Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
The protocol described here has been implemented to determine the delivery and uptake of metal-based anticancer drugs containing either Pt or Ru. Although these methods have already been published, this protocol discusses important considerations and details to adapt this methodology for a range of compounds. The OECD protocol coupled with tissue digestion and ICPMS analysis allowed us to determine that PMC79 was more potent than cisplatin and resulted in disparate tissue accumulation, suggesting separate mechanisms. Fur…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
References
- Rehman, K., Fatima, F., Waheed, I., Akash, M. S. H. Prevalence of exposure of heavy metals and their impact on health consequences. Journal of Cellular Biochemistry. 119 (1), 157-184 (2018).
- Anyanwu, B. O., Ezejiofor, A. N., Igweze, Z. N., Orisakwe, O. E. Heavy metal mixture exposure and effects in developing nations: an update. Toxics. 6 (4), 65 (2018).
- Doherty, C. L., Buckley, B. T. Translating analytical techniques in geochemistry to environmental health. Molecules. 26 (9), 2821 (2021).
- Boros, E., Dyson, P. J., Gasser, G. Classification of metal-based drugs according to their mechanisms of action. Chem. 6 (1), 41-60 (2020).
- Robertson, J., Barr, R., Shulman, L. N., Forte, G. B., Magrini, N. Essential medicines for cancer: WHO recommendations and national priorities. Bulletin of the World Health Organization. 94 (10), 735-742 (2016).
- Wheate, N. J., Walker, S., Craig, G. E., Oun, R. The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Transactions. 39 (35), 8113-8127 (2010).
- Brown, A., Kumar, S., Tchounwou, P. B. Cisplatin-based chemotherapy of human cancers. Journal of Cancer Science & Therapy. 11 (4), 97 (2019).
- Ghosh, S. Cisplatin: The first metal based anticancer drug. Bioorganic Chem. 88, 102925 (2019).
- Abid, M., Shamsi, F., Azam, A. Ruthenium complexes: an emerging ground to the development of metallopharmaceuticals for cancer therapy. Mini Reviews in Medicinal Chemistry. 16 (10), 772-786 (2016).
- Alessio, E., Messori, L. NAMI-A and KP1019/1339, two iconic ruthenium anticancer drug candidates face-to-face: a case story in medicinal inorganic chemistry. Molecules. 24 (10), 1995 (2019).
- Alessio, E., Mestroni, G., Bergamo, A., Sava, G. Ruthenium antimetastatic agents. Current Topics in Medicinal Chemistry. 4 (15), 1525-1535 (2004).
- Lin, K., Zhao, Z. -. Z., Bo, H. -. B., Hao, X. -. J., Wang, J. -. Q. Applications of ruthenium complex in tumor diagnosis and therapy. Frontiers in Pharmacology. 9, 1323 (2018).
- Howe, K., et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 496 (7446), 498-503 (2013).
- Wiley, D. S., Redfield, S. E., Zon, L. I. Chemical screening in zebrafish for novel biological and therapeutic discovery. Methods in Cell Biology. 138, 651-679 (2017).
- Bambino, K., Chu, J. Zebrafish in toxicology and environmental health. Current Topics in Developmental Biology. 124, 331-367 (2017).
- Rubinstein, A. L. Zebrafish assays for drug toxicity screening. Expert Opinion on Drug Metabolism & Toxicology. 2 (2), 231-240 (2006).
- Cassar, S., et al. Use of zebrafish in drug discovery toxicology. Chemical Research in Toxicology. 33 (1), 95-118 (2020).
- Westerfield, M. . The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th edition. , (2000).
- Material safety data sheet: cisplatin injection). Pfizer Available from: https://cdn.pfizer.com/pfizercom/products/material_safety_data/PZ01470.pdf (2011)
- Nasiadka, A., Clark, M. D. Zebrafish breeding in the laboratory environment. ILAR Journal. 53 (2), 161-168 (2012).
- OECD. Test No. 236: Fish embryo acute toxicity (FET) test. OECD Guidelines for the Testing of Chemicals Available from: https://www.oecd-ilibrary.org/environment/test-no-236-fish-embryo-acute-toxicity-fet-test_9789264203709-en (2013)
- EMD Millipore Corporation. Material Safety Data Sheet: OmniTrace Nitric Acid. EMD Millipore Corporation. , (2013).
- Safety data sheet: Hydrogen peroxide 30% Suprapur. EMD Millipore Corporation Available from: https://www.merckmillipore.com/IN/en/product/Hydrogen-peroxide-300-0 (2014)
- Karas, B. F., et al. A novel screening method for transition metal-based anticancer compounds using zebrafish embryo-larval assay and inductively coupled plasma-mass spectrometry analysis. Journal of Applied Toxicology. 39 (8), 1173-1180 (2019).
- Henn, K., Braunbeck, T. Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP. 153 (1), 91-98 (2011).
- Mandrell, D., et al. Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens. Journal of Laboratory Automation. 17 (1), 66-74 (2012).
- Karas, B. F., Hotz, J. M., Buckley, B. T., Cooper, K. R. Cisplatin alkylating activity in zebrafish causes resistance to chorionic degradation and inhibition of osteogenesis. Aquatic Toxicology. 229, 105656 (2020).
