Name: analysis of the ambient particulate matter-induced chromosomal aberrations using an in vitro system
Uploaded: 2016-12-21T15:00:00.000+00:00
Duration: 8 min 48 s
Description: Watch this Scientific Journal Video about Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System at JoVE.com

这项工作是支持的，部分由生物学卓越研究[授权号1P20GM109005]，阿肯色空间格兰特财团通过美国国家航空和航天局[授权号NNX15AK32A]卫生中心的国家研究所和美国国家职业安全和健康研究所（NIOSH）授权号码2T420H008436。笔者想感谢克里斯托弗Fettes校对和编辑这篇稿子。

1.颗粒收集和准备<ol><li> 高容量总悬浮微粒采样器的校准 <ol><li>断开质量流量控制器的采样器马达和马达连接到一个稳定的交流电源（ 图1）。 注:在此过程中不使用的过滤器。 </li><li>校准器安装孔和顶部装载转接板到采样。拧紧顶部装载适配器压紧螺母牢固，以确保没有空气泄漏是否存在。允许采样马达升温到其正常的操作温度（约10-15分钟）。 </li><li>覆盖与手口在管口和测压管顶部的孔进行泄漏测试。侦听由逃逸的空中发出高亢的尖叫声。由于存在泄漏如果听到声音，再拧紧顶部装载适配器压紧螺母。如果声音是较低时，泄漏是靠近在sys其他垫圈之一透射电镜。 </li><li>水压力计的一侧连接到压力抽头上用橡胶真空管孔口的一侧。离开压力计通向大气的相对侧。保持一个压力计垂直，以确保准确的读数。攻连续流动记录的背面，将有助于居中笔，并提供准确的读数。 </li><li>重复步骤1.1.2使用从30至60英尺/分钟（大约每5-6英尺3 /分钟），代表工作流率5流速。调整对可变孔旋钮五个不同位置，采取五种不同的读数。 </li><li>记录周围空气的温度，环境大气压，取样序号，孔口序列号，孔口斜率和截距与日期最后认证，日期，场地的位置，和操作者的对校准片缩写。 </li></ol></li><li> 总悬浮颗粒物样品采集和重分析 <ol><li>一个包裹8"×10"，在铝箔的两层石英纤维过滤器。放置在炉中的卷绕的过滤器，并设置在550℃的温度。烤滤波器为至少4小时。让炉冷并检索滤波器。放置过滤器与碳酸钙的干燥器。 </li><li>权衡为10微克精度微量天平过滤器。在一天重复测量三次。单独的测量应在25微克。如果不这样实现的，被包装的过滤器返回到干燥器并重复此过程次日。 </li><li>打开盖子住所（ 图2）。松开四个蝶形螺母，使铜螺栓和垫圈，翻下出的方式删除过滤器保持框。从打开铝箔过滤器，并使用清洁镊子小心地居中，粗糙的一面朝上，支持屏幕上。正确对齐屏幕上的过滤器。 </li><li>在画面上放置框架和保护它黄铜螺栓和垫圈而施加足够的压力，以避免在边缘处的空气泄漏。擦掉过滤器支架周围的任何灰尘堆积用干净的布。关闭住所盖子仔细并固定。 </li><li>确保所有线均已插入其合适的插座插座及鼓风机马达压力水龙头，连续流动记录之间的管道连接。 </li><li>准备流量记录。按下笔臂升降，以提高笔尖，并插入一个新的图表。对齐图表记录仪的驱动毂的选项卡，然后按拇指，以降低图表上中心枢纽。通过直至图表正确的时间与时间索引指针对准旋转驱动轮毂顺时针设定的时间。 </li><li>手动切换的采样，并开始收集。监控记录，以确保它是正确着墨和经过时间指示器，以确保其工作正常。 </li><li>在采样周期结束时，关掉采样器，记录的经过时间，并检索该图表。删除框架，露出过滤器。轻轻保持它在与镊子的末端移除所述支撑屏幕的露出滤波器。纵向折叠过滤器，使样品接触的样品两次，在原有的铝箔包裹。 </li><li>与碳酸钙称重之前存储过滤干燥器中至少24小时。重复步骤1.2.2取样后测量过滤器的重量。放置过滤器在一拉链塑料袋中并储存在-80℃直至分析。 </li></ol></li><li> 收集的总悬浮颗粒物采样分析 <ol><li>检索存储的过滤器，展开铝箔和一个干净的表面特氟龙转移过滤器。使用镊子和手术刀，切开过滤器外（白色）的一部分，处理。 <ol><li>接着，切断该过滤器在0.5"×0.5"件和地点片在100毫升烧瓶中。加入50mL超纯水。放置烧瓶在超声浴中并在33±3千赫超声60分钟。 </li></ol></li><li>通过0.45μm聚丙烯注射器过滤器的100毫升圆底烧瓶中过滤提取物。在50℃下蒸发使用压力下的旋转蒸发仪中的水，直至约5ml提取物保留在烧瓶中。 <ol><li>蒸发剩余的水在已知重量的15毫升梨形瓶中。权衡与干提取物的烧瓶中，并记录干提取物的质量。 </li></ol></li><li>通过超声处理15分钟溶解用400μlD 2 O中的干燥样品，并转移到一个5毫米的标准NMR管。在12,000 xg离心离心5分钟，在室温下，以除去任何未溶解的物质。 </li><li>重复用300μlD 2的O.添加10微升NaN 3的1.4％（终浓度:0.02％重量/体积）在NMR管。添加10微升（32微克）总悬浮颗粒物（TSP）〜D4（3.2毫克/毫升）的在NMR管D 2 O中（终浓度0.258毫摩尔）。 </li><li>通过收购在600.17兆赫运行的核磁共振波谱仪1核磁共振光谱。 <ol><li>将NMR管进入三重共振冷冻探针。校准仪器按以下顺序:锁，调整垫片，90°脉冲的长度校准器和接收增益调整。 </li><li>测量在25℃的光谱，使用在数字四路检测模式180水选择性脉冲，8,192扫描，4虚设扫描，8012.57赫兹的扫描宽度的1个D激励雕刻顺序，频率为水信号抑制设定在4.70 ppm的，1.70偏移秒的采集时间，32,768时域数据点（TD），1毫秒的时间梯度（P16），后梯度（D16）100微秒恢复延迟。 </li></ol></li><li>过程1通过2（65,536个数据点）的一个因素施加1赫兹和零填充的谱线增宽H-NMR光谱。手动校正阶段和基线和整合。在0.0 ppm的设置TSP-D4高峰。 </li></ol></li></ol> 2.粒子曝光<ol><li> 文化成立 <ol><li>含有10％胎牛血清（FBS）和青霉素 - 链霉素溶液（100单位/毫升），0.25％胰蛋白酶溶液和钙镁自由磷酸盐缓冲盐水（PBS）在水浴（37℃）预热完全生长培养基为培养开始前至少30分钟。 </li><li>取出从含有培养的RAW264.7细胞，5％CO 2培养箱一个T75烧瓶中并用2ml的预温热的PBS两次洗涤细胞。 </li><li>逐出的组织培养容器中的用1ml预温热胰蛋白酶溶液，接着刮底部粘附RAW264.7细胞。 </li><li>收集来自组织培养容器中的细胞，并通过反复吹打单细胞悬浮液。算用锥虫蓝和板细胞的数量在预先温热完整的媒体100毫米组织培养皿一个8000 /厘米2（500,000个细胞总数）密度。调节介质体积10毫升50,000个细胞的终浓度每毫升。 注:加倍板的数量，如果也进行DNA甲基化分析。 </li><li>放置新镀RAW264.7细胞回在5％CO 2培养箱中在37℃，并允许以24小时连接。 </li><li>此外，在生物安全柜，过滤悬浮颗粒。例如，以0.5毫克/毫升无菌PBS为5微克/毫升的治疗剂量的浓度稀释的颗粒，并以5毫克/毫升为50微克/毫升的治疗剂量。稀释可以事先准备最多一个月并储存在-80℃。 </li></ol></li><li> 治疗用微粒物 <ol><li>取出从5％CO 2培养箱的细胞和使用用于细胞生长，细胞形态，培养条件，和污染在倒置显微镜下10倍放大镜检查。之一应该期望具有至少30％汇合的细胞在这个阶段。 </li><li>无菌，使用消毒吸管吸媒体并用2ml 37℃的PBS洗涤培养容器两次以完全去除媒体和浮动或死亡的细胞。 </li><li>添加的颗粒物质预先确定的剂量，用新鲜培养基在培养容器和在5％CO 2培养箱中 ，在37°C孵育所需的时间。在这项研究中，治疗RAW264.7细胞用PM 72小时为并行用于并发基因毒性和epigenotoxic分析曝光。 注:细胞毒性可在细胞遗传学分析的质量产生负面影响。因此，如细胞毒性，DNA甲基化，Western印迹，或基因表达分析的附加测定在处理过的细胞中通常进行的。暴露的细胞所描述和与期望的特定测定法进行。没有经验的用户也可能希望包括阳性对照，如甲基甲磺酸，以测试他们检测的CA的能力。 </li></ol></li></ol> 3.细胞遗传学分析<ol><li> 治疗逮捕在中期细胞 <ol><li>暖秋水仙碱这样lution（10微克/毫升）在37℃水浴中30分钟。擦拭用70％酒精盖生物安全柜下，以尽量减少污染的风险</li><li>完全无菌删除媒体并用2ml预热的PBS两次洗培养容器。 </li><li>添加预热的新鲜培养基（2毫升培养基/ 10 6个细胞）中含有培养容器秋水仙胺溶液（5微升/ ml）和孵育2小时。 </li></ol></li><li> 收获细胞 <ol><li>温暖的胰蛋白酶溶液和PBS的水浴中保持在37℃30分钟。从该步骤起，就没有必要保持无菌状态。 </li><li>彻底去除媒体，用2毫升37℃PBS洗涤培养容器两次，加预热的胰蛋白酶溶液所需体积（通常为2毫升至60mm培养盘或T25瓶和4毫升100毫米的菜或T75烧瓶），以及培养容器放回在5％CO 2培养箱中在37℃下1分钟。 </li><li>德Ë出培养容器，分离用刮刀的细胞，并加入10毫升的PBS以停止胰蛋白酶作用。 </li><li>移液器上下至少十倍，使单细胞悬浮液并转移细胞在15ml圆锥形底离心管中。 </li><li>在室温下摆动出来离心机在400×g离心5分钟离心细胞悬浮液。 </li><li>除去上清液，通过轻轻敲击打破细胞沉淀，加入10毫升的PBS，并再次离心五分钟。 </li></ol></li><li> 低渗处理和固定 <ol><li>在37℃水中至少30分钟浴预暖0.075 1M氢氯化物溶液。 </li><li>而不干扰细胞沉淀除去上清液，留下大约为0.5毫升的PBS。 </li><li>轻轻打破细胞沉淀，并用P200移液管使单细胞悬浮液。 </li><li>通过轻轻摇动滴加入4毫升预热氯化钾溶液滴。 </li><li>孵育低渗P中的细胞在37℃水浴中20分钟otassium氯化物溶液。 </li><li>加入3份的甲醇与1部分冰醋酸做出新的acetomethanol解决方案。保持这个新鲜配制固定液在室温下。 </li><li>低渗处理后，添加固定剂的等体积（4毫升）中的管和通过反转所述管将溶液轻轻混匀。 </li><li>离心在400 xg离心在室温下5分钟，并除去上清液。在此阶段细胞沉淀尺寸增大，变得发白和更明显。 </li><li>去除上清液，加入4毫升的新鲜固定液，并保持在室温下30分钟。 </li><li>离心机在400 XG 5分钟，除去上清液，并给出两个洗涤用4ml固定液。 </li></ol></li><li> 滑动清洗和中期扩展准备 <ol><li>完全在10％的液体洗涤剂浸渍的玻璃载玻片30分钟。 </li><li>彻底冲洗载玻片在冷的流动的自来水30分钟。 </li><li>凛本身用蒸馏水滑动三次以彻底除去洗涤剂，在蒸馏水浸泡，并在冰箱中以备将来使用存储。 注:清洁幻灯片便于使用前均匀传播，提高质量的中期。 </li><li>滴上冷却湿滑板10微升细胞悬浮液，并允许它空气中在室温下干燥过夜。 </li></ol></li><li> 染色和安装 <ol><li>用PBS的一个部分混合姬姆萨染色解决方案的一部分，准备50毫升姬姆萨染色解决方案，并倒在一个罐子科普林。 </li><li>浸入染色溶液中的玻片20分钟。 </li><li>冲洗载玻片在蒸馏水以除去过量染色剂和立即干燥用吹风机的幻灯片。 </li><li>留在室温下将载玻片过夜。 </li><li>适用于滑动安装平台的单个下降，轻轻地放在一个盖玻片安装介质上，允许介质慢慢滑动传播，并取出用纸巾过量介质。允许安装型滑轨，以至少1小时移动或在显微镜下观察，以避免盖玻片运动之前干燥。 注意:要仔细放置盖玻片而不在该步骤中形成的任何气泡是非常重要的。建议不要使用过多的热量，以消除气泡。 </li></ol></li><li> 显微观察和畸变类型 <ol><li>检查结构的CA在中期使用高质量的亮场显微镜放大100倍的利差。 </li><li>拿下至少50至100中期利差为诱导大量的CA治疗各治疗组。对于较小的影响大小，高达300中期利差的分析建议。 </li></ol></li></ol>

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Sci. 148 (2), 473-487 (2015).</li><li>Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+instability+in+cancer+development.">Genomic instability in cancer development.</a> Genome Instability in Cancer Development. , 363-392 (2005).</li><li>Michael, S., Montag, M., Dott, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-inflammatory+effects+and+oxidative+stress+in+lung+macrophages+and+epithelial+cells+induced+by+ambient+particulate+matter.">Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells induced by ambient particulate matter.</a> Environ Pollut. 183, 19-29 (2013).</li><li>Li, N., 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=Induction+of+heme+oxygenase-1+expression+in+macrophages+by+diesel+exhaust+particle+chemicals+and+quinones+via+the+antioxidant-responsive+element.">Induction of heme oxygenase-1 expression in macrophages by diesel exhaust particle chemicals and quinones via the antioxidant-responsive element.</a> J Immunol. 165 (6), 3393-3401 (2000).</li><li>Pathak, R., Ramakumar, A., Subramanian, U., Prasanna, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radio-sensitivities+of+human+chromosomes+1+and+2+in+one+donor+in+interphase-+and+metaphase-spreads+after+60Co+gamma-irradiation.">Differential radio-sensitivities of human chromosomes 1 and 2 in one donor in interphase- and metaphase-spreads after 60Co gamma-irradiation.</a> BMC Med. Phys. 9, 6649 (2009).</li><li>Pathak, R., Dey, S. K., Sarma, A., Khuda-Bukhsh, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxic+effects+in+M5+cells+and+Chinese+hamster+V79+cells+after+exposure+to+7+Li-beam+(LET=+60keV/&#181;m)+and+correlation+of+their+survival+dynamics+to+nuclear+damages+and+cell+death.">Genotoxic effects in M5 cells and Chinese hamster V79 cells after exposure to 7 Li-beam (LET= 60keV/&#181;m) and correlation of their survival dynamics to nuclear damages and cell death.</a> Mutat. Res. Genet. Toxicol. Environ. Mutagen. 628 (1), 56-66 (2007).</li><li>Pathak, R., Dey, S. K., Sarma, A., Khuda-Bukhsh, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+killing,+nuclear+damage+and+apoptosis+in+Chinese+hamster+V79+cells+after+irradiation+with+heavy-ion+beams+of+16+O,+12+C+and+7+Li.">Cell killing, nuclear damage and apoptosis in Chinese hamster V79 cells after irradiation with heavy-ion beams of 16 O, 12 C and 7 Li.</a> Mutat. Res. Genet. Toxicol. Environ. Mutagen. 632 (1), 58-68 (2007).</li><li>Chalbot, M. C., Kavouras, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+spectroscopy+for+determining+the+functional+content+of+organic+aerosols:+a+review.">Nuclear magnetic resonance spectroscopy for determining the functional content of organic aerosols: a review.</a> Environ. Pollut. 191, 232-249 (2014).</li></ol>

The authors have nothing to disclose.

细胞遗传学研究或数字或染色体，主要是在中期扩展的结构，微观分析，为预测，风险评估和治疗各种疾病的关键信息。它现在公认该细胞遗传学异常与几种疾病，包括癌症的进展和发展相关。迄今为止，中国科学院已经在所有主要类型的肿瘤中发现。可诱导各种类型的细胞遗传学改变16的主要外部危险因素之一-的CA可以自发或通过外部或内部的刺激，如电离辐射（IR）的出现。 CA的辅助?...

据估计，暴露于颗粒物（PM）导致每年超过300万过量死亡，主要是从心肺疾病和肺癌1。的确，PM被确认为致癌由国际癌症研究机构（组1）人类，如肺癌的暴露的水平提高到下午的风险增加已经显示2。有趣的是，几乎所有的肿瘤细胞海港数值和/或结构染色体异常。颗粒有机碳是一种变型的，复杂的，和非均相混合物，其组成和大小分布取决于排放以及物理和化学变化。它占来自城市的PM2.5质量的35-55％（PM 2.5微米尺寸和更小）以及农村和大陆背景的PM 2.5质量3,4的60％以上。水溶性部分占有机气溶胶的30-90％。大量有机化合物已经确定，包括脂族烃，多环芳烃，和它们的氧化和硝化的衍生物，脂族醛和醇，游离脂肪酸和它们的盐，二羧酸，多官能的化合物，蛋白质和类腐殖质大分子（HULIS）使用色谱法加上质谱技术5-8。这些化合物代表微粒的有机碳小于10-20％，因此大多数的有机碳是未知9。 实验证据表明，细胞毒性，氧化应激和炎症中涉及的PM-有关的病理状态的发展。最近已显示，但是，暴露于PM也导致一些外遗传改变的，包括在重复元件的DNA甲基化的改变，在这两个实验在体外系统以及在人受试者10-12。特别感兴趣的是的效果PM卫星DNA - 主要和次要的卫星 - 这是在围绕染色体着丝粒的异色地区发现的。结果表明，这些效果可能是由自然持久的，因为他们可以在至少72小时曝光12后进行检测。改变DNA甲基化，特别是围绕着丝粒，可以在细胞分裂过程导致的卫星DNA的mRNA转录物，折衷染色体完整性累积，并随后导致的各种病理状态13的发展。 为CA的分析所造成的PM后生改建的终点细胞遗传学方法的适应性，因此具有很高的重要性。在这里，我们报告使用小鼠巨噬细胞RAW264.7模型系统中的CA 体外照射和分析环境PM收集和准备的办法。巨噬细胞包括防御的第一线对吸入异物，因此，叔他的细胞系用作建立和粒子毒理学11，12，14，15中的最常用的模型。

<table><tbody><tr><td>Total suspended particulate sampler</td><td>Tisch Environmental</td><td>TE-5170</td><td></td></tr><tr><td>Bruker Avance III NMR spectrometer&amp;nbsp;</td><td>Bruker</td><td>NA</td><td></td></tr><tr><td>TopSpin 3.5/pl2 software</td><td>Bruker</td><td>NA</td><td></td></tr><tr><td>ACD/NMR Processor Academic Edition</td><td>ACD/Labs</td><td>NA</td><td></td></tr><tr><td>RAW264.7 murine macrophages</td><td>ATCC</td><td>ATCC TIB-71</td><td></td></tr><tr><td>High glucose DMEM GlutaMAX media</td><td>ThermoFisher</td><td>10569010</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoFisher</td><td>16000044</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>ThermoFisher</td><td>15140163</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>ThermoFisher</td><td>25200056</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>PBS, pH 7.4</td><td>ThermoFisher</td><td>10010049</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>KaryoMAX Colcemid Solution in PBS</td><td>ThermoFisher</td><td>15212012</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>KaryoMAX Potassium Chloride Solution</td><td>ThermoFisher</td><td>10575090</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>Methanol (HPLC)</td><td>Fisher Scientific</td><td>A452N1-19</td><td></td></tr><tr><td>Acetic Acid, Glacial</td><td>Fisher Scientific</td><td>BP1185-500</td><td></td></tr><tr><td>Decon Contrad 70 Liquid Detergent</td><td>Fisher Scientific</td><td>04-355-1</td><td></td></tr><tr><td>Wright and Wright-Giemsa Stain Solutions</td><td>Fisher Scientific</td><td>23-200733</td><td></td></tr><tr><td>Permount Mounting Medium</td><td>Fisher Scientific</td><td>SP15-100</td><td></td></tr><tr><td>Axio Imager 2</td><td>Zeiss</td><td>NA</td><td></td></tr></tbody></table>

analysis of the ambient particulate matter-induced chromosomal aberrations using an in vitro system

暴露于颗粒物质（PM）是一个主要的全球健康问题，这可能会损坏的各种细胞组分，包括核遗传物质。为了评估PM的核遗传完整性的影响，染色体结构畸变的得分在小鼠巨噬细胞RAW264.7细胞的中期扩展。 PM被从环境空气中具有高体积总悬浮颗粒采样收集。所收集的材料被溶解，并过滤以保持水溶性，细部。所述颗粒的特征在于用于通过核磁共振（NMR）谱的化学组成。不同浓度的颗粒悬浮液中加入到RAW264.7小鼠巨噬细胞的体外培养72小时的总暴露时间，与未处理的对照细胞一起。在曝光结束后，文化与秋水仙胺治疗逮捕细胞分裂中期。细胞，然后收获，用低渗溶液，定格在acetomethanol，D处理ropped到玻片上，最后用吉姆萨溶液染色。幻灯片进行检查，以评估使用亮场显微镜的结构染色体畸变（CA）的分裂中期利差在放大1000倍。 50至100中期扩展被打进每个治疗组。该技术适于检测结构染色体畸变（CA）的，如染色单体型断裂，染色单体型交流，偏心片段，双着丝粒和环染色体，双分钟，核内复制，和罗伯逊易位体外暴露于PM之后的。这是一个行之有效的细胞遗传学端点后天的改变相关联的有效方法。

极大，应注意在选择TSP采样器的位置，以及当进行收集一年中的时间。的化学组成，以及颗粒的尺寸可以基本上影响结果。收集的材料应该是对白色滤波器可见。普通鼠标中期扩展将有40偏心染色体。该技术的目的是要证明在异常染色体中处理的细胞与对照相比的比例的变化（或没有）。那么这种改变可以被量化（染色体异常的数量）和合格（类型异常）。 <p class="jove_...

Watch this Scientific Journal Video about Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System at JoVE.com

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

该协议与周围空气的微粒物质处理后描述了在RAW264.7小鼠巨噬细胞体外量化和染色体畸变的表征技术。

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

analysis-ambient-particulate-matter-induced-chromosomal-aberrations

Universidad San Sebastian

Research

JoVE Journal

Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

8.6K 次观看.  University of Arkansas for Medical Sciences. 该协议与周围空气的微粒物质处理后描述了在RAW264.7小鼠巨噬细胞体外量化和染色体畸变的表征技术。 

视频: Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

遗传学

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

免疫与感染

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

生物工程

The Lambda Select cII Mutation Detection System

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (&#955;) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

The Lambda Select cII Mutation Detection System

We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells.

Lambda 选择cII突变检测系统

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

生物学

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

环境细颗粒物小鼠组织中三种 DNA 损伤的质谱定量及水平评价

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

研究DNA损伤检验点<em&gt;爪蟾</em&gt;卵提取物

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

染色体制备培养细胞

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

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.

测量DNA损伤与修复小鼠脾细胞慢性后<em&gt;在体内</em&gt;暴露于β和γ辐射剂量很低

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

使用了大气颗粒物引起的染色体畸变分析

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