+sulfate+sulfatases:+characteristics+and+biological+roles.">Steroid sulfotransferases and steroid sulfate sulfatases: characteristics and biological roles.</a> Can J Biochem Cell Biol. 63 (11), 1127-1144 (1985).</li></ol>

None of the authors have competing interests or conflicting interests. The information in these materials is not a formal dissemination of information by FDA and does not represent agency position or policy.

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，?...

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&gt; 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。

<table><tbody><tr><td>Coverslips (No. 1, 24 x 50 mm)&amp;nbsp;</td><td>Fisher</td><td>12-544-14</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher</td><td>12-550-123</td><td></td></tr><tr><td>Dimethylsulfoxide (DMSO)&amp;nbsp;</td><td>Fisher</td><td>67-68-5</td><td></td></tr><tr><td>EDTA, Disodium</td><td>Fisher</td><td>BP120-1</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Fisher</td><td>ICN1860454</td><td></td></tr><tr><td>1x Hanks Balanced Salt Solution (HBSS) (Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt; free)</td><td>HyClone</td><td>SH30588.02</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-1</td><td></td></tr><tr><td>Low Melting Point Agarose (LMP)</td><td>Lonza</td><td>50081</td><td>NuSieve GTG Agarose</td></tr><tr><td>Normal Melting Agarose (NMA)&amp;nbsp;</td><td>Fisher</td><td>BP1356-100</td><td></td></tr><tr><td>pH testing paper strips (pH 7.5-14)</td><td>Fisher</td><td>M95873</td><td></td></tr><tr><td>Potassium Cloride</td><td>Fisher</td><td>7447-40-7</td><td></td></tr><tr><td>Potassium Hydroxide</td><td>Fisher</td><td>1310-58-3</td><td></td></tr><tr><td>slide labels (0.94 x 0.5 in.)</td><td>Fisher</td><td>NC9822036</td><td></td></tr><tr><td>Sodium Chloride (NaCl)&amp;nbsp;</td><td>Fisher</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)&amp;nbsp;</td><td>Fisher</td><td>1310-73-2</td><td></td></tr><tr><td>SYBR&amp;trade; Gold&amp;nbsp;</td><td>Invitrogen</td><td>S11494</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher</td><td>9002-93-1</td><td></td></tr><tr><td>Trizma Base&amp;nbsp;</td><td>Fisher</td><td>77-86-1</td><td></td></tr><tr><td>2.0 ml microcentrifuge tubes</td><td>Fisher</td><td>05-402-6</td><td></td></tr><tr><td>Cell strainer (40 &amp;micro;m)</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Endonuclease III (Nth)&amp;nbsp;</td><td>New England Biolabs</td><td>M0268S</td><td>Dilution 1:1,000</td></tr><tr><td>hOGG1&amp;nbsp;</td><td>New England Biolabs</td><td>M0241S</td><td>Dilution 1:1,000</td></tr></tbody></table>

in vivo alkaline comet assay and enzyme-modified alkaline comet assay for measuring dna strand breaks and oxidative dna damage in rat liver

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.

结合进行用酶改性的彗星试验在体内碱性彗星试验以测量在用醋酸环丙孕酮（CPA）5处理的大鼠的肝直接和氧化性DNA损伤。 CPA是一种人工合成的激素药物诱导大鼠肝脏肿瘤特定性别的方式，用五倍诱导雄性大鼠肝脏肿瘤相比，女24例需要更高剂量。我们发现，通过单次转换在雄性和雌性大鼠的肝脏中产生的直接的DNA损伤具有相同性别特异性图案作为?...

Watch this Scientific Journal Video about In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver at JoVE.com

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

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

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.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

in-vivo-alkaline-comet-assay-enzyme-modified-alkaline-comet-assay-for

Research

JoVE Journal

Biology

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

15.3K 次观看.  US FDA/National Center for Toxicological Research. 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.

视频: In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

利用彗星试验评价体外DNA 损伤

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

科研

癌症研究

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其对32 P-postlabeling 的测定

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

生物化学

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip:一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

生物工程

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

在彗星分析中使用冷冻组织评估DNA损伤

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

遗传学

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

彗星试验测定DNA损伤的海马神经元

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

神经科学

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

使用DNA纤维测定检测人细胞中复制后间隙的积累和修复

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...

研究 DNA 复制过程中发生的 DNA 断裂程度 

Detection of DNA Breaks in Dividing Human Cells by Neutral Comet Assay

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during genome duplication can stall replisomes and convert replication forks into double-strand breaks. If left unrepaired, these toxic DNA breaks can trigger chromosomal rearrangements, leading to heightened genome instability and an increased likelihood of cellular transformation. Additionally, cancer cells exhibit persistent replication stress, making the targeting of replication fork vulnerabilities in tumor cells an attractive strategy for chemotherapy. A highly versatile and powerful technique to study DNA breaks during replication is the comet assay. This gel electrophoresis technique reliably detects the induction and repair of DNA breaks at the single-cell level. Herein, a protocol is outlined that allows investigators to measure the extent of DNA damage in mitotically dividing human cells using fork-stalling agents across multiple cell types. Coupling this with automated comet scoring facilitates rapid analysis and enhances the reliability in studying induction of DNA breaks.

作者聚焦:揭示 SNF2L 在复制叉稳定性和基因组复制中的作用

This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress reagents.

通过中性彗星测定法检测分裂人类细胞中的 DNA 断裂

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

彗星试验系统性氧化应激的间接测量

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

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

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利用彗星试验评价体外DNA 损伤

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其^对32 P-postlabeling 的测定

CometChip:一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

在彗星分析中使用冷冻组织评估DNA损伤

彗星试验测定DNA损伤的海马神经元

使用DNA纤维测定检测人细胞中复制后间隙的积累和修复

通过中性彗星测定法检测分裂人类细胞中的 DNA 断裂

通过中性彗星测定法检测分裂人类细胞中的 DNA 断裂

通过中性彗星测定法检测分裂人类细胞中的 DNA 断裂

彗星试验系统性氧化应激的间接测量