The knowledge on potential harmful effects of metallic nanomaterials lags behind their increased use in consumer products and therefore, the safety data on various nanomaterials applicable for risk assessment are urgently needed. In this study, 11 metal oxide nanoparticles (MeOx NPs) prepared using flame pyrolysis method were analyzed for their toxicity against human alveolar epithelial cells A549, human epithelial colorectal cells Caco2 and murine fibroblast cell line Balb/c 3T3. The cell lines were exposed for 24 h to suspensions of 3-100 μg/mL MeOx NPs and cellular viability was evaluated using. Neutral Red Uptake (NRU) assay. In parallel to NPs, toxicity of soluble salts of respective metals was analyzed, to reveal the possible cellular effects of metal ions shedding from the NPs. The potency of MeOx to produce reactive oxygen species was evaluated in the cell-free assay. The used three cell lines showed comparable toxicity responses to NPs and their metal ion counterparts in the current test setting. Six MeOx NPs (Al2O3, Fe3O4, MgO, SiO2, TiO2, WO3) did not show toxic effects below 100 µg/mL. For five MeOx NPs, the averaged 24 h IC50 values for the three mammalian cell lines were 16.4 µg/mL for CuO, 22.4 µg/mL for ZnO, 57.3 µg/mL for Sb2O3, 132.3 µg/mL for Mn3O4 and 129 µg/mL for Co3O4. Comparison of the dissolution level of MeOx and the toxicity of soluble salts allowed to conclude that the toxicity of CuO, ZnO and Sb2O3 NPs was driven by release of metal ions. The toxic effects of Mn3O4 and Co3O4 could be attributed to the ROS-inducing ability of these NPs. All the NPs were internalized by the cells according to light microscopy studies but also proven by TEM, and internalization of Co3O4 NPs seemed to be most prominent in this aspect. In conclusion, this work provides valuable toxicological data for a library of 11 MeOx NPs. Combining the knowledge on toxic or non-toxic nature of nanomaterials may be used for safe-by-design approach.
The use of metal based nanoparticles (NPs) is increasing which leads to their release in water bodies via various waste streams and warrants risk assessment. Consistent biological effect data on NPs for environmentally relevant test species that are accompanied by thorough characterization of NPs are scarce but indispensable for understanding possible risks of NPs. We composed and tested a library of 12 metal-based nanoparticles () using alga Pseudokirchneriella subcapitata, three bacterial species (Vibrio fischeri, Escherichia coli, Staphylococcus aureus) and protozoan Tetrahymena thermophila. The NPs were characterized for physico-chemical properties, solubility and abiotic reactive oxygen species (ROS) production. Also, respective soluble salts were analysed for toxic effects. The algal growth inhibition assay proved the most sensitive and yielded EC 50 values for 10 NPs ranging from 0.1 to 58 mg/l. Algal toxicity correlated with abiotic ROS production of NPs and the majority of NPs formed agglomerates that entrapped algal cells. Despite of different sensitivity, there was a common trend in the toxicity of NPs across different species and test formats: CuO and ZnO had highest toxicity (EC 50 values below 1 mg/l) to all organism groups except protozoa. The high toxicity was mostly due to shedding of toxic concentrations of Zn and Cu ions; for most of the test species Al 2 O 3 , SiO 2 , WO 3 and Sb 2 O 3 were not toxic below 100 mg/l and MgO showed no adverse effect below 100 mg/l to any test species in any test setting. Nano impactThe lack of good quality nanotoxicity data for environmentally relevant test species accompanied by physico-chemical characterization of nanoparticles (NPs) severely hampers risk assessment. This study provides biological response data of a thoroughly characterized library of metal-based NPs using algal, bacterial and protozoan tests. It includes correlations between NP properties and toxicity, as well as results obtained in deionized water, thus eliminating medium-specific effects. Similar trends across species are shown, although algae proved the most sensitive. EC 50 values of 10 NPs ranged from 0.1 to 58 mg/l. The values may be used for toxicity modelling or directly for risk assessment, as the 72 h algal growth inhibition data are mandatory for the registration of chemicals in the European Union.EC 50 -half effective concentration; MBC -Minimal Biocidal Concentration, i.e., the lowest tested nominal concentration of NPs which completely inhibited the formation of visible colonies after sub-culturing on toxicant-free agarised growth medium. Prior subculturing bacteria were incubated with NPs for 24 h at 25 °C in deionised water.
IntroductionSilver nanoparticles (AgNP) are widely used in consumer products and in medicine, mostly due to their excellent antimicrobial properties. One of the generally accepted antibacterial mechanisms of AgNP is their efficient contact with cells and dissolution in the close vicinity of bacterial cell envelope. Yet, the primary mechanism of cell wall damage and the events essential for bactericidal action of AgNP are not elucidated.Materials and methodsIn this study we used a combination of various assays to differentiate the adverse effects of AgNP on bacterial cell envelope: outer membrane (OM) and plasma membrane (PM).ResultsWe showed that PM was the main target of AgNP in gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa: AgNP depolarized PM, induced the leakage of the intracellular K+, and inhibited cellular respiration. The results of bacterial bioluminescence inhibition assay in combination with AgNP dissolution and oxidation assays demonstrated that the adverse effects of AgNP occurred at concentrations 7–160 µM. These toxic effects occurred already within the first few seconds of contact of bacteria and AgNP and were driven by dissolved Ag+ ions targeting bacterial PM. However, the irreversible inhibition of bacterial growth detected after 1-hour exposure occurred at 40 µM AgNP for P. aeruginosa and at 320 µM AgNP for E. coli. In contrast to effects on PM, AgNP and Ag+ ions had no significant effect on the permeability and integrity of bacterial OM, implying that AgNP indeed targeted mainly PM via dissolved Ag+ ions.ConclusionAgNP exhibited antibacterial properties via rapid release of Ag+ ions targeting the PM and not the OM of gram-negative bacteria.
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