Metallic Nano‐Composite Toxicity Evaluation by Zebrafish Embryo Toxicity Test with Identification of Specific Exposure Biomarkers

Roberta Pecoraro1, Antonio Salvaggio2, Fabio Marino3, Gianfranco Di Caro3, Fabiano Capparucci3, Bianca Maria Lombardo1, Giuseppina Messina1, Elena Maria Scalisi1, Maurizio Tummino1, Francesco Loreto1, Giusi D'Amante1, Roberto Avola4, Daniele Tibullo4, Maria Violetta Brundo1

1 Department of Biological, Geological and Environmental Science, University of Catania, Catania, 2 Experimental Zooprophylactic Institute of Sicily, Catania, 3 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, 4 Biological and Biotechnological Sciences, University of Catania, Catania
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 1.14
DOI:  10.1002/cptx.34
Online Posting Date:  November, 2017
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Nanomaterials (NM) have different shapes and can be composed of different materials such as carbon, silicon, and some metals like gold, silver, and titanium. They are used as fillers, catalysts, semiconductors, cosmetics, drug carriers in medicine, energy storage systems, and antifriction coatings. NM are the parent compounds of nanoparticles (NPs), which may be divided into two groups: fullerenes and engineered nanoparticles (ENPs). After crossing the cell membrane, NPs may be stored in vesicles, mitochondria, and additional organelles within epithelial cells. They may generate reactive oxygen species, oxidative stress, and cytotoxicity, as well as denaturation of proteins, apoptosis, and necrosis. Nowadays, new toxicological data are required to assess the potential exposure of the environment and human beings to pollutants. The aim of the present investigation is to evaluate the toxicity of the metallic nano‐composite by the zebrafish embryo toxicity test (ZFET). The methods described here can be easily adapted to other nanocomposites or nanomaterials with some variations. © 2017 by John Wiley & Sons, Inc.

Keywords: Danio rerio; biomarkers; embryonic; toxicity; animal testing; nanoparticles; nanomaterials; metallothioneins

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Table of Contents

  • Introduction
  • Basic Protocol 1: Zebrafish Embryo Toxicity Test
  • Support Protocol 1: Immunohistochemical Analysis
  • Support Protocol 2: Detection of Protein Expression by Western Blotting
  • Support Protocol 3: Determination of Gene Expression by Real‐Time PCR
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Zebrafish Embryo Toxicity Test

  • Test compound stock solutions (e.g., AuNPs or TiO 2NPs at different concentrations in 5 ml of fresh water)
  • 3,4‐dichloroaniline (DCA; Sigma‐Aldrich, cat. no. 437778‐5G)
  • Zebrafish
  • Glass vessels to prepare different test concentrations and dilution water or to collect eggs
  • Standard 24‐well plates with a depth of ∼20 mm
  • Probe sonicator
  • Fish tanks made of chemically inert material (e.g., glass) and with 10 liters capacity
  • Glass marbles or other inert material to protect the eggs laid
  • Binocular microscope with a capacity of at least 60/80‐fold magnification
  • Pipet with widened openings to collect eggs
  • Incubator (26 ± 1°C)
  • Additional reagents and equipment for use of zebrafish in toxicological studies (unit 1.7; Zhang, Willett, & Fremgen, )

Support Protocol 1: Immunohistochemical Analysis

  • Zebrafish larvae
  • Phosphate buffered saline (prepared using PBS tablet; Sigma)
  • 4% paraformaldehyde (J.T. Baker) in PBS
  • Triton X‐100 (Chemsolute)
  • Blocking solution (Abcam, cat. no. ab189136)
  • Primary antibody: anti‐metallothionein (anti‐mouse MT; use at 1:500 dilution; Abcam)
  • Secondary antibody: FITC‐conjugated anti–rabbit (goat anti‐mouse IgG; Sigma‐Aldrich)
  • PBS–Tween 20 (J.T Baker)
  • 70%, 80%, and 95% (v/v) ethanol
  • Vectashield antifade mounting medium with DAPI (Vector Laboratories)
  • Rubber cement
  • Microscope slides
  • Coverslips
  • Zeiss AXIO Observer Z1 microscope with Apotome2 system, equipped with the ZEN PRO software

Support Protocol 2: Detection of Protein Expression by Western Blotting

  • Zebrafish larvae
  • Liquid nitrogen
  • Lysis buffer (BioRad, cat. no. 1632086)
  • 20× BoltTM MES or MOPS SDS running buffer (Thermo Fisher Scientific)
  • Odyssey protein marker
  • Licor blocking buffer (Licor Biosciences)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Tween 20
  • Primary antibody: anti‐metallothionein (FL‐61): sc‐11377 (Santa Cruz Biotechnology)
  • IRDye® 800CW goat anti–rabbit IgG (Licor Biosciences)
  • Dissecting tools
  • Round‐bottom microcentrifuge tubes
  • Glass Dounce tissue homogenizer
  • 70°C water bath or heat block
  • Novex® Mini Gels, Bolt® Bis‐Tris Plus Gels, NuPAGE® Mini Gels
  • iBlot® Transfer Stack, nitrocellulose and western blotting system (Thermo Fisher Scientific)
  • Rocking platform
  • Sealable containers, preferably sealable sandwich bags
  • Odyssey scanner (Odyssey® Fc Imaging System; Licor Biosciences)
  • Additional reagents and equipment for electrophoresis ( appendix 3F; Gallagher, )

Support Protocol 3: Determination of Gene Expression by Real‐Time PCR

  • High‐Capacity cDNA Reverse Transcription Kit (Life Technologies)
  • RNA sample (RNA sample extract from larvae)
  • SYBR Green PCR master mix, 200 reactions (Life Technologies)
  • Oligonucleotide primers, gene specific for metallothionein:
  • All primers are desalted and both UV absorbance and capillary electrophoresis are used to assess the quality of primer synthesis
  • 96‐well optical plates and adhesive covers (Life Technologies)
  • Centrifuge with plate adapter
  • 7900HT Fast Real‐Time PCR System (Life Technologies)
  • GraphPad Prism software
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Literature Cited

Literature Cited
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