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.

Funding: NJAES-Rutgers NJ01201, NIEHS Training Grant T32-ES 007148, NIH-NIEHS P30 ES005022. Additionally, Brittany Karas is supported by training grant T32NS115700 from NINDS, NIH. The authors acknowledge Andreia Valente and the Portuguese Foundation for Science and Technology (Fundac&#807;a&#771;o para a Cie&#770;ncia e Tecnologia, FCT; PTDC/QUI-QIN/28662/2017) for the supply of PMC79.

The AB strain zebrafish (Danio rerio) were used for all experiments (see the Table of Materials), and the husbandry protocol (#08-025) was approved by the Rutgers University Animal Care and Facilities Committee.
1. Zebrafish husbandry
<ol>
	<li>Breed and maintain the zebrafish in a recirculating aquatic habitat system on a 14 h light:10 h dark cycle.
	<ol>
		<li>Purify municipal tap water through sand and carbon filtration to obtain fish system wate...

<ol>
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Z., Bo, H. -. B., Hao, X. -. J., Wang, J. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+ruthenium+complex+in+tumor+diagnosis+and+therapy.">Applications of ruthenium complex in tumor diagnosis and therapy.</a> Frontiers in Pharmacology. 9, 1323 (2018).</li><li>Howe, K., 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=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Wiley, D. S., Redfield, S. E., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+screening+in+zebrafish+for+novel+biological+and+therapeutic+discovery.">Chemical screening in zebrafish for novel biological and therapeutic discovery.</a> Methods in Cell Biology. 138, 651-679 (2017).</li><li>Bambino, K., Chu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+in+toxicology+and+environmental+health.">Zebrafish in toxicology and environmental health.</a> Current Topics in Developmental Biology. 124, 331-367 (2017).</li><li>Rubinstein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+assays+for+drug+toxicity+screening.">Zebrafish assays for drug toxicity screening.</a> Expert Opinion on Drug Metabolism &amp; Toxicology. 2 (2), 231-240 (2006).</li><li>Cassar, S., 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=Use+of+zebrafish+in+drug+discovery+toxicology.">Use of zebrafish in drug discovery toxicology.</a> Chemical Research in Toxicology. 33 (1), 95-118 (2020).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th edition. , (2000).</li><li>Material safety data sheet: cisplatin injection). Pfizer Available from: <a target="_blank" href="https://cdn.pfizer.com/pfizercom/products/material_safety_data/PZ01470.pdf">https://cdn.pfizer.com/pfizercom/products/material_safety_data/PZ01470.pdf</a> (2011)</li><li>Nasiadka, A., Clark, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+breeding+in+the+laboratory+environment.">Zebrafish breeding in the laboratory environment.</a> ILAR Journal. 53 (2), 161-168 (2012).</li><li>OECD. Test No. 236: Fish embryo acute toxicity (FET) test. OECD Guidelines for the Testing of Chemicals Available from: <a target="_blank" href="https://www.oecd-ilibrary.org/environment/test-no-236-fish-embryo-acute-toxicity-fet-test_9789264203709-en">https://www.oecd-ilibrary.org/environment/test-no-236-fish-embryo-acute-toxicity-fet-test_9789264203709-en</a> (2013)</li><li>EMD Millipore Corporation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Material+Safety+Data+Sheet:+OmniTrace+Nitric+Acid.">Material Safety Data Sheet: OmniTrace Nitric Acid.</a> EMD Millipore Corporation. , (2013).</li><li>Safety data sheet: Hydrogen peroxide 30% Suprapur. EMD Millipore Corporation Available from: <a target="_blank" href="https://www.merckmillipore.com/IN/en/product/Hydrogen-peroxide-300-0">https://www.merckmillipore.com/IN/en/product/Hydrogen-peroxide-300-0</a> (2014)</li><li>Karas, B. 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Toxicology &amp; Pharmacology: CBP. 153 (1), 91-98 (2011).</li><li>Mandrell, D., 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=Automated+zebrafish+chorion+removal+and+single+embryo+placement:+optimizing+throughput+of+zebrafish+developmental+toxicity+screens.">Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens.</a> Journal of Laboratory Automation. 17 (1), 66-74 (2012).</li><li>Karas, B. F., Hotz, J. M., Buckley, B. T., Cooper, K. 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The protocol described here has been implemented to determine the delivery and uptake of metal-based anticancer drugs containing either Pt or Ru. Although these methods have already been published, this protocol discusses important considerations and details to adapt this methodology for a range of compounds. The OECD protocol coupled with tissue digestion and ICPMS analysis allowed us to determine that PMC79 was more potent than cisplatin and resulted in disparate tissue accumulation, suggesting separate mechanisms. Fur...

Metals and metal-based compounds continue to have pharmacological and toxicological relevance. The prevalence of heavy metal exposure and its impact on health has exponentially increased scientific investigation since the 1960s and reached an all-time high in 2021. The concentrations of heavy metals in drinking water, air pollution, and occupational exposure exceeds regulatory limits worldwide and remain an issue for arsenic, cadmium, mercury, chromium, lead, and other metals. Novel methods to quantify environmental exposure and analyze pathological development continue to be in high demand1,2,3.
Conversely, the medical field has harnessed the physiochemical properties of various metals for clinical treatment. Metal-based drugs or metallodrugs have a rich history of medicinal purposes and have shown activity against a range of diseases, with the highest success as chemotherapeutics4. The most famous of metallodrugs, cisplatin, is a Pt-based anticancer drug deemed by the World Health Organization (WHO) as one of the world&#39;s essential drugs5. In 2010, cisplatin and its Pt derivatives had up to a 90% success rate in several cancers and were used in approximately 50% of chemotherapy regimens6,7,8. Although Pt-based chemotherapeutics have had irrefutable success, the dose-limiting toxicity has set in motion investigations of alternative metal-based drugs with refined biological delivery and activity. Of these alternatives, Ru-based compounds have become the most popular9,10,11,12.
Novel models and methodology are required to keep pace with the rate of need for metal pharmaco- and toxicokinetic studies. The zebrafish model lies at the intersection of complexity and throughput, being a high-fecundity vertebrate with 70% conserved gene homology13. This model has been an asset in pharmacology and toxicology, with extensive screenings for various compounds for lead discovery, target identification, and mechanistic activity14,15,16,17. However, high-throughput screening of chemicals typically relies on waterborne exposures. Given that uptake can be variable based on the physicochemical properties of the compound in solution (i.e., photodegradation, solubility), this can be a major limitation of correlating dose delivery and response.
To overcome this limitation for comparison of dose to higher vertebrates, a methodology was designed to analyze trace metal concentrations in zebrafish larval tissue. Here, dose-response curves of lethal and sublethal endpoints were evaluated for cisplatin and novel Ru-based anticancer compounds. Lethality and delayed hatching were evaluated for nominal concentrations of 0, 3.75, 7.5, 15, 30, and 60 mg/L cisplatin. Pt accumulation in organism tissue was determined by ICPMS analysis, and organism uptake of respective doses were 0.05, 8.7, 23.5, 59.9, 193.2, and 461.9 ng (Pt) per organism. Additionally, zebrafish larvae were exposed to 0, 3.1, 6.2, 9.2, 12.4 mg/L of PMC79. These concentrations were analytically determined to contain 0, 0.17, 0.44, 0.66, and 0.76 mg/L of Ru. This protocol also allowed for the distinguishment of concentrations of Pt sequestered in the chorion of the larvae compared with the zebrafish tissue. This methodology was able to provide reliable, robust data for comparisons of pharmaco- and toxicokinetic activity between a well-established chemotherapeutic and a novel compound. This method can be applied to a wide range of metals and metal-based compounds.

<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

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the toxicokinetics of these compounds have both historical and modern-day relevance. Zebrafish have become an attractive model organism in elucidating pharmaco- and toxicokinetics in environmental exposure and clinical translation studies. Although zebrafish studies have the benefit of being higher-throughput than rodent models, there are several significant constraints to the model.
One such limitation is inherent in the waterborne dosing regimen. Water concentrations from these studies cannot be extrapolated to provide reliable internal dosages. Direct measurements of the metal-based compounds allow for a better correlation with compound-related molecular and biological responses. To overcome this limitation for metals and metal-based compounds, a technique was developed to digest zebrafish larval tissue after exposure and quantify metal concentrations within tissue samples by inductively coupled plasma mass spectrometry (ICPMS).
ICPMS methods were used to determine the metal concentrations of platinum (Pt) from cisplatin and ruthenium (Ru) from several novel Ru-based chemotherapeutics in zebrafish tissue. Additionally, this protocol distinguished concentrations of Pt that were sequestered in the chorion of the larval compared with the zebrafish tissue. These results indicate that this method can be applied to quantitate the metal dose present in larval tissues. Further, this method may be adjusted to identify specific metals or metal-based compounds in a broad range of exposure and dosing studies.

These results have been previously published24. Tissue uptake studies were conducted with waterborne exposures of cisplatin and a novel Ru-based anticancer compound, PMC79. Lethality and delayed hatching were evaluated for nominal concentrations of cisplatin 0, 3.75, 7.5, 15, 30, and 60 mg/L cisplatin. Pt accumulation in organism tissue was determined by ICPMS analysis, and organism tissue contained respective doses of 0.05, 8.7, 23.5, 59.9, 193.2, and 461.9 ng (Pt) per organism (...

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

The increased rate of pharmaco- and toxicokinetic analyses of metals and metal-based compounds in zebrafish can be advantageous for environmental and clinical translation studies. The limitation of unknown waterborne exposure uptake was overcome by conducting trace metal analysis on digested zebrafish tissue using inductively coupled plasma mass spectrometry.

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

Zebrafish are an attractive model, but scientists don't know how much the organism is uptaking with waterborne dosing. We developed a technique to digest tissue after exposure and quantify metals via ICPMS. This technique allows us to accurately quantify and validate the dose response using ICPMS.This method has a lot of potential. Pharmacological or environmental metal exposures can be applied from the cellular level all the way to entire organ systems in animal models. Add approximately 0.25 milliliters of high purity nitric acid to the 15 milliliter polypropylene centrifuge tubes for up to 10 larvae.Ultrasonicate for one hour to predigests the samples. For ultrasonication, perform short cycles of tissue digestion with five minute intervals in an acid safe microwave digester until all tissue is visibly oxidized, producing a uniform, clear yellow solution. Monitor the integrity of the tubes carefully to avoid rupture and conduct short spins in the centrifuge between cycles.Once the tissue is visibly oxidized, dilute the samples in a fume hood to 3.5%nitric acid using 6.75 milliliters of high purity water and vortex to mix thoroughly. Then to conduct a matrix-matched, 7-point calibration curve to account for any potential isobaric interferences. Using a stock concentration of the aqueous certified elemental standard, take a 0.1 milliliter aliquot, and by pipet into a new 15 milliliter centrifuge tube.Dilute with 3.5%nitric acid to a final volume of 10 milliliters to produce 10 parts per billion standard solutions. Using the 10 parts per billion stock, make the serial dilutions 0.1, 1.0, and 5.0 parts per billion standard solutions in 3.5%nitric acid. Using the 0.1 stock, make the serial dilutions 0.001, 0.005, and 0.01 parts per billion standard solutions in 3.5%nitric acid.Then before preparing the ICPMS instrument for sample analysis, ensure that the argon gas valve is open, all tubing is securely connected and clean, 5%nitric acid is open for rinsing tubing and glassware between sample analyses. Check the condition of the torch and cones and ensure that the torch box is securely latched. And the spray chamber drainage tube is connected correctly to the peripump.Open the software, check the vacuum readings, and ensure all turbo pumps are running at 100%Click START in the Plasma Control system status window to initiate the start sequence. Turn on the plasma pump and plasma chiller, perch the nebulizer and light the plasma. Wait for the plasma to be lit and stable when the Status window indicates that the startup sequence is complete.At this point, observe the green dots on the system state window that indicate that all power supplies are on. In the menu bar, click on Control, Autosampler in the dropdown menu. Enter the autosampler rack position for the tube containing 5%nitric acid, allow the acid to enter the plasma.In the menu bar, click on Scans, Magnet in the dropdown menu. In the MagnetScan window, type 115 into the Marker Mass Position and click enter. Allow the magnet to scan across the mass range for 30 minutes while the instrument warms up.After 30 minutes, use the autosampler control to move to the position of a one parts per billion multielement tuning solution. Aspirate the tuning solution and tune the instrument to optimize the signal reading. Adjust the torch position for X, Y, Z coordinates, such that the torch aligns with the center of the cones and nebulizer flow rate in the Plasma Control window.Make the necessary adjustments in the Ion Optics Tuning window for the Source, Detector, and Analyzer. Once the signal reading is optimized, click Stop in the Magnet Scan window, click Calibrate Magnet and select a Low Resolution in the popup window. Click OK And open the Mass Calibration smc file to calibrate the magnet.Click Save, Use to apply the current magnet calibration to the analyses. When measuring unknown samples with a huge range of concentrations, perform a detector calibration to compare ion pulse count signals at low concentrations to attenuated ion signals produced at higher concentrations. Initiate sample analysis by clicking on Data Acquisition, then click on Method Setup in the dropdown menu.Use an existing method provided by the manufacturer or create a method based on the elements of interest. If necessary, adjust the Analysis Mode, Dwell Time, Switch Delay, Number of Sweeps and Cycles, Resolution, Detection Mode and the Park Mass for deflector settings. Click Save to record the method settings.Optimize the parameters for each metal and isotype. In the menu bar, click on Data Acquisition. Click on Batch Run in the dropdown menu.Alternatively, click on the BATCH icon below the menu bar, import the batch parameters from a spreadsheet or create a sequence in the Batch Run window. Enter the sample type, the autosampler rack position, transfer time, wash time, replicates, sample ID, and method file. Arrange the batch run for standard solutions for calibration curve, followed by a quality control standard, then unknown samples.Monitor the instrument drift and sample reproducibility by including a 0.5 parts per billion quality control standard every 5 to 10 samples. Tissue uptake studies were conducted with waterborne exposures of cisplatin and a novel ruthenium-based anti-cancer compound PMC79. Lethality and delayed hatching were evaluated for nominal concentrations of cisplatin.0, 3.75, 7.5 15, 30 and 60 milligrams per liter of cisplatin. Platinum accumulation in organism tissue was determined by ICPMS analysis and organism tissue contained respective doses of 0.05, 8.7, 23.5, 59.9, 193.2, and 461.9 nanograms per organism. Delayed hatching was observed at all cisplatin concentrations.Post dechorionation, chorions were collected and analyzed for platinum separately. Nonlethal doses of cisplatin used for dechorionation studies determined that 93 to 96%of the total delivered dose of cisplatin had accumulated in the chorion, with the remaining dose within the larval tissue. Zebrafish larvae were exposed to 0, 3.1, 6.2, 9.2, and 12.4 milligrams per liter of PMC79.These concentrations were analytically determined to contain 0, 0.17, 0.44, 0.66, and 0.76 milligrams per liter of ruthenium. Unlike this cisplatin, delayed hatching was not observed in the PMC79 exposed larvae. Chorions were not included in ruthenium analysis as they naturally degraded prior to larval collection.The massive metal within larval tissues analyzed at each concentration was 0.19, 0.41, and 0.68 nanograms of ruthenium per larva. Any of the reagents added to the exposure protocol give the potential for isobaric interferences. When possible, consider alternatives like rapid cooling compared to tricaine.This method allows metal dose quantification for toxicity, efficacy, experiments to be compared across higher vertebrates. We were especially excited that our threshold dose was within an order of magnitude given to patients.

dose-uptake-platinum-ruthenium-based-compound-exposure-zebrafish

Research

JoVE Journal

Chemistry

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

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

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

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

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

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

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

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

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

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

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

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

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

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 large variety of analytical methods are available to characterize particles in aerosols and suspensions. The choice of the appropriate technique depends on the properties to be determined. In many fields information about particle size and chemical composition are of great importance. While in aerosol techniques particle size distributions of gas-borne particles are determined online, their elemental composition is commonly analyzed offline after an appropriate sampling and preparation procedure. To obtain both types of information online and simultaneously, a hyphenated setup was recently developed, including a Scanning Mobility Particle Sizer (SMPS) and an Inductively Coupled Plasma Mass Spectrometer (ICPMS). This allows first to classify the particles with respect to their mobility diameter, and then to determine their number concentration and elemental composition in parallel. A Rotating Disk Diluter (RDD) is used as the introduction system, giving more flexibility regarding the use of different aerosol sources. In this work, a practical guide is provided describing the different steps for establishing this instrumentation, and how to use this analysis tool. The versatility of this hyphenated technique is demonstrated in example measurements on three different aerosols generated out of a) a salt solution, b) a suspension, and c) emitted by a thermal process.

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

In this work a practical guide is provided, describing the different steps to establish the coupling of SMPS and ICPMS systems, and how to use them. Three descriptive examples are presented.

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

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

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

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 this compound commences from [Ru(NH3)5Cl]Cl2 in 1 M NH4OH in a closed container, yielding a green solution. Purification is accomplished with cation-exchange chromatography. This compound is characterized and verified to be pure by UV-vis and IR spectroscopy. The mitochondrial calcium uptake inhibitory properties are assessed in permeabilized HeLa cells by fluorescence spectroscopy.

A protocol for the synthesis, purification, and characterization of a ruthenium-based inhibitor of mitochondrial calcium uptake is presented. A procedure to evaluate its efficacy in permeabilized mammalian cells is demonstrated.

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

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

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

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

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

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) for Quantification of Iron Redox Species (Fe(II), Fe(III))

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative conditions. Excessive iron results in free redox-active Fe(II) and can cause devastating effects within the cell like oxidative stress (OS) and death by lipid peroxidation known as ferroptosis (FPT). Therefore, quantitative measurements of ferrous (Fe(II)) and ferric (Fe(III)) iron rather than total Fe-determination is the key for closer insight into these detrimental processes. Since Fe(II)/(III) determinations can be hampered by fast redox-state shifts and low concentrations in relevant samples, like cerebrospinal fluid (CSF), methods should be available that analyze quickly and provide low limits of quantification (LOQ). Capillary electrophoresis (CE) offers the advantage of fast Fe(II)/Fe(III) separation and works without a stationary phase, which could interfere with the redox balance or cause analyte sticking. CE combined with inductively coupled plasma mass spectrometry (ICP-MS) as a detector offers further improvement of detection sensitivity and selectivity. The presented method uses 20 mM HCl as a background electrolyte and a voltage of +25 kV. Peak shapes and concentration detection limits are improved by conductivity-pH-stacking. For reduction of 56[ArO]+, ICP-MS was operated in the dynamic reaction cell (DRC) mode with NH3 as a reaction gas. The method achieves a limit of detection (LOD) of 3 &#181;g/L. Due to stacking, higher injection volumes were possible without hampering separation but improving LOD. Calibrations related to peak area were linear up to 150 &#181;g/L. Measurement precision was 2.2% (Fe(III)) to 3.5% (Fe(II)). Migration time precision was &#60;3% for both species, determined in 1:2 diluted lysates of human neuroblastoma (SH-SY5Y) cells. Recovery experiments with standard addition revealed accuracy of 97% Fe(III) and 105 % Fe(II). In real-life bio-samples like CSF, migration time can vary according to varying conductivity (i.e., salinity). Thus, peak identification is confirmed by standard addition.

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry CE-ICP-MS for Quantification of Iron Redox Species FeII, FeIII

This iron redox speciation method is based on capillary electrophoresis-inductively coupled plasma mass spectrometry with sample stacking combined with short analysis in one run. The method quickly analyzes and provides low limits of quantification for iron redox species across a diverse range of tissues and biofluid samples.

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative ...