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Toxicity of metallic nanoparticles to algae is mediated by agglomeration and reactive oxygen species formation
Abstracts / Toxicology Letters 238S (2015) S56–S383
P08-057 Toxicity of metallic nanoparticles to algae is mediated by agglomeration and reactive oxygen species formation V. Aruoja ∗ , M. Sihtmäe, A. Kahru National Institute of Chemical Physics and Biophysics, Laboratory of Environmental Toxicology, Tallinn, Estonia Increasing use of metal containing nanoparticles (NP) in consumer products leads to their discharge into aquatic environments with potentially harmful consequences. The toxicity of metallic NPs has been associated with bioavailable metal ions leaching from the particles (eg in the case of ZnO, CuO, Ag), however some of the metal oxide and metal NPs are highly toxic to aquatic organisms despite no evidence of dissolution. In this study a thoroughly characterized library of NPs consisting of ZnO, Pd, CuO, Co3 O4 , TiO2 , Mn3 O4 , Fe3 O4 , Al2 O3 , SiO2 , WO3 , MgO and Sb2 O3 was analyzed in terms of toxicity to the microalga Pseudokirchneriella subcapitata. Heteroagglomerates of nanoparticles and algal cells that form during the algal growth inhibition assay were investigated using fluorescence microscopy and the potential of NPs to create reactive oxygen species (ROS) was determined using fluorescent probes 2,7-dichlorofluorescein diacetate and 3 -(p-hydroxyphenyl) florescein. Most of the studied NPs formed heteroagglomerates causing entrapment and flocculation of most or all of the algal cells. The NPs that consistently produced ROS in abiotic conditions (TiO2 , Mn3 O, Co3 O4 , Pd, CuO, Fe3 O4 ) were the most toxic to algae. The results suggest a causal relationship between ROS production and toxicity that is enhanced by the close contact of NP-s with algal cells in the heteroagglomerates. This research was supported by the IUT 23-5 project and the EU 7th Framework Programme under Grant Agreement No. 309314 (MODERN). http://dx.doi.org/10.1016/j.toxlet.2015.08.637
P08-058 In vitro toxicity screening of silica-coated superparamagnetic iron oxide nanoparticles in glial cells C. Costa 1,2,∗ , F. Brandão 1 , M.J. Bessa 1 , S. Costa 1,2 , V. Valdiglesias 3 , G. Kilic¸ 3,4 , N. Fernández-Bertólez 3,4 , E. Pásaro 3 , B. Laffon 3 , J. Teixeira 1,2 1
Portuguese National Institute of Health, Environmental Health Department, Porto, Portugal 2 EPIUnit - Institute of Public Health, Porto, Portugal 3 Universidade da Coru˜ na, DICOMOSA Group, A Coru˜ na, Spain 4 Universidade da Coru˜ na, Department of Cell and Molecular Biology, A Coru˜ na, Spain Nanotechnology industry is progressing with prospects of substantial benefits to economics and science. Superparamagnetic iron oxide nanoparticles (ION) have been showing excellent magnetic properties, biocompatibility and biodegradability, broadening their potential applications and importance in the biomedical field. Nevertheless, there are increasing concerns as to the potential adverse effects on human health and environment and, currently, data on the effects of ION on the human nervous system are controversial. The present study aims to evaluate cytotoxicity of silica-coated ION in human glioblastoma (A172) cell line by using the classical in vitro cytotoxicity assays, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red uptake
S215
(NRU) and Alamar Blue (AB). Glioblastoma cells were exposed to different concentrations of ION (range 5–300 g/ml), prepared in either complete or incomplete (serum-free) cell culture medium for three exposure times (3, 6, 24 h). In addition, ION were characterized for their size and zeta potential by DLS (dynamic light scattering). The mean hydrodynamic diameter of silica-coated ION (0.3 mg/ml) in water, complete medium, and incomplete medium was 93.3 nm, 222.6 nm and 185.7 nm, respectively. A decrease in viability of A172 cells was observed at all studied times of exposure assessed by MTT, NRU and AB assay, after exposure to silicacoated ION in incomplete medium. Similar results were obtained in complete medium although, in this condition, observed cell viability was consistently higher than that observed after exposure in incomplete medium. Results presented here suggest that silicacoated ION can cause adverse cellular effects on glioblastoma cells in certain conditions and, in consequence, further studies on this matter are now in progress to understand the underlying mechanism for the cytotoxicity observed. Acknowledgments: This work was supported by Xunta de Galicia (EM 2012/079), the project NanoToxClass (ERA ERASIINN/001/2013) [funded by FCT/MCTES (PIDDAC) and co-funded by the European Regional Development Fund (ERDF) through the COMPETE Programme], and by TD1204 MODENA COST Action. http://dx.doi.org/10.1016/j.toxlet.2015.08.638
P08-059 Cell type dependence of Si/SiO2 quantum dots uptake and toxicity L. Stanca 1,∗ , A.I. Serban 1 , A. Dinischiotu 2 1
University of Agronomic Sciences and Veterinary Medicine, Preclinical Sciences, Bucharest, Romania 2 University of Bucharest, Biochemistry and Molecular Biology, Bucharest, Romania Question: The intense fluorescence emission and photobleaching resistance make quantum dots (QDs) promising tools for live-cell imaging, although, at present toxicity issues limit their use. While Si/SiO2 QDs possess lower elemental toxicity compared to other types of QDs, their interaction with cells might generate unexpected cytotoxicity, which we investigated. Materials and methods: Cells were exposed to 5 nm Si/SiO2 QDs for 6 and 24 h and viability was tested by MTT. Cell morphology was observed by fluorescence and bright field microscopy. SOD 1 and Nrf2 protein expression were assessed by immunoblot, while intracellular superoxide radical and extracellular LDH levels were quantified by colorimetric assays. Results: Dose response curves for cell viability with maximal QDs doses up to the saturating effect for each cell line (60% viability for HepG2 and 11.5% for RAW 264.7) showed that IC50 after 24 h were 111 g/ml QDs and 15.3 g/ml for HepG2 and RAW 264.7 cells respectively. Further experiments were performed using the IC50 for each cell line. Although QDs surrounded the HepG2 clusters, microscopic analysis revealed no apparent uptake, while RAW 264.7 internalized the QDs, which were observed as perinuclear aggregates. Vacuolization (after 6 h), multinucleated osteoclast-like giant cells and necrosis (after 24 h) were observed in RAW 264.7 cultures, whereas in HepG2 the stress fibers displayed diffuse staining, suggesting microfilaments disorganization. Superoxide radical levels increased 1.83 and 1.2 fold after 24 h in RAW 264.7 and HepG2 cells respectively. The expression of 61 kDa Nrf2 increased after 6 h of QDs exposure in RAW 264.7 and after 24 h in HepG2 cells, while the ubiquitinated 100 kDa form was detected only in QDs exposed macrophages. SOD 1 expression
P08-057 Toxicity of metallic nanoparticles to algae is mediated by agglomeration and reactive oxygen species formation V. Aruoja ∗ , M. Sihtmäe, A. Kahru National Institute of Chemical Physics and Biophysics, Laboratory of Environmental Toxicology, Tallinn, Estonia Increasing use of metal containing nanoparticles (NP) in consumer products leads to their discharge into aquatic environments with potentially harmful consequences. The toxicity of metallic NPs has been associated with bioavailable metal ions leaching from the particles (eg in the case of ZnO, CuO, Ag), however some of the metal oxide and metal NPs are highly toxic to aquatic organisms despite no evidence of dissolution. In this study a thoroughly characterized library of NPs consisting of ZnO, Pd, CuO, Co3 O4 , TiO2 , Mn3 O4 , Fe3 O4 , Al2 O3 , SiO2 , WO3 , MgO and Sb2 O3 was analyzed in terms of toxicity to the microalga Pseudokirchneriella subcapitata. Heteroagglomerates of nanoparticles and algal cells that form during the algal growth inhibition assay were investigated using fluorescence microscopy and the potential of NPs to create reactive oxygen species (ROS) was determined using fluorescent probes 2,7-dichlorofluorescein diacetate and 3 -(p-hydroxyphenyl) florescein. Most of the studied NPs formed heteroagglomerates causing entrapment and flocculation of most or all of the algal cells. The NPs that consistently produced ROS in abiotic conditions (TiO2 , Mn3 O, Co3 O4 , Pd, CuO, Fe3 O4 ) were the most toxic to algae. The results suggest a causal relationship between ROS production and toxicity that is enhanced by the close contact of NP-s with algal cells in the heteroagglomerates. This research was supported by the IUT 23-5 project and the EU 7th Framework Programme under Grant Agreement No. 309314 (MODERN). http://dx.doi.org/10.1016/j.toxlet.2015.08.637
P08-058 In vitro toxicity screening of silica-coated superparamagnetic iron oxide nanoparticles in glial cells C. Costa 1,2,∗ , F. Brandão 1 , M.J. Bessa 1 , S. Costa 1,2 , V. Valdiglesias 3 , G. Kilic¸ 3,4 , N. Fernández-Bertólez 3,4 , E. Pásaro 3 , B. Laffon 3 , J. Teixeira 1,2 1
Portuguese National Institute of Health, Environmental Health Department, Porto, Portugal 2 EPIUnit - Institute of Public Health, Porto, Portugal 3 Universidade da Coru˜ na, DICOMOSA Group, A Coru˜ na, Spain 4 Universidade da Coru˜ na, Department of Cell and Molecular Biology, A Coru˜ na, Spain Nanotechnology industry is progressing with prospects of substantial benefits to economics and science. Superparamagnetic iron oxide nanoparticles (ION) have been showing excellent magnetic properties, biocompatibility and biodegradability, broadening their potential applications and importance in the biomedical field. Nevertheless, there are increasing concerns as to the potential adverse effects on human health and environment and, currently, data on the effects of ION on the human nervous system are controversial. The present study aims to evaluate cytotoxicity of silica-coated ION in human glioblastoma (A172) cell line by using the classical in vitro cytotoxicity assays, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red uptake
S215
(NRU) and Alamar Blue (AB). Glioblastoma cells were exposed to different concentrations of ION (range 5–300 g/ml), prepared in either complete or incomplete (serum-free) cell culture medium for three exposure times (3, 6, 24 h). In addition, ION were characterized for their size and zeta potential by DLS (dynamic light scattering). The mean hydrodynamic diameter of silica-coated ION (0.3 mg/ml) in water, complete medium, and incomplete medium was 93.3 nm, 222.6 nm and 185.7 nm, respectively. A decrease in viability of A172 cells was observed at all studied times of exposure assessed by MTT, NRU and AB assay, after exposure to silicacoated ION in incomplete medium. Similar results were obtained in complete medium although, in this condition, observed cell viability was consistently higher than that observed after exposure in incomplete medium. Results presented here suggest that silicacoated ION can cause adverse cellular effects on glioblastoma cells in certain conditions and, in consequence, further studies on this matter are now in progress to understand the underlying mechanism for the cytotoxicity observed. Acknowledgments: This work was supported by Xunta de Galicia (EM 2012/079), the project NanoToxClass (ERA ERASIINN/001/2013) [funded by FCT/MCTES (PIDDAC) and co-funded by the European Regional Development Fund (ERDF) through the COMPETE Programme], and by TD1204 MODENA COST Action. http://dx.doi.org/10.1016/j.toxlet.2015.08.638
P08-059 Cell type dependence of Si/SiO2 quantum dots uptake and toxicity L. Stanca 1,∗ , A.I. Serban 1 , A. Dinischiotu 2 1
University of Agronomic Sciences and Veterinary Medicine, Preclinical Sciences, Bucharest, Romania 2 University of Bucharest, Biochemistry and Molecular Biology, Bucharest, Romania Question: The intense fluorescence emission and photobleaching resistance make quantum dots (QDs) promising tools for live-cell imaging, although, at present toxicity issues limit their use. While Si/SiO2 QDs possess lower elemental toxicity compared to other types of QDs, their interaction with cells might generate unexpected cytotoxicity, which we investigated. Materials and methods: Cells were exposed to 5 nm Si/SiO2 QDs for 6 and 24 h and viability was tested by MTT. Cell morphology was observed by fluorescence and bright field microscopy. SOD 1 and Nrf2 protein expression were assessed by immunoblot, while intracellular superoxide radical and extracellular LDH levels were quantified by colorimetric assays. Results: Dose response curves for cell viability with maximal QDs doses up to the saturating effect for each cell line (60% viability for HepG2 and 11.5% for RAW 264.7) showed that IC50 after 24 h were 111 g/ml QDs and 15.3 g/ml for HepG2 and RAW 264.7 cells respectively. Further experiments were performed using the IC50 for each cell line. Although QDs surrounded the HepG2 clusters, microscopic analysis revealed no apparent uptake, while RAW 264.7 internalized the QDs, which were observed as perinuclear aggregates. Vacuolization (after 6 h), multinucleated osteoclast-like giant cells and necrosis (after 24 h) were observed in RAW 264.7 cultures, whereas in HepG2 the stress fibers displayed diffuse staining, suggesting microfilaments disorganization. Superoxide radical levels increased 1.83 and 1.2 fold after 24 h in RAW 264.7 and HepG2 cells respectively. The expression of 61 kDa Nrf2 increased after 6 h of QDs exposure in RAW 264.7 and after 24 h in HepG2 cells, while the ubiquitinated 100 kDa form was detected only in QDs exposed macrophages. SOD 1 expression