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 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.
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'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.
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
2. Zebrafish dose-response protocol (Figure 1)
3. Tissue digestion and ICPMS evaluation (Figure 2)
Watts | Power | Minutes |
300 | 50% | 5 |
300 | 75% | 5 |
300 | 0% | 5 |
300 | 75% | 5 |
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.
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.
Cisplatin | PMC79 | |||||
Nominal (mg/L) | µM | Pt (ng) / organism | Analytical Ru (mg/L) | µM | Ru (ng) / organism | |
LC50/LD50 | 31 (95% CI: 20.5-34.0) | 158 (95% CI: 105-174) | 193 (± 130) | 0.79 (95% CI: 0.43-1.20) | 7.8 (95% CI: 4.2-11.8) | NA |
EC50 | 4.6 | 12.5 | NA | NA | NA | NA |
LOAEL | 3.75 | 15.3 | 8.7 (± 4) | 0.17 | 1.7 | 0.19 (± 0.05) |
Table 3: Determination of solution and metallodrug uptake associated with toxicological endpoints. LD50 was determined by metal equivalent analysis of Pt and Ru for cisplatin and PMC79, respectively. The LC50 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 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 = Lethal Concentration for 50% of the population; LD50 = Lethal Dose for 50% of the population; EC50 = Effective Concentration for 50% of the population; LOAEL = lowest observed adverse effect level.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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: >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. Please click here to view a larger version of this figure.
Figure 4: Comparison of Pt (ng) present in the larvae and the chorion after exposure to 7.5 or 15 mg/L. Composite >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 < 0.001 between larvae and chorion for both doses. This figure has been modified from 24. Please click here to view a larger version of this figure.
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 >4 composite samples per dose. Two experimental replicates were conducted, the ranges of which are displayed. This figure has been modified from 24. Please click here to view a larger version of this figure.
Dwell Time per Peak | 4 ms |
Switch Delay/ Peak (x10micros) | 2 |
Number of Sweeps | 350 |
Number of Cycles | 1 |
Instrument Resolution | 300 |
Detection Mode | Attenuated, Deflector Jump |
Park Mass | 98.90594 |
Element (isotopes) | Pt (192, 194, 195, 196), Ru (99, 100, 101, 102) Sr (84) |
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.
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.
The authors have nothing to disclose.
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 (Fundação para a Ciência e Tecnologia, FCT; PTDC/QUI-QIN/28662/2017) for the supply of PMC79.
AB Strain Zebrafish (Danio reri) | Zebrafish International Resource Center | Wild-Type AB | Wild-Type AB Zebrafish |
ACS Grade Nitric Acid | VWR BDH Chemicals | BDH3130-2.5LP | Nitric Acid (68-70%); used to make 10% HNO3 acid-bath solution for soaking/pre-celaning centrifuge tubes |
Aquatox Fish Diet (Flake) | Zeigler Bros, Inc. | Flake food to be mixed in a 1:4 ratio of Aquatox Fish Diet to TetraMin Tropical Flakes and used as feed | |
Artemia cysts, brine shrimp | PentairAES | BS90 | Brine shrimp eggs sold in 15-ozz, vacuum-packed cans to be hatched and used as feed |
ASX-510 Autosampler for ICPMS | Teledyne CETAC | 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. | |
Centrifuge | Thermo Scientific | CL 2 | 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 |
Centrifuge tubes | VWR | 21008-105 | Ultra high performance polypropylene centrifuge tubes with flat cap; 15 mL volume; leak-proof with conical bottom |
Class A Clear Glass Threaded Vials | Fisherbrand | 03-339-25B | Individual glass vials for exposure containment |
Dimethyl Sulfoxide | Millipore Sigma | D8418 | Solvent or vehicle for hydrophobic compounds |
Fixed Speed Vortex Mixer | VWR | 10153-834 | Vortex mixer; used to homogenize sample after acid digestion and dilution |
High Purity Hydrogen Peroxide | Merk KGaA, EDM Millipore | 1.07298.0250 | Suprapur Hydrogen peroxide (30%); used for sample digestion |
High Purity Nitric Acid | EDM Millipore | NX0408-2 | Omni Trace Ultra Nitric Acid (69%); used for sample digestion |
Instant Ocean Sea Salt | Spectrum Brands, Inc. | Instant Ocean® Sea Salt | Egg water solution contains instand ocean sea salt with a final concentration of 60 µg/ml |
Mars X Microwave Digestion System | CEM, Matthews, NC | 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). | |
Multi-element Solution 3 | SPEX CertiPREP | CLMS-3 | 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 |
Nu Instruments AttoM High Resolution Inductively Coupled Plasma Mass Spectrometer (HR-ICP-MS) | Nu Instruments/Amatek | 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. | |
Platinum (Pt) standard solution, NIST 3140 | National Institute of Standards and Technology | 3140 | Prepared from ampoule containing 9.996 mg/g Pt in 10% HCl; ; used as a quality control standard for Pt analyses |
Platinum (Pt) standard solution, single-element | High Purity Standards | 100040-2 | Contains 1000 mg/L Pt in 5% HCl |
Ruthenium (Ru) standard solution, single-element | High Purity Standards | 100046-2 | Contains 1000 mg/L Ru in 2% HCl |
TetraMin Tropical Flakes | Tetra | 77101 | Flake food to be mixed in a 1:4 ratio of Aquatox Fish Diet to TetraMin Tropical Flakes and used as feed |
Trace Metal Grade Nitric Acid | VWR BDH Chemicals | 87003-261 | Aristar Plus Nitric Acid (67-70%); used for rinse solution in ASX-510 Autosampler |
Ultrasonic water bath | VWR | B2500A-DTH | Ultrasonic water bath used to aid in acid digestion prior to microwave digestion |