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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.
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.
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 assessment1,2,3,4. 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. These combined effects may not be predictable based solely on chemical analysis5.
In recent years, the ecotoxicology of pollutants of emerging concern (emerging pollutants) represents a field where effect-based methods can be useful tools for detecting exposure and assessing the impact on the biota1,5,6,7. Several effect-based methods use bivalve mollusks as test organisms in environmental monitoring and assessment8,9. Some characteristics make these organisms suitable for ecotoxicological studies, such as their wide distribution, their filter-feeding nature, their sessile lifestyle, the capability of bioaccumulation of a wide range of environmental pollutants and to develop detectable responses to pollutants, the possibility of working with different life stages, and to maintain under laboratory conditions7. They are highly sensitive to pollution exposure and show a variety of responses to toxic contaminants depending on species, life stage, and environmental conditions8,9,10. Therefore, several environmental guidelines use bivalve species as standardized test species10,11.
Among the bivalve mollusks, the widespread Mytilus galloprovincialis is one of the most used species in the ecotoxicological field due to its ability to develop early detectable responses to chemical pollution exposure, including metallothionein induction, antioxidant enzyme alteration, lysosomal membrane destabilization, lipid peroxidation, lipofuscin accumulation, increased micronuclei frequency, carbonic anhydrase induction12,13,14,15. Hemocytes, the immunocompetent hemolymphatic cells, are widely used to study the toxicological impacts of environmental pollutants in bivalve mollusks4,13,16,17. These cells are crucial to the organism's immune response, carrying out several important functions of cell-mediated innate immunity. These include the elimination of microbes through phagocytosis and various cytotoxic reactions, such as the release of lysosomal enzymes, anti-microbial peptides, and the production of oxygen metabolites during the respiratory burst18,19,20. Hemocytes are intrinsically motile cells21,22,23 able to migrate to the site of infection during the early stage of the organism's immune response. In general, motility is a fundamental feature that characterizes all immune cells since it enables the immunosurveillance of these cells to protect the body24. Research across various molluscan species demonstrates that hemocyte motility is a critical component of their immune response, wound healing, and interaction with pathogens. This motility is regulated by specific molecular pathways, highlighting the complexity and specialization of hemocyte functions in molluscs21,25,26,27.
Despite the fundamental importance of motility in the physiology of hemocytes, very few studies have investigated the sensitivity of hemocyte motility to environmental chemical pollutants23,28,29,30. Recently, our group characterized the spontaneous movement of Mytilus galloprovincialis hemocytes in a tissue culture-treated polystyrene 96-well microplate and examined the sensitivity of hemocyte motility to in vitro exposure to paracetamol23. M. galloprovincialis hemocytes showed a random-like cell movement based on lamellipodia and fast shape changes, as previously found in another mussel species, Mytilus edulis21,22,23,28, and already described in human immune cells31. Hemocyte motility has recently been demonstrated to be sensitive to chemical stressors23,28. Based on these previous findings, 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 motility of M. galloprovincialis hemocytes and its alterations, through velocimetric analysis of cell motility (quantification of mean velocity, migrated distance, Euclidean distance, and directness). The method offers the possibility to in vitro screen the toxicity of several substances either in short-term assays (lasting 1-4 h) or prolonged exposure assays, lasting 24-48 h.
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 artificial seawater (ASTM) or filtered natural seawater
2. Animal acclimation
3. Reagent preparation for hemocyte motility assessment
4. Hemolymph sampling
5. Hemocyte plating and culture
6. Short-term assay
7. Prolonged exposure assay
8. Cell motility assessment by time-lapse microscopy
9. Cell tracking and velocimetric parameters calculation
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...
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 cells21,22,23,37,38, therefore any ...
The authors have nothing to disclose.
This research was funded by the project "Dipartimento di Eccellenza" awarded to DiSTeBA by the Italian Ministry of University and Research, CUP: F85D18000130001, and by NBFC (National Biodiversity Future Center) funded by European Union NextGenerationEU, PNRR, project n. CN00000033. We also thank BIOforIU infrastructure at the Department of Biological and Environmental Sciences and Technologies of the University of Salento.
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 |
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