title: Negligible cytotoxicity induced by different titanium dioxide nanoparticles in fish cell lines.
abstract: Titanium dioxide nanoparticles (TiO2-NPs) have a wide number of applications in cosmetic, solar and paint industries due to their photocatalyst and ultraviolet blocking properties. The continuous increase in the production of TiO2-NPs enhances the risk for this manufactured nanomaterial to enter water bodies through treated effluents or agricultural amendments. TiO2-NPs have shown very low toxicity in a number of aquatic organisms. However, there are no conclusive data about their deleterious effects and on their possible mechanisms of toxic action. At this level, in vitro cell culture systems are a useful tool to gain insight about processes underlying the toxicity of a wide variety of substances, including nanomaterials. Differences in the physiology of different taxa make advisable the use of cells coming from the taxon of interest, but collecting data from a variety of cellular types allows a better understanding of the studied processes. Taking all this into account, the aim of the present study was to assess the toxicity of three types of TiO2-NP, rutile hydrophobic (NM-103), rutile hydrophilic (NM-104) and rutile-anatase (NM-105), obtained from the EU Joint Research Centre (JRC) repository, using various fish cell lines (RTG-2, PLHC-1, RTH-149, RTL-W1) and rainbow trout primary hepatocytes. For comparative purposes, the effect of different dispersion protocols, end-point assays and extended exposure time was studied in a fish cell line (RTG-2) and in the rat hepatoma cell line (H4IIE). TiO2-NPs dispersions showed a variable degree of aggregation in cell culture media. Disruption of mitochondrial metabolic activity, plasma membrane integrity and lysosome function was not detected in any cell line after exposure to TiO2-NPs at any time and concentration ranges tested. These results are indicative of a low toxicity of the TiO2-NPs tested and show the usefulness of fish cells maintained in vitro as high throughput screening methods that can facilitate further testing in the framework of integrated testing strategies.
authors.affiliation: Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Departamento de Medio Ambiente, Ambiente, Carretera de la Coruña, Km 7.5, 28040 Madrid, Spain. Electronic address: bermejo.azucena@inia.es.
authors.forename: Azucena
authors.initials: A
authors.lastname: Bermejo-Nogales
authors.affiliation: Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Departamento de Medio Ambiente, Ambiente, Carretera de la Coruña, Km 7.5, 28040 Madrid, Spain; Heriott-Watt University, School of Life Sciences, Edinburgh, UK. Electronic address: mc29@hw.ac.uk.
authors.forename: Mona
authors.initials: M
authors.lastname: Connolly
authors.affiliation: Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Departamento de Medio Ambiente, Ambiente, Carretera de la Coruña, Km 7.5, 28040 Madrid, Spain. Electronic address: prosenkranz@gmx.net.
authors.forename: Philipp
authors.initials: P
authors.lastname: Rosenkranz
authors.affiliation: Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Departamento de Medio Ambiente, Ambiente, Carretera de la Coruña, Km 7.5, 28040 Madrid, Spain. Electronic address: fcruz@inia.es.
authors.forename: María-Luisa
authors.initials: ML
authors.lastname: Fernández-Cruz
authors.affiliation: Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Departamento de Medio Ambiente, Ambiente, Carretera de la Coruña, Km 7.5, 28040 Madrid, Spain. Electronic address: jmnavas@inia.es.
authors.forename: José M
authors.initials: JM
authors.lastname: Navas
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects*; D022781:Hepatocytes; D057927:Hydrophobic and Hydrophilic Interactions; D008247:Lysosomes / Q000187:drug effects*; D053768:Metal Nanoparticles / Q000633:toxicity*; D008928:Mitochondria / Q000187:drug effects*; D017686:Oncorhynchus mykiss; D051381:Rats; D014025:Titanium / Q000633:toxicity*
chemical_list: C009495:titanium dioxide; D014025:Titanium
keywords: Cytotoxicity; Hepatocyte; Nanomaterials; Rainbow trout; Titanium dioxide; Topminnow fish