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 water. Maintain the aquatic system water at 28 &#176;C, &#60;0.05 ppm nitrite, &#60;0.2 ppm ammonia, and pH between 7.2 and 7.7.</li>
		<li>Feed the zebrafish a diet of hatched Artemia cysts, brine shrimp, and fish diet flake food.</li>
	</ol>
	</li>
</ol>
2. Zebrafish dose-response protocol (Figure 1)
<ol>
	<li>Prepare zebrafish egg water solution, either E3 medium or egg water made from stock sea salt at a concentration of 60 &#181;g/mL dissolved in deionized water18. Avoid the use of methylene blue. 
	NOTE: Through ICPMS, isobaric interference of zebrafish egg water was identified for strontium oxides, which overlapped with an isotope of ruthenium. Careful rinsing of the larvae before downstream analysis ameliorated this issue. E3 medium may be an easier choice for some due to the proprietary makeup of commercial sea salts.</li>
	<li>Dissolve the metal or metal-based compounds in E3 or egg water. Vortex to break up any aggregate material and homogenize the solution. 
	NOTE: In the experiment outlined below, PMC79 and cisplatin were dissolved at maximum concentrations of 12.4 mg/L and 60 mg/L with a maximum concentration of 0.5% dimethyl sulfoxide (DMSO) to resist precipitation.
	<ol>
		<li>Dilute heavy metal or metal-based compounds, such as PMC79 and cisplatin, with E3 or egg water, preparing at least 5 concentration doses.
		<ol>
			<li>Begin with low concentrations of stock solutions in pure vehicle (i.e., DMSO), then dilute with E3 or egg water. Consider the final vehicle concentrations carefully. 
			NOTE: Certain metal-based compounds, such as cisplatin, degrade rapidly, and their solutions must be made fresh daily. CAUTION: Handle heavy metals and chemotherapeutics with care. Review the specific material safety data sheet (MSDS) for the metal of interest. Cisplatin may cause eye and skin irritation, be fatal if swallowed, and can cause toxicity in kidneys, blood, blood-forming organs, and fetal tissue. Avoid breathing fumes and contact with eyes, skin, and clothing. Wear impervious gloves and clothing, and safety glasses or goggles19.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Set up breeding tanks the afternoon prior to the experiment in the ideal ratio of 2 females to 1 male with a divider in place between the sexes20.
	<ol>
		<li>Pull the divider when the lights come on for the morning cycle.</li>
		<li>Allow the zebrafish to breed. 
		NOTE: The length of time for breeding depends on the initial exposure stage required. For 3 h postfertilization, allow breeding for approximately 2 h. The eggs will reach 3 hpf after cleaning and segregating eggs.</li>
		<li>Move the breeding fish to a clean tank.</li>
		<li>Collect the eggs by pouring the tank water through a strainer.</li>
		<li>Invert the strainer over a Petri dish and use a squirt bottle filled with E3 or egg water to rinse the eggs into the dish.</li>
		<li>Clean the dish of food and waste prior to experimental use.</li>
	</ol>
	</li>
	<li>Randomize approximately twenty 3 hpf embryos per dose into individual glass vials using a transfer pipette and a small quantity of water.</li>
	<li>After all embryos are in vials, remove all egg water and replace with enough dosing solution so that there is approximately 1 inch of solution above the height of the egg. 
	NOTE: The necessity of dechorionation should be carefully considered. See the discussion section for more details.</li>
	<li>Observe the embryos daily for lesions or lethality. Due to the rapid development of embryonic zebrafish, obtain daily images using any brightfield microscope/camera setup to identify minor lesions between days.</li>
	<li>At the termination of the dose response (4-5 days post fertilization [dpf], per Organization Economic Cooperation and Development [OECD] guideline21), combine 3-5 larvae prior to euthanization for composite sampling. Euthanize by rapid cooling by snap-freezing in liquid nitrogen. 
	NOTE: Euthanasia through an overdose of MS-222 or tricaine methanesulfonate may potentially interfere with ICPMS analysis downstream. Rapid-cooling euthanasia methods are encouraged for this protocol to reduce the possibility of interference.</li>
	<li>Conduct 3 washes of tissue with high-purity water (such as reverse osmosis) to remove the excess compound from the exterior of the tissue.</li>
	<li>Move the samples to acid- and microwave-safe 15 mL polypropylene centrifuge tubes. Be careful to remove all excess water as any remaining liquid may dilute the nitric acid, and therefore, the oxidizing potential of the acid during tissue degradation. 
	NOTE: At this point, tissue may be stored at -20 &#176;C until further analysis. For naturally abundant metals, cleaning the tubes in a 5% nitric acid bath prior to use will ameliorate ambient background levels.</li>
</ol>
3. Tissue digestion and ICPMS evaluation (Figure 2)
<ol>
	<li>Add approximately 0.25 mL to the 15 mL polypropylene centrifuge tubes for up to 10 larvae (~100 &#181;g) of high-purity nitric acid (69%). Ultrasonicate for 1 h to predigest the samples using the following settings: ultrasonic bath output: 85 W; 42 kHz &#177; 6%; temperature range: 19-27 &#176;C. 
	CAUTION: Wear ear protection during sonication. Nitric acid causes serious respiratory tract, eye, and skin burns. Wear full protective equipment and work in the fume hood or in places with adequate ventilation. It can be flammable with other materials. Nitric acid should be handled exclusively in a fume hood to prevent exposure to vapors produced during digestion. Do not inhale or ingest.
	<ol>
		<li>Perform short cycles of tissue digestion (5 min intervals) in an acid-safe microwave digester until all tissue is visibly oxidized (i.e., uniform, clear-yellow solution). 
		NOTE: The microwave protocol in Table 1 is recommended to be performed three times and includes a 5 min cooldown interval between each heating step.</li>
		<li>Monitor the integrity of the tubes carefully to avoid rupture and conduct short spins in the centrifuge (313 &#215; g for 1 min) between cycles to move acid condensation to the bottom of the tube. 
		NOTE: If the tissue is difficult to digest (especially chorions), 30% high-purity hydrogen peroxide can be used after acid digestion. Use the hydrogen peroxide (6.75 mL) to dilute the acid concentration to 3.5%, and allow the samples to sit overnight in a fume hood. Hydrogen peroxide will decompose to H2O and is suitable for ICPMS analysis. Further, alternative metals may dissolve better in hydrochloric acid or a mixture of hydrochloric acid and nitric acid (i.e., aqua regia). Hydrogen peroxide is harmful if swallowed and causes serious eye damage. Wear skin and eye protection22,23.</li>
	</ol>
	</li>
	<li>Once the tissue is visibly oxidized (i.e., uniform, clear-yellow solution), dilute the samples in a fume hood to 3.5% nitric acid using 6.75 mL of high-purity water and vortex to mix thoroughly. 
	NOTE: At this point, the samples can be stored at room temperature. This step is unnecessary if hydrogen peroxide was added to aid digestion in step 3.1.</li>
	<li>Conduct a matrix-matched, 7-point calibration curve (concentration range of 0.001-10 ppb) using a certified elemental standard (i.e., Pt or Ru, depending on the assay) with the metal of interest and optimal isotope(s) to account for any potential isobaric interferences.
	<ol>
		<li>Using a stock concentration of the aqueous, certified elemental standard (Ru, Pt = 1000 ppb), take a 0.1 mL aliquot and pipet into a new 15 mL centrifuge tube. Dilute with 3.5% nitric acid to a final volume of 10 mL to produce a 10 ppb standard solution.</li>
		<li>Using the 10 ppb stock, make the following serial dilutions: 0.1, 1.0, and 5.0 ppb standard solutions in 3.5% nitric acid.</li>
		<li>Using the 0.1 stock, make the following serial dilutions: 0.001, 0.005, and 0.01 ppb standard solutions in 3.5% nitric acid.</li>
	</ol>
	</li>
	<li>Prepare the ICPMS instrument (see the Table of Materials) for sample analysis as follows:
	<ol>
		<li>Prior to starting the instrument, 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.</li>
		<li>Check the condition of the torch and cones, and make sure the torch box is securely latched and the spray chamber drainage tube is properly connected to the peripump.</li>
		<li>Open the software (see the Table of Materials).</li>
		<li>Check the vacuum readings and ensure all turbo pumps are running at 100%.</li>
		<li>Click START in the Plasma Control System Status window to initiate the start sequence, turn on the plasma pump, plasma chiller, purge the nebulizer, and light the plasma. Wait for the plasma to be lit and stable when the status window will indicate 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.</li>
		<li>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.</li>
		<li>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 115In (114.6083 to 115.3749) for 30 min while the instrument warms up.</li>
		<li>After 30 min, use the autosampler control to move to the position of a 1 ppb multielement tuning solution. Aspirate the tuning solution and tune the instrument to optimize the signal reading. Adjust the torch position (X, Y, Z) such that the torch is aligned with the center of the cones and nebulizer flow rate (~ 30 psi) in the Plasma control window. Make the necessary adjustments in the Ion Optics Tuning window for the Source, Detector, and Analyzer.</li>
		<li>Once the signal reading is optimized (~1.2 &#215; 106&#160;counts/s for 1 ppb on 115In), click Stop in the Magnet Scan window.
		<ol>
			<li>Click Calibrate Magnet and select Low Resolution in the popup window.</li>
			<li>Click OK and open the &#34;Mass Calibration (Low Resolution).smc&#34; file to calibrate the Magnet. 
			NOTE: The magnet calibration will measure the counts/s and fit a curve through the following mass range: 7Li to 238U.</li>
			<li>Click Save | Use to apply the current magnet calibration to the analyses. If measuring unknown samples with a huge range of concentrations, perform a Detector Calibration to compare ion pulse counts signals at low concentrations to attenuated ion signals produced at higher concentrations. Analyze the samples once tuning and calibration are complete.</li>
		</ol>
		</li>
		<li>In the menu bar, click on Data Acquisition.
		<ol>
			<li>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/cycles, resolution, detection mode, and the park mass for deflector settings.</li>
			<li>Click Save to record the method settings. Optimize the parameters for each metal and isotope. See Table 2 for the specific operation parameters used in this study.</li>
		</ol>
		</li>
		<li>In the menu bar, click on Data Acquisition.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Arrange the batch run in the following order: standard solutions for the calibration curve (0.001-10 ppb Pt or Ru), followed by a quality control standard, then unknown samples. 
		NOTE: Standard solutions are measured as counts/s on the ICPMS, and a linear regression is fit through the standards with a relative standard deviation (RSD) &#62; 0.999. Unknowns are also measured as counts/s and solved for concentration in ppb using the linear regression of the calibration curve, y = mx + b. Data processing can also be completed using the referenced software.</li>
		<li>Monitor instrument drift and sample reproducibility by including a 0.5 ppb quality control standard every 5-10 samples. 
		NOTE: Suitable quality control standards should be a certified standard reference material different from the standard used in the calibration curve.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Watts</td>
			<td>Power</td>
			<td>Minutes</td>
		</tr>
		<tr>
			<td>300</td>
			<td>50%</td>
			<td>5</td>
		</tr>
		<tr>
			<td>300</td>
			<td>75%</td>
			<td>5</td>
		</tr>
		<tr>
			<td>300</td>
			<td>0%</td>
			<td>5</td>
		</tr>
		<tr>
			<td>300</td>
			<td>75%</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1: Microwave digestion protocol for larval tissue mass. Zebrafish larval samples were digested in 0.25 mL of nitric acid. This table has been modified from 24.

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There are no conflicts of interest to be disclosed by any of the authors.

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. Furthermore, because the delivered dose of cisplatin was quantified, dose-response results were extrapolated to patient populations. Sublethal doses (e.g., LOAEL) were comparable to intravenous dosing concentrations in patients24.
Although this method may be applied to a broad spectrum of metals and metal-based compounds, careful investigation of the physicochemical properties of the analyte must be taken into consideration. Metal-based compounds may be very difficult to dissolve, and various vehicles can be used to avoid this. Vehicle concentrations, such as DMSO, may need to be at higher concentrations than recommended in the OECD protocol. As such, it is important to maintain a nontoxic dose by closely monitoring the development of controls; continuously rocking the embryos during exposure mitigates precipitation. Additionally, organometallic compounds may not be stable in aqueous solution. If the degradation process is unknown, studies that involve 24 h solution renewal may be considered or compared to nonrenewal dose-response curves.
It is recommended to follow the OECD Fish Acute Embryo Toxicity Test (FET) Number 23621. However, modifications can be made to suit specific purposes. Glass containers avoid confounding toxicological variables, such as plastic and plasticizers, and do not adsorb metals as strongly, which would remove analytes from the egg water. For compounds that photodegrade, such as cisplatin, it may be beneficial to conduct the exposure without a light cycle.
There is much discussion in the literature regarding the need for dechorionation in zebrafish dose-response studies25,26,27. Arguments for dechorionation at 24 hpf suggest that the chorion limits the permeability of compounds, thus generating false-negative results or augmented dose-response curves. Although these points have merit, conducting studies without dechorionation may provide mechanistic insight. These studies suggest cisplatin accumulates in the chorion of the embryos due to its alkylating activity (Figure 2). The resulting adducts reinforce the structure, resulting in delayed hatching. However, PMC79 and other Ru-based anticancer drugs did not cause this phenomenon27. Although many chemotherapeutics enact their anticancer activity by alkylation, the lack of delayed hatching post PMC79 exposure indicated a disparate mechanism. Studies with or without dechorionation must be carefully considered or conducted in parallel.
Downstream tissue digestion and ICPMS analysis must be continuously considered. It is suggested to avoid using any reagents that may cause isobaric interferences and implement alternative methods. Reagents used during the dose-response studies may impact or react with the nitric acid and its oxidizing potential or contribute to isobaric interferences. It was discovered that the salt solution used to make egg water generated strontium (Sr) oxides, which overlapped with a specific isotope of Ru24. Lowering salt concentrations or carefully cleaning the larvae can ameliorate this issue. For these reasons, it is suggested to avoid the antimicrobial methylene blue or the euthanizing agent, tricaine. Instead, autoclave and subsequently aerate the egg water to remove microbes or euthanize the larvae by rapid cooling. It is important at this step to achieve linear isotopic standard curves with minimal isobaric interferences for the analyte of interest.
An important limitation to this protocol is that organometallic compounds will be oxidized such that only the metal remains. As such, metabolism studies cannot be conducted. Although the protocol can be considered medium-throughput, the dose-response portion may be expedited with the aid of automatic chemical delivery systems and imaging. This protocol is a nascent methodology that may be modified and refined for a broad spectrum of metal and metal-based compounds for pharmaco- and toxicokinetic studies.

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><tbody><tr><td>AB Strain Zebrafish (&lt;em&gt;Danio reri&lt;/em&gt;)</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% HNO&lt;sub&gt;3&lt;/sub&gt; 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.&amp;nbsp;&amp;nbsp;</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&amp;reg; Sea Salt</td><td>Egg water solution contains instand ocean sea salt with a final concentration of 60 &amp;micro;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).&amp;nbsp;</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% HNO&lt;sub&gt;3&lt;/sub&gt;; 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.&amp;nbsp;</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 (Figure 3). Analytical determination of the nominal concentrations for cisplatin was not assessed, given the known stability of cisplatin.
Delayed hatching was observed at all cisplatin concentrations. Additional experiments were conducted for Pt concentrations with and without manual dechorionation. Post dechorionation, chorions were collected and analyzed for Pt separately. Nonlethal doses of cisplatin used for dechorionation studies determined that 93-96% of the total delivered dose of cisplatin had accumulated in the chorion with the remaining dose within the larval tissue (Figure 4).
Zebrafish larvae were exposed to 0, 3.1, 6.2, 9.2, 12.4 mg/L of PMC79. These doses were selected by determining the derivatives of an IC50, as described previously16. These concentrations were analytically determined to contain 0, 0.17, 0.44, 0.66, and 0.76 mg/L of Ru. Unlike the cisplatin dose-response curve, delayed hatching was not observed in PMC79-exposed larvae. Chorions were not included in Ruthenium analysis as they naturally degraded prior to larval collection. Researchers may include chorion analysis without delayed hatching by dechorionating and collecting chorions at 24 dpf. The mass of metal within larval tissues analyzed at each concentration was 0.19, 0.41, and 0.68 ng (Ru) per larva (Figure 5). A summary of the toxicological endpoints, including lethal concentrations and/or doses for 50% of the population (LC50/LD50), effective concentrations, or doses for 50% of the populations (EC50/ED50), and the lowest observed adverse effect level (LOAEL) can be found in Table 3.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Cisplatin</td>
			<td colspan="3">PMC79</td>
		</tr>
		<tr>
			<td></td>
			<td>Nominal (mg/L)</td>
			<td>&#181;M</td>
			<td>Pt (ng) / organism</td>
			<td>Analytical Ru (mg/L)</td>
			<td>&#181;M</td>
			<td>Ru (ng) / organism</td>
		</tr>
		<tr>
			<td>LC50/LD50</td>
			<td>31 (95% CI: 20.5-34.0)</td>
			<td>158 (95% CI: 105-174)</td>
			<td>193 (&#177; 130)</td>
			<td>0.79 (95% CI: 0.43-1.20)</td>
			<td>7.8 (95% CI: 4.2-11.8)</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>EC50</td>
			<td>4.6</td>
			<td>12.5</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>LOAEL</td>
			<td>3.75</td>
			<td>15.3</td>
			<td>8.7 (&#177; 4)</td>
			<td>0.17</td>
			<td>1.7</td>
			<td>0.19 (&#177; 0.05)</td>
		</tr>
	</tbody>
</table>
Table 3: Determination of solution and metallodrug uptake associated with toxicological endpoints. LD50&#160;was determined by metal equivalent analysis of Pt and Ru for cisplatin and PMC79, respectively. The LC50&#160;concentrations for PMC79 were analytically determined. However, analytical determination of nominal cisplatin concentrations was not conducted; given the known stability of cisplatin in solution, it was assumed that nominal and measured concentrations in solution would be equivalent. The delayed hatching endpoint for cisplatin exposure was evaluated in terms of ED50&#160;and LOAEL. The LOAEL concentrations of PMC79 were analytically determined. The LOAEL included lesions such as hemorrhaging along the caudal vein and tail artery, spinal curvature, and yolk sac edema. All 95% confidence intervals were calculated using the Litchfield Wilcoxon method. This table has been modified from 24. Abbreviations: CI = confidence interval; LC50&#160;= Lethal Concentration for 50% of the population; LD50&#160;= Lethal Dose for 50% of the population; EC50&#160;= Effective Concentration for 50% of the population; LOAEL = lowest observed adverse effect level.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63587/63587fig01.jpg" /> 
Figure 1: Zebrafish dose-response protocol. This protocol uses a modified approach adapted from the OECD FET. Made with Biorender. Abbreviation: OECD = Organization Economic Cooperation and Development; FET = fish embryo acute toxicity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63587/63587fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63587/63587fig02.jpg" /> 
Figure 2: Tissue digestion and ICPMS evaluation. The digestion protocol is effective for digesting a composite sample of zebrafish larvae. Abbreviation: ICPMS = inductively coupled plasma mass spectrometry. Created with Biorender. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63587/63587fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63587/63587fig03.jpg" /> 
Figure 3: Cisplatin dose-response. (A) Percentage mean delayed hatching at 5 dpf correlated to the mean Pt equivalents determined per organism. (B) Percentage mean lethality at 5 dpf correlated to the mean Pt equivalents per organism. Percentage means: N = 40 per dose. Pt (ng) per organism: &#62;4 composite samples per dose. Two experimental replicates were conducted, the ranges of which are displayed. This figure has been modified from 24. Abbreviation: dpf = days post fertilization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63587/63587fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63587/63587fig04.jpg" /> 
Figure 4: Comparison of Pt (ng) present in the larvae and the chorion after exposure to 7.5 or 15 mg/L. Composite &#62;3 larvae or chorions per sample; from left to right N = 13, 10, 10, and 11. Error bars represent standard deviation. Mann-Whitney rank-sum test P &#60; 0.001 between larvae and chorion for both doses. This figure has been modified from 24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63587/63587fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63587/63587fig05.jpg" /> 
Figure 5: PMC79 dose-response. (A) Percentage mean lethality was correlated to the analytically determined mean Ru equivalents in solution (mg/L). (B) Percentage mean lethality at 5 days post fertilization from the same experiment was correlated to the mean Ru equivalents per larva. Lethality: N = 40 per dose. Ru (mg/L): N = 6 composite samples per dose. Ru (ng) per larva &#62;4 composite samples per dose. Two experimental replicates were conducted, the ranges of which are displayed. This figure has been modified from 24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63587/63587fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dwell Time per Peak</td>
			<td>4 ms</td>
		</tr>
		<tr>
			<td>Switch Delay/ Peak (x10micros)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Number of Sweeps</td>
			<td>350</td>
		</tr>
		<tr>
			<td>Number of Cycles</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Instrument Resolution</td>
			<td>300</td>
		</tr>
		<tr>
			<td>Detection Mode</td>
			<td>Attenuated, Deflector Jump</td>
		</tr>
		<tr>
			<td>Park Mass</td>
			<td>98.90594</td>
		</tr>
		<tr>
			<td>Element (isotopes)</td>
			<td>Pt (192, 194, 195, 196), Ru (99, 100, 101, 102) Sr (84)</td>
		</tr>
	</tbody>
</table>
Table 2: ICPMS method parameters. Parameters for analysis of Pt and Ru isotopes to determine tissue concentrations of cisplatin and PMC79, respectively. Sr was included to monitor isobaric interferences associated with the tank water composition. This table has been modified from 24. ICPMS = inductively coupled plasma mass spectrometry.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

2.3K Views.  Rutgers University. Zebravissen zijn een aantrekkelijk model, maar wetenschappers weten niet hoeveel het organisme opneemt met watergedragen dosering. We ontwikkelden een techniek om weefsel na blootstelling te verteren en metalen te kwantificeren via ICPMS. Deze techniek stelt ons in staat om de dosisrespons nauwkeurig te kwantificeren en te valideren met behulp van ICPMS.Deze methode heeft veel potentie. Farmacologische of milieutechnische metaalblootstellingen kunnen worden toegepast vanaf cellulair ni...

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

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Cancer Research

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Immunology and Infection

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

Developmental Biology

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Medicine

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the ...

Biology

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

Zebrafish Xenograft Assay: A Method to Assess the Anti-cancer Efficacy of Therapeutics on Tumorigenicity in Zebrafish In Vivo

Source: Lee, J. Y et al.,<a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57490/preclinical-assessment-bioactivity-anticancer-coumarin-ot48-spheroids">Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts.</a>&#160;J. Vis. Exp. (2018)
This video describes the zebrafish xenograft assay to assess in vivo tumor formation and study the effect of therapeutic agents.

Source: Lee, J. Y et al.,Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish ...

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

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