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.
