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 demand^1,2,

Protocol

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 zebra....

Results

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 (.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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