S282 Abstracts / Toxicology Letters 196S (2010) S37–S351 Recent literature revealed that some specific nanomaterials interact with these assays because they interfere with absorp- tion/fluorescence measurements. Nevertheless, these assays are still commonly used cytotoxicity assessment techniques in nan- otoxicology. In this study, we tested the cytotoxicity of bare and hyaluronic acid coated gold nanoparticles by means of WST-1 and LDH assays in porcine proximal tubule cells (LLC-PK1) and human hepatocar- cinoma cells (Hep G2) which represents potential target organs following systemic administration. An increase in absorbance values of the LDH assay of hyaluronic acid coated gold was observed in the most concentrated samples, which indicates a possible positive response, but no reduction in the WST- 1 assay associated absorbance was noted at assayed con- centrations, indicating a non toxic response. Although media containing nanoparticles is removed in WST-1 assay, we suspected that cells had internalized gold nanoparti- cles interfering in the absorbance readings. We confirm that cells internalized bare and coated gold nanoparticles (or were strongly adhered in the cell membrane) in an uptake assay measured by ICP-MS quantification. We also carried out the evaluation with flu- orescent microscopy. So, no conclusive results can be obtained nor with LDH nor with WST-1 assays, and, a cellular testing method which does not employ absorbance measurement should be developed to assess cytotoxicity of this kind of engineered nanoparticles. This work has been funded by the Spanish Ministry of Science and Innovation NANOSOST project PSE-420000-2008-WP3. doi:10.1016/j.toxlet.2010.03.1152 P303-032 Cellular toxicity and haemolytic potential of functionalized fullerene and multi-walled carbon nanotubes R. Bonafè, S. Bussi, A. Maiocchi Bracco Imaging SpA, Italy Nanomaterials find a wide range of applications in a variety of prod- ucts thanks to their extremely small size and high surface area. They are capable of entering the human body by inhalation, ingestion, skin penetration, intravenous injections or medical devices, and have the potential to interact with intracellular macromolecules with possible implication in human health. Our work was focused on an early toxicological screening based on in vitro cytotoxicity (MTT and LDH assays) and haemolysis tests (human and rat blood) performed on functionalized nanomaterials. In vitro cytotoxicity was assayed on six cell lines: HepG2, NHDF, HUVEC, CaCo2, J774 A1, and U937. Cytotoxicity test performed on the selected cell lines always revealed a stronger effect for ShortMWCNT-PEI and a moderate effect for the remaining compounds, except for Full-Peg10000 that did not showed any toxic sign. At MTT test, Short MWCNT- Peg5000, Short MWCNT-Peg10000, Short MWCNT-COOH, and Short MWCNT-OH showed a slight to moderate toxicity on all cell lines except on J774 A1, where the effects were more severe, and U937 where the effects were slight. Haemolysis was mea- sured by spectrophotometrical reading of plasma cleared from nanoparticles by centrifugation on filtering plates. In accordance with haemolytic potential classification no haemolytic effect was observed on human blood; only ShortMWCNT-COOH revealed a slight effect on rat blood, correlating with test ranking on human blood. It can be concluded that functionalized nanomaterials, except fullerenes, delineated a complex toxicity pattern, which can be summarized in a severe cellular toxicity of Short MWCNT-PEI, not correlating with haemolytic data. doi:10.1016/j.toxlet.2010.03.1153 P303-033 Comparative toxicity of copper and titanium oxide nanoparticles on human alveolar epithelial cells P. Mantecca 1 , E. Moschini 1 , M. Gualtieri 1 , E. Longhin 1 , U. Fascio 2 , M. Camatini 1 1 Università Milano Bicocca, Italy, 2 Università Milano, Italy Metal oxide NPs, abundantly used in industrial nanotech devices, have been previously tested in vitro. Besides, lack of information exists in associating their cytotoxicity to the differential capacity to interact with cell structures. In this research human pulmonary cells, A549, were exposed to CuO and TiO 2 NPs to analyse their effects on cell viability and cell cycle, and their oxidative potential. Ultrastructural analysis evi- denced cell–particle interactions and morphological modifications. Laser scanning microscopy (LSM), by probing organelles, mem- branes, ROS and nuclei, was able to couple NPs biological effects with their intracellular localization by confocal reflection contrast. TiO 2 produced not significant effects, while CuO induced heavy cytotoxicity. Cell viability diminished in dose- and time-dependent manner, oxidative stress and the frequency of cells arrested in G2/M transition phase significantly augmented, as confirmed by the altered microtubules assembly, which failed to form the spin- dle. The soluble fraction of CuO NP suspensions did not induce comparable effects. Such strong cytotoxic and genotoxic responses to CuO did not seem related to the amount of NP internalized in cells, as shown by LSM. TiO 2 abundantly adhered and pene- trated the cells in clusters ranging in hundreds nm. Such clusters generated at cell membrane level and were usually internalized by phagocytic machinery, and they were always found associated to the membrane systems. CuO showed high biological reactivity, and cell membranes displayed instability and lyses. CuO was often observed in cells in a very finely dispersed manner, while clusters were detected only rarely. At ultrastructural level CuO NPs were present in membrane bound vacuoles, free in the cytoplasm, in mitochondria and sporadically in the nuclei too. CuO and TiO 2 behaved similarly in water suspension, but greatly differed when in the presence of cells: their different surface reactivity and internalization processes brought to different bio- chemical responses and toxicological outputs. doi:10.1016/j.toxlet.2010.03.1154 P303-034 Behavior of gold nanoparticles coated with hyaluronan: Citotoxicity, cell internalization and rat biodistribution M. Borras 1 , J. Sendra 2 , C. Di Guglielmo 1 , H. Parkkola 2 , M. Ramis 2 , J. De Lapuente 1 , C. Porredon 1 1 UTOX, United States, 2 ENDOR Nanotechnologies, Spain The properties of nanoconjugates make them of biological and medical interest as local nanocarriers for diagnostic and drug deliv- ery. To better understand their behavior with biological organisms and encompass them in a broader project, we designed 12 nm gold nanoparticles coated with 5 kDa MW hyaluronan (Au-HA). We studied their citotoxicity by WST-1 assay, their cell internalization and their biodistribution.