Name: dose uptake of platinum- and ruthenium-based compound exposure in zebrafish by inductively coupled plasma mass spectrometry with broader applications
Uploaded: 2022-04-21T13:10:00
Description: Watch this Scientific Journal Video about Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications at JoVE.

资助：NJAES-Rutgers NJ01201，NIEHS培训补助金T32-ES 007148，NIH-NIEHS P30 ES005022。此外，布列塔尼卡拉斯还得到了NIDS，NIH的培训补助金T32NS115700的支持。作者感谢Andreia Valente和葡萄牙科学技术基金会（Fundação para a Ciência e Tecnologia， FCT;PTDC/QUI-QIN/28662/2017）供应PMC79。

AB菌株斑马鱼（Danio rerio）用于所有实验（见 材料表），饲养方案（#08-025）由罗格斯大学动物护理和设施委员会批准。
1. 斑马鱼养殖
<ol><li>在循环水生生境系统中繁殖和维持斑马鱼，采用14小时光照：10小时黑暗循环。<ol>
<li>通过沙子和碳过滤净化市政自来水，获得鱼类系统水。将水生系统水保持在28°C，<0.05 ppm亚硝酸盐，<0.2 ppm氨，pH值在7.2?...

<ol>
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这里描述的方案已经实施以确定含有Pt或Ru的金属基抗癌药物的递送和摄取。尽管这些方法已经发表，但该协议讨论了使该方法适用于一系列化合物的重要考虑因素和细节。经合组织方案与组织消化和ICPMS分析相结合，使我们能够确定PMC79比顺铂更有效，并导致不同的组织积累，这表明了不同的机制。此外，由于顺铂的递送剂量被量化，剂量反应结果被外推到患者群体。亚致死剂量（例如LOAEL）与患...

金属和金属基化合物继续具有药理学和毒理学相关性。自20世纪60年代以来，重金属暴露的流行及其对健康的影响呈指数级增长，并在2021年达到历史最高水平。饮用水，空气污染和职业暴露中重金属的浓度超过了全球范围内的监管限度，并且仍然是砷，镉，汞，铬，铅和其他金属的问题。量化环境暴露和分析病理发展的新方法继续处于高需求状态1，2，3。
相反，医学领域已经利用各种金属的物理化学特性进行临床治疗。金属基药物或金属药物具有丰富的药用历史，并显示出对一系列疾病的活性，其中最有效的是化疗药物4。最著名的金属药物顺铂是一种基于Pt的抗癌药物，被世界卫生组织（WHO）认为是世界上必不可少的药物之一5.2010年，顺铂及其Pt衍生物在几种癌症中的成功率高达90%，并用于约50%的化疗方案6，7，8。尽管基于Pt的化疗药物取得了无可辩驳的成功，但剂量限制性毒性已经启动了对具有精制生物递送和活性的替代金属基药物的研究。在这些替代品中，Ru类化合物已成为最受欢迎的9，10，11，12。
需要新的模型和方法来跟上金属药代动力学和毒代动力学研究的需求速度。斑马鱼模型位于复杂性和通量的交叉点，是一种高繁殖力的脊椎动物，具有70%保守的基因同源性13。该模型一直是药理学和毒理学的资产，对各种化合物进行了广泛的筛选，以进行先导物发现，靶标鉴定和机理活性14，15，16，17。然而，化学品的高通量筛选通常依赖于水性暴露。鉴于摄取可以根据溶液中化合物的物理化学性质（即光降解，溶解度）而变化，这可能是相关剂量递送和反应的主要限制。
为了克服与高等脊椎动物比较剂量的这一局限性，设计了一种方法来分析斑马鱼幼虫组织中的痕量金属浓度。在这里，评估了顺铂和新型基于Ru的抗癌化合物的致死和亚致死终点的剂量 - 反应曲线。对标称浓度为0、3.75、7.5、15、30和60mg/L顺铂的致死性和延迟孵化进行评估。通过ICPMS分析测定微生物组织中Pt的积累，生物体对各剂量的摄取量分别为0.05、8.7、23.5、59.9、193.2和461.9ng（Pt）。此外，斑马鱼幼虫暴露于0，3.1，6.2，9.2，12.4mg / L的PMC79。分析测定这些浓度含有0，0.17，0.44，0.66和0.76mg / L Ru。该方案还允许与斑马鱼组织区分幼虫绒毛膜中螯合的Pt浓度。该方法能够为比较完善的化疗药物和新型化合物之间的药代动力学和毒代动力学活性提供可靠，可靠的数据。该方法可应用于各种金属和金属基化合物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AB Strain Zebrafish (Danio reri)</td>
			<td>Zebrafish International Resource Center</td>
			<td>Wild-Type AB</td>
			<td>Wild-Type AB Zebrafish</td>
		</tr>
		<tr>
			<td>ACS Grade Nitric Acid</td>
			<td>VWR BDH Chemicals</td>
			<td>BDH3130-2.5LP</td>
			<td>Nitric Acid (68-70%); used to make 10% HNO3 acid-bath solution for soaking/pre-celaning centrifuge tubes</td>
		</tr>
		<tr>
			<td>Aquatox Fish Diet (Flake)</td>
			<td>Zeigler Bros, Inc.</td>
			<td></td>
			<td>Flake food to be mixed in a 1:4 ratio of Aquatox Fish Diet to TetraMin Tropical Flakes and used as feed</td>
		</tr>
		<tr>
			<td>Artemia cysts, brine shrimp</td>
			<td>PentairAES</td>
			<td>BS90</td>
			<td>Brine shrimp eggs sold in 15-ozz, vacuum-packed cans to be hatched and used as feed</td>
		</tr>
		<tr>
			<td>ASX-510 Autosampler for ICPMS</td>
			<td>Teledyne CETAC</td>
			<td></td>
			<td>Automatic sampler with conifgurable XYZ movement, flowing rinse station, and 0.3 mm inner dimension probe. Compatible with Nu AttoLab software for programmable batch analyses.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>CL 2</td>
			<td>Thermo Scientific CL 2 compact benchtop centrifuge with variable speed range up to 5200 rpm; used to bring sample and acid condensate to the bottom of the centrifuge tube bewteen microwave digestion intervals; aids in sample retention</td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>VWR</td>
			<td>21008-105</td>
			<td>Ultra high performance polypropylene centrifuge tubes with flat cap; 15 mL volume; leak-proof with conical bottom</td>
		</tr>
		<tr>
			<td>Class A Clear Glass Threaded Vials</td>
			<td>Fisherbrand</td>
			<td>03-339-25B</td>
			<td>Individual glass vials for exposure containment</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Millipore Sigma</td>
			<td>D8418</td>
			<td>Solvent or vehicle for hydrophobic compounds</td>
		</tr>
		<tr>
			<td>Fixed Speed Vortex Mixer</td>
			<td>VWR</td>
			<td>10153-834</td>
			<td>Vortex mixer; used to homogenize sample after acid digestion and dilution</td>
		</tr>
		<tr>
			<td>High Purity Hydrogen Peroxide</td>
			<td>Merk KGaA, EDM Millipore</td>
			<td>1.07298.0250</td>
			<td>Suprapur Hydrogen peroxide (30%); used for sample digestion</td>
		</tr>
		<tr>
			<td>High Purity Nitric Acid</td>
			<td>EDM Millipore</td>
			<td>NX0408-2</td>
			<td>Omni Trace Ultra Nitric Acid (69%); used for sample digestion</td>
		</tr>
		<tr>
			<td>Instant Ocean Sea Salt</td>
			<td>Spectrum Brands, Inc.</td>
			<td>Instant Ocean&#174; Sea Salt</td>
			<td>Egg water solution contains instand ocean sea salt with a final concentration of 60 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Mars X Microwave Digestion System</td>
			<td>CEM, Matthews, NC</td>
			<td></td>
			<td>Microwave acid digestion system used to digest and homogenize samples under uniform conditions. For this methodology the open vessel digestion method was completed using single-use polypropylene centrifuge tubes at low power (300 W).&#160;</td>
		</tr>
		<tr>
			<td>Multi-element Solution 3</td>
			<td>SPEX CertiPREP</td>
			<td>CLMS-3</td>
			<td>Contains 10 mg/L Au, Hf, Ir, Pd, Pt, Fu, Sb, Sr, Te, Sn in 10% HCl/1% HNO3; used as a quality control standard for Pt and Ru analyses</td>
		</tr>
		<tr>
			<td>Nu Instruments AttoM High Resolution Inductively Coupled Plasma Mass Spectrometer (HR-ICP-MS)</td>
			<td>Nu Instruments/Amatek</td>
			<td></td>
			<td>Double focussing magnetic sector inductively coupled plasma mass spectrometer with flexible low to high resolution slit system, and dynamic range detector system. Data processing and quantification is done using NuQuant companion software.&#160;</td>
		</tr>
		<tr>
			<td>Platinum (Pt) standard solution, NIST 3140</td>
			<td>National Institute of Standards and Technology</td>
			<td>3140</td>
			<td>Prepared from ampoule containing 9.996 mg/g Pt in 10% HCl; ; used as a quality control standard for Pt analyses</td>
		</tr>
		<tr>
			<td>Platinum (Pt) standard solution, single-element</td>
			<td>High Purity Standards</td>
			<td>100040-2</td>
			<td>Contains 1000 mg/L Pt in 5% HCl</td>
		</tr>
		<tr>
			<td>Ruthenium (Ru) standard solution, single-element</td>
			<td>High Purity Standards</td>
			<td>100046-2</td>
			<td>Contains 1000 mg/L Ru in 2% HCl</td>
		</tr>
		<tr>
			<td>TetraMin Tropical Flakes</td>
			<td>Tetra</td>
			<td>77101</td>
			<td>Flake food to be mixed in a 1:4 ratio of Aquatox Fish Diet to TetraMin Tropical Flakes and used as feed</td>
		</tr>
		<tr>
			<td>Trace Metal Grade Nitric Acid</td>
			<td>VWR BDH Chemicals</td>
			<td>87003-261</td>
			<td>Aristar Plus Nitric Acid (67-70%); used for rinse solution in ASX-510 Autosampler</td>
		</tr>
		<tr>
			<td>Ultrasonic water bath</td>
			<td>VWR</td>
			<td>B2500A-DTH</td>
			<td>Ultrasonic water bath used to aid in acid digestion prior to microwave digestion</td>
		</tr>
	</tbody>
</table>

dose uptake of platinum- and ruthenium-based compound exposure in zebrafish by inductively coupled plasma mass spectrometry with broader applications

金属和金属基化合物包括多种药活性和毒理学异种生物。从重金属毒性到化疗药物，这些化合物的毒代动力学具有历史和现代相关性。斑马鱼已成为环境暴露和临床转化研究中阐明药代动力学和毒代动力学的有吸引力的模式生物。虽然斑马鱼研究具有比啮齿动物模型更高的通量的好处，但该模型存在几个显着的限制。
水源性给药方案固有的一个这样的限制是固有的。这些研究的水浓度不能外推以提供可靠的内部剂量。直接测量金属基化合物可以与化合物相关的分子和生物反应更好地相关。为了克服金属和金属基化合物的这种局限性，开发了一种技术，用于在暴露后消化斑马鱼幼虫组织，并通过电感耦合等离子体质谱（ICPMS）量化组织样品中的金属浓度。
使用ICPMS方法测定斑马鱼组织中几种新型基于Ru的化疗药物中顺铂（Pt）和钌（Ru）的金属浓度。此外，该方案区分了与斑马鱼组织相比，螯合在幼虫绒毛膜中的Pt浓度。这些结果表明，该方法可用于定量幼虫组织中存在的金属剂量。此外，该方法可以调整以在广泛的暴露和剂量研究中鉴定特定的金属或金属基化合物。

这些结果之前已经发布了24。组织摄取研究是在顺铂和一种新型基于Ru的抗癌化合物PMC79的水源暴露下进行的。评估顺铂 0、3.75、7.5、15、30 和 60 mg/L 顺铂标称浓度的致死性和延迟孵化。通过ICPMS分析测定微生物组织中Pt的积累，生物体组织中每个生物体的含量分别为0.05，8.7，23.5，59.9，193.2和461.9ng（Pt） （图3）。鉴于顺铂的已知稳?...

Watch this Scientific Journal Video about Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications at JoVE.

Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications

斑马鱼中金属和金属基化合物的药代动力学和毒代动力学分析速率的提高对于环境和临床转化研究是有利的。通过使用电感耦合等离子体质谱对消化的斑马鱼组织进行痕量金属分析，克服了未知水性暴露摄取的局限性。

通过电感耦合等离子体质谱法对斑马鱼中铂基和钌基化合物暴露的剂量吸收，具有更广泛的应用

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the ...

斑马鱼是一个有吸引力的模型，但科学家们不知道这种生物在水源性剂量下吸收了多少。我们开发了一种技术，可以在暴露后消化组织并通过ICPMS量化金属。该技术使我们能够使用ICPMS准确量化和验证剂量反应。这种方法有很大的潜力。药理学或环境金属暴露可以从细胞水平一直到动物模型中的整个器官系统。向15毫升聚丙烯离心管中加入约0.25毫升高纯度硝酸，最多可灭10只幼虫。超声处理一小时以预消化样品。对于超声分析，在酸安全的微波蒸煮器中每隔五分钟进行短周期的组织消化，直到所有组织都明显氧化，产生均匀，透明的黄色溶液。仔细监测管子的完整性，以避免破裂，并在循环之间在离心机中进行短旋转。一旦组织明显氧化，使用6.75毫升高纯水和涡旋将通风橱中的样品稀释至3.5%硝酸以充分混合。然后进行矩阵匹配的7点校准曲线，以考虑任何潜在的等压干扰。使用水性认证元素标准的储备浓度，取0.1毫升等分试样，并通过移液管进入新的15毫升离心管中。用3.5%硝酸稀释至最终体积为10毫升，以生产十亿分之十一的标准溶液。使用10亿分之10的储备液，在3.5%硝酸中连续稀释0.1，1.0和5.0的十亿分之一标准溶液。使用0.1储备液，在3.5%硝酸中连续稀释0.001，0.005和0.01十亿分之一的标准溶液。然后在准备用于样品分析的ICPMS仪器之前，确保氩气阀打开，所有管子连接牢固且清洁，5%硝酸打开用于冲洗管和玻璃器皿之间的样品分析。检查割炬和锥体的状况，并确保割炬盒已牢固锁定。并且喷雾室引流管正确连接到围膜上。打开软件，检查真空读数，并确保所有涡轮泵都以100%的速度运行，单击等离子控制系统状态窗口中的“启动”以启动启动顺序。打开等离子泵和等离子体冷却器，栖息雾化器并点燃等离子体。等待等离子体点亮且稳定，此时状态窗口指示启动序列已完成。此时，观察系统状态窗口上的绿点，这些绿点指示所有电源都已打开。在菜单栏中，单击下拉菜单中的控制，自动进样器。进入含有5%硝酸的管的自动进样器机架位置，让酸进入血浆。在菜单栏中，单击下拉菜单中的扫描，磁铁。在“磁扫描”窗口中，在“标记质量位置”中键入 115，然后单击 Enter。让磁体在整个质量范围内扫描 30 分钟，同时仪器预热。30 分钟后，使用自动进样器控件移动到十亿分之一多元素调整解决方案的位置。吸出调谐解决方案并调谐仪器以优化信号读数。调整 X、Y、Z 坐标的割炬位置，使割炬与等离子体控制窗口中锥体的中心和雾化器流速对齐。在“离子光学调谐”窗口中对源、检测器和分析仪进行必要的调整。优化信号读数后，单击磁体扫描窗口中的停止，单击校准磁体，然后在弹出窗口中选择低分辨率。单击“确定”并打开“质量校准”smc 文件以校准磁体。单击保存，使用将电流磁体校准应用于分析。当测量具有较大浓度范围的未知样品时，执行检测器校准以将低浓度下的离子脉冲计数信号与较高浓度下产生的衰减离子信号进行比较。通过单击“数据采集”启动样品分析，然后单击下拉菜单中的方法设置。使用制造商提供的现有方法或基于感兴趣的元素创建方法。如有必要，可调整偏转器设置的分析模式、停留时间、开关延迟、扫描和周期数、分辨率、检测模式和停车质量。单击“保存”以记录方法设置。优化每种金属和同种型的参数。在菜单栏中，单击“数据采集”。单击下拉菜单中的“批量运行”。或者，单击菜单栏下方的 BATCH 图标，从电子表格导入批处理参数或在“批处理运行”窗口中创建序列。输入样品类型、自动进样器架位置、转移时间、洗涤时间、重复、样品 ID 和方法文件。安排校准曲线的标准溶液的批次运行，然后是质量控制标准，然后是未知样品。通过每 5 到 10 个样品包含一个 0.5/00 的质量控制标准来监控仪器漂移和样品再现性。组织摄取研究是在顺铂和一种新型钌基抗癌化合物PMC79的水源暴露下进行的。对致死性和延迟孵化进行顺铂名义浓度评估。0、3.75、7.5、15、30和60毫克每升顺铂。通过ICPMS分析测定了生物体组织中铂的积累，生物体组织中的浓度分别为每生物体0.05、8.7、23.5、59.9、193.2和461.9纳克。在所有顺铂浓度下均观察到延迟孵化。去皮后，分别收集绒毛膜并分析铂金。用于去皮研究的非致死剂量顺铂确定，顺铂总递送剂量的93%至96%已积聚在绒毛膜中，其余剂量在幼虫组织内。斑马鱼幼虫暴露于每升PMC79的0，3.1，6.2，9.2和12.4毫克。分析确定这些浓度含有每升0，0.17，0.44，0.66和0.76毫克的钌。与这种顺铂不同，在PMC79暴露的幼虫中没有观察到延迟孵化。绒毛膜未包含在钌分析中，因为它们在幼虫收集之前自然降解。在每种浓度下分析的幼虫组织中的块状金属为每只幼虫0.19，0.41和0.68纳克钌。添加到暴露方案中的任何试剂都可能导致等压干扰。如果可能的话，考虑与三卡因相比，快速冷却等替代方案。这种方法允许金属剂量定量的毒性，功效，实验在高等脊椎动物之间进行比较。我们特别兴奋的是，我们的阈值剂量在给予患者的一个数量级之内。

Research

JoVE Journal

Chemistry

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

增强型氟烷烃的化学气相沉积等离子通过疏水金属 - 有机骨架的制备氨的去除

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

科研

化学

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Improved In-gel Reductive ÃƒÅ½Ã‚Â²-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

汞（II）与Dicysteinyl四肽的络合的电喷雾质​​谱研究

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

环境质谱可视化纹影摄影的使用

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

A Practical Guide on Coupling a Scanning Mobility Sizer and Inductively Coupled Plasma Mass Spectrometer (SMPS-ICPMS)

A Practical Guide on Coupling a Scanning Mobility Sizer and Inductively Coupled Plasma Mass Spectrometer SMPS-ICPMS

耦合扫描流动性测定仪和电感耦合等离子体质谱仪（SMPS-ICPMS）的实用指南

A large variety of analytical methods are available to characterize particles in aerosols and suspensions. The choice of the appropriate technique depends ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

钌线粒体钙吸收抑制剂的合成与评价

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

综合结构质谱分析蛋白质结构和蛋白质配体配合物

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...