title: In vitro exposure of haemocytes of the clam Ruditapes philippinarum to titanium dioxide (TiO2) nanoparticles: nanoparticle characterisation, effects on phagocytic activity and internalisation of nanoparticles into haemocytes.
abstract: The continuous growth of nanotechnology and nano-industries, the considerable increase of products containing nanoparticles (NPs) and the potential release of NPs in aquatic environments suggest a need to study NP effects on aquatic organisms. In this context, in vitro assays are commonly used for evaluating or predicting the negative effects of chemicals and for understanding their mechanisms of action. In this study, a physico-chemical characterisation of titanium dioxide NPs (n-TiO2) was performed, and an in vitro approach was used to investigate the effects of n-TiO2 on haemocytes of the clam Ruditapes philippinarum. In particular, the effects on haemocyte phagocytic activity were evaluated in two different experiments (with and without pre-treatment of haemocytes) by exposing cells to P25 n-TiO2 (0, 1 and 10 μg/mL). In addition, the capability of n-TiO2 to interact with clam haemocytes was evaluated with a transmission electron microscope (TEM). In this study, n-TiO2 particles showed a mean diameter of approximately 21 nm, and both anatase (70%) and rutile (30%) phases were revealed. In both experiments, n-TiO2 significantly decreased the phagocytic index compared with the control, suggesting that NPs are able to interfere with cell functions. The results of the TEM analysis support this hypothesis. Indeed, we observed that TiO2 NPs interact with cell membranes and enter haemocyte cytoplasm and vacuoles after 60 min of exposure. To the best of our knowledge, this is the first study demonstrating the internalisation of TiO2 NPs into R. philippinarum haemocytes. The present study can contribute to the understanding of the mechanisms of action of TiO2 NPs in bivalve molluscs, at least at the haemocyte level.
authors.affiliation: Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy. Electronic address: ilaria.marisa@studenti.unipd.it.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Marisa
authors.affiliation: Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy.
authors.forename: Maria Gabriella
authors.initials: MG
authors.lastname: Marin
authors.affiliation: Department of Biology, Electron Microscopy Laboratory, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Caicci
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy.
authors.forename: Erica
authors.initials: E
authors.lastname: Franceschinis
authors.affiliation: Industrial Engineering Department and INSTM, University of Padova, Via Marzolo 9, 35131 Padova, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Martucci
authors.affiliation: Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy.
authors.forename: Valerio
authors.initials: V
authors.lastname: Matozzo
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000187:drug effects*; D006434:Hemocytes / Q000187:drug effects; D053768:Metal Nanoparticles / Q000633:toxicity*; D046529:Microscopy, Electron, Transmission; D010316:Particle Size; D010587:Phagocytosis / Q000187:drug effects*; D014025:Titanium / Q000633:toxicity*; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D014874:Water Pollutants, Chemical; C009495:titanium dioxide; D014025:Titanium
keywords: Bivalve; Haemocyte; Internalisation; Nanoparticles; Phagocytosis; Titanium dioxide; Transmission electron microscope