A Toxicological and Ecotoxicological Assay Based on Mussel (Mytilus galloprovincialis) Hemocytes Motility

Summary

The article proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on the motility of Mytilus galloprovincialis hemocytes. The method aims to contribute to the development of more ethical and sensitive toxicological and ecotoxicological exposure tests.

Abstract

Hemocytes are the circulating immune-competent cells in bivalve mollusks and play a key role in several important functions of cell-mediated innate immunity. During the early stages of the immune response, hemocytes actively migrate to the site of infection. This inherent motility is a fundamental characteristic of these cells. It represents a key cellular function that integrates multiple processes, such as cell adhesion, cell signaling, cytoskeletal dynamics, and changes in cell volume. Therefore, alterations in cell motility following exposure to drugs or pollutants can serve as a useful toxicological endpoint. Despite the fundamental role of cell motility in cellular physiology, it has been poorly investigated from a toxicological perspective. This work proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on evaluating the hemocyte motility of Mytilus galloprovincialis. We developed a cell motility assay on hemocytes adhering to the bottom of a 96-well polystyrene microplate. Following exposure to increasing concentrations of drugs, cell trajectories, and velocities were quantified by cell tracking under time-lapse microscopy, allowing us to measure the effects on hemocyte motility. Due to the ease of hemocyte collection from the animals in a relatively non-invasive manner, the proposed method offers an alternative test for screening the effects and mechanisms of action of pollutants and drugs. It aligns with the 3Rs (Replacement, Reduction, and Refinement) criteria, addressing ethical concerns and contributing to the reduction of vertebrate in vivo animal testing.

Introduction

Effect-based methods, such as in vitro and in vivo bioassays, represent innovative tools for the detection of the effects of environmental chemical pollutants in living organisms and for their use as tools in environmental monitoring and risk assessment^1,2,3,4. They complement the classical analytical chemical approach by overcoming some of its limitations. For instance, effect-based methods can assess the bioavailability of pollutants, their impact on organism health, and the combined toxicological effects of mixtures. The....

Protocol

All experiments were performed under the Italian Animal Welfare legislation (D.L.26/2014) that implemented the European Committee Council Directive (2010/63 EEC). Mytilus galloprovincialis is a filter-feeding bivalve, commonly known as the Mediterranean mussel. It is native to the Mediterranean Sea and the Atlantic coast of southern Europe. It was introduced and is widespread in Western North America, Asia, and South Africa. It is an important commercial fishery species in several parts of the world. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Preparation of art....

Results

The study introduces a novel in vitro method for quickly and sensitively assessing the toxicity and ecotoxicity of pollutants, utilizing the motility of Mytilus galloprovincialis hemocytes. Figure 1A-C shows representative time-lapse imaging of hemocytes after 30 min attachment to the bottom of the well. The cells in the figure were stained with Neutral Red just before the motility assessment. Cell movements were monitored using optical mic.......

Discussion

The protocol described in this work represents a novel in vitro method suitable for the rapid and sensitive assessment of the toxicity of drugs and pollutants based on evaluating the motility of M. galloprovincialis hemocytes and its alterations. Motility is a peculiar aspect of the immune function of these cells^{21,22,23,37,38}, therefore any .......

Materials

Name	Company	Catalog Number	Comments
0.2 µm filter (diameter 25 mm)	ABLUO		labware
2.5 ml hypodermic syringe needdle 22G	Rays	2522CM32	labware
96-well flat-bottom polystyrene TC-treated microplate	Corning	3916	labware
CaCl2.2H2O	Merk (Sigma - Aldrich)	C3881-1KG	Chemical
Chemotaxis and Migration Tool software	(Ibidi GmbH)		software
Cytation 5	Agilent BioTeck	Cytation 5	Equipment: Cell imaging multimode reader
Dimethyl sulfoxide (DMSO)	Merk (Sigma - Aldrich)	472301	Solvent
Falcon 15 mL Tube Conical Bottom	Corning	352196	labware
H3BO3	Merk (Sigma - Aldrich)	B0394	Chemical
Hemocytometer Fast read 102	Biosigma	BVS100	labware
HEPES (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid)	Merk (Sigma - Aldrich)	H3375-500G	Chemical
ImageJ software	NIH		software
KBr	Merk (Sigma - Aldrich)	P9881	Chemical
KCl	Merk	104936	Chemical
L-glutamine	Merk (Sigma - Aldrich)	G7513	Essential amino acid for cell culture medium
MgCl2·6H2O	Merk (Sigma - Aldrich)	M2670	Chemical
MgSO4	Merk (Sigma - Aldrich)	M7506	Chemical
Microscope Nikon Eclipse E600	Nikon		Equipment: Cell imaging
Na2SO4	Riedel-de Haen	31481	Chemical
NaCl	Merk (Sigma - Aldrich)	31434-1KG-R	Chemical
NaF	Fluka	71519 500g	Chemical
NaHCO3	Merk (Sigma - Aldrich)	S5761-1KG	Chemical
Neutral Red	Merk (Sigma - Aldrich)	N4638-1G	Vital cell dye
Penicillin/Streptomycin	Merk (Sigma - Aldrich)	P0781-100ML	Antibiotics for cell culture
SrCl2·6H2O	Merk (Sigma - Aldrich)	255521	Chemical
Trypan blue	Merk (Sigma - Aldrich)	T8154	Cell dye