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

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

1. Breed and maintain the zebrafish in a recirculating aquatic habitat system on a 14 h light:10 h dark cycle.
   1. Purify municipal tap water through sand and carbon filtration to obtain fish system water. Maintain the aquatic system water at 28 °C, <0.05 ppm nitrite, <0.2 ppm ammonia, and pH between 7.2 and 7.7.
   2. Feed the zebrafish a diet of hatched Artemia cysts, brine shrimp, and fish diet flake food.

2. Zebrafish dose-response protocol (Figure 1)

1. Prepare zebrafish egg water solution, either E3 medium or egg water made from stock sea salt at a concentration of 60 µg/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.
2. 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.
   1. Dilute heavy metal or metal-based compounds, such as PMC79 and cisplatin, with E3 or egg water, preparing at least 5 concentration doses.
      1. 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.
3. 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.
   1. Pull the divider when the lights come on for the morning cycle.
   2. 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.
   3. Move the breeding fish to a clean tank.
   4. Collect the eggs by pouring the tank water through a strainer.
   5. 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.
   6. Clean the dish of food and waste prior to experimental use.
4. Randomize approximately twenty 3 hpf embryos per dose into individual glass vials using a transfer pipette and a small quantity of water.
5. 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.
6. 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.
7. 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.
8. Conduct 3 washes of tissue with high-purity water (such as reverse osmosis) to remove the excess compound from the exterior of the tissue.
9. 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 °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.

3. Tissue digestion and ICPMS evaluation (Figure 2)

1. Add approximately 0.25 mL to the 15 mL polypropylene centrifuge tubes for up to 10 larvae (~100 µg) 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 ± 6%; temperature range: 19-27 °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.
   1. 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.
   2. Monitor the integrity of the tubes carefully to avoid rupture and conduct short spins in the centrifuge (313 × 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 protection^22,23.
2. 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.
3. 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.
   1. 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.
   2. 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.
   3. 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.
4. Prepare the ICPMS instrument (see the Table of Materials) for sample analysis as follows:
   1. 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.
   2. 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.
   3. Open the software (see the Table of Materials).
   4. Check the vacuum readings and ensure all turbo pumps are running at 100%.
   5. 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.
   6. 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.
   7. 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.
   8. 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.
   9. Once the signal reading is optimized (~1.2 × 10⁶ counts/s for 1 ppb on ¹¹⁵In), click Stop in the Magnet Scan window.
      1. Click Calibrate Magnet and select Low Resolution in the popup window.
      2. Click OK and open the "Mass Calibration (Low Resolution).smc" 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.
      3. 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.
   10. In the menu bar, click on Data Acquisition.
       1. 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.
       2. 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.
   11. In the menu bar, click on Data Acquisition.
       1. 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.
   12. 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) > 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.
   13. 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.

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.