Approved Abstracts

Hidroxyapatite nanobiomaterials only produce toxic effects in fish cell lines after a long-term exposure



Author(s): Hernández-Moreno D; Navas JM; Fernández-Cruz ML;
Presenter: David Hernández-Moreno

Nanobiomaterials (NBMs) are nanostructured materials for biomedical applications. The use of NBMs is receiving much attention because of their specific interaction with the cell membranes and their applications (e.g. drug delivery system with remote activation, cell therapy, gene therapy, tissue regeneration, etc.). In vitro assays are being evaluated as useful tools to assess the toxic effect of emerging contaminants, like NBMs in order to reduce the number of experiments performed with animals.
In the present study, the short and long-term toxicity of different NBMs based on hydroxyapatite (HA) were evaluated in vitro in different rainbow trout (Oncorhynchus mykiss) cell lines. The RTgill-W1 (gills), RTL-W1 (liver) and RTS-11 (spleen) were exposed for 24h to a range of concentrations (0.78 – 100 μg/mL) of CaHA-P, FeHA, TiHA and TiHA-Alg. CaHA-P consists of calcium phosphate mineral prepared by means of a neutralization process between calcium hydroxyde and phosphoric acid. Metal doped NMs were developed following the same process adding Fe2+/3+ (Fe-HA) or titanium isopropoxide solution (Ti-HA). Finally, TiHA-Alg consisted in a HA doped with Ti4+ nucleated on alginate fiber. The RTgill-W1 was used for the long-term exposure (28d exposure, weekly cell renewal) to 50 μg/mL of CaHA-P or FeHA and to 100 μg/mL of the four NBMs. Recovery was also tested after 14d exposure and 14d recovery periods.
None of the NBMs studied provoked cytotoxicity after 24h exposure at the tested concentrations (IC50>100 μg/mL). However, an IT50 (time to cause a 50% decrease in cell viability) was reached during the long-term assays. In this sense, CaHA-p and FeHA showed an IT50 of 25 and 22 days at 100 μg/mL, respectively, finding also the inhibition of viability after 25 days of exposure to 50 μg/mL of FeHA. The cytotoxicity was higher for both titanium NBMs with IT50 values of 15 and 12 days for TiHA and TiHA-Alg, respectively. Cells previously exposed to the four NBMs for 14 days did not show a recovery after 14 days in clean medium but, at least, they were able to remain stable and their viability did not continue decreasing.
These results evidence the need to test the long-term toxicity of NBMs and show differences in cytotoxicity for different NBMs probably associated to different mechanisms of toxic action. Rtgill-W1 cells are appropriate to screen short and long-term toxicity of NBMs providing valuable information about NBMs toxicity in fish.
Acknowledgements: This work was funded from the European Union’s Horizon 2020 Programme under Grant Agreement No 760928 (BIORIMA - BIOmaterial RIsk MAnagement). Instituto di Scienza e Tecnologia dei Materiali Ceramici (Istec. Italy) supplied the NBMs.


Keywords: long-term toxicity; fish cell line; nanobiomaterial

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