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Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy.

Farcal L, Torres Andón F, Di Cristo L, Rotoli BM, Bussolati O, Bergamaschi E, Mech A, Hartmann NB, Rasmussen K, Riego-Sintes J, Ponti J, Kinsner-Ovaskainen A, Rossi F, Oomen A, Bos P, Chen R, Bai R, Chen C, Rocks L, Fulton N, Ross B, Hutchison G, Tran L, Mues S, Ossig R, Schnekenburger J, Campagnolo L, Vecchione L, Pietroiusti A, Fadeel B - PLoS ONE (2015)

Bottom Line: Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (NM-200) and pyrogenic (NM-203).Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'.A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103).

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.

No MeSH data available.


Related in: MedlinePlus

Heat map representation of IC50 values.The IC50 values of the six representative NMs in different cellular systems calculated at different time points (24 to 72h or 10 days). The heat map shows a higher toxicity of ZnO NMs, followed by SiO2 and TiO2. The highest sensitivity after short term exposure to NMs was noted in the case of murine alveolar macrophages (MH-S) while Calu-3 were the most resistant cells. The long term exposure (10 days) of NIH3T3 and mES cells to NMs induced also significant cytotoxic effect.
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pone.0127174.g003: Heat map representation of IC50 values.The IC50 values of the six representative NMs in different cellular systems calculated at different time points (24 to 72h or 10 days). The heat map shows a higher toxicity of ZnO NMs, followed by SiO2 and TiO2. The highest sensitivity after short term exposure to NMs was noted in the case of murine alveolar macrophages (MH-S) while Calu-3 were the most resistant cells. The long term exposure (10 days) of NIH3T3 and mES cells to NMs induced also significant cytotoxic effect.

Mentions: The cytotoxicity results obtained after the exposure of these cell systems to all six NMs allowed us to calculate the IC50 for each NM and to compare the results obtained at different time points, between different cellular systems and with different assays (Fig 3). In addition, a hazard ranking of these NMs could be established. The concentrations tested in these studies were generally between 1 and 100 μg/ml, with lower and higher limits at 0.125 and 200 μg/ml, respectively. Therefore, the concentrations to induce maximum effect (100% cell death) could not always be established, as the administration of too high doses of NMs could in some cases cause interferences with the assays. Furthermore, we have used the IC50 index in order to establish a hazard ranking of NMs. For this reason we have divided the results in six categories of toxicity, depending on the calculated IC50 value: <10, 10.1–30.0, 30.1–50.0, 50.1–70.0, 70.1–100 and >100 μg/ml. These IC50 values were calculated from the cell viability data by GraphPad Prism Software. The biological assays applied in this study allowed also the evaluation of different cytotoxicity mechanisms by assessing the damage of cell membrane (i.e. lactate dehydrogenase (LDH) assay), lysosomal integrity (Neutral Red Uptake) or the cellular metabolism (i.e. resazurin assay, water soluble tetrazolium—WST-1 and WST-8 assays). In addition to these colorimetric assays, we have evaluated the cytotoxicity and cytostatic effects of NMs by performing the colony forming efficiency (CFE) assay in Caco-2 cells after long term/repeated exposure. The transepithelial electrical resistance (TEER) of Calu-3 and Caco-2 cells was measured in order to evaluate epithelial barrier damages caused by exposure to NMs. The potential toxicity of all six NMs on embryonic tissues was also evaluated, by performing the Embryonic Stem Cell Test (EST). Prior to the toxicity testing, the evaluation of lipopolysaccharides (LPS) contamination using the conventional Limulus Amebocyte Lysate (LAL) chromogenic assay was performed for all NMs; the results showed that the LPS values for all samples were below the maximum admissible limit of 0.5 EU/ml [24, 25]. We also evaluated the possible interferences between the NMs and the assays reagents or the readouts and we did, indeed, observe some interference especially in the case of TiO2 NMs. However, this was found to not affect the outcome of the toxicity assays performed.


Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy.

Farcal L, Torres Andón F, Di Cristo L, Rotoli BM, Bussolati O, Bergamaschi E, Mech A, Hartmann NB, Rasmussen K, Riego-Sintes J, Ponti J, Kinsner-Ovaskainen A, Rossi F, Oomen A, Bos P, Chen R, Bai R, Chen C, Rocks L, Fulton N, Ross B, Hutchison G, Tran L, Mues S, Ossig R, Schnekenburger J, Campagnolo L, Vecchione L, Pietroiusti A, Fadeel B - PLoS ONE (2015)

Heat map representation of IC50 values.The IC50 values of the six representative NMs in different cellular systems calculated at different time points (24 to 72h or 10 days). The heat map shows a higher toxicity of ZnO NMs, followed by SiO2 and TiO2. The highest sensitivity after short term exposure to NMs was noted in the case of murine alveolar macrophages (MH-S) while Calu-3 were the most resistant cells. The long term exposure (10 days) of NIH3T3 and mES cells to NMs induced also significant cytotoxic effect.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440714&req=5

pone.0127174.g003: Heat map representation of IC50 values.The IC50 values of the six representative NMs in different cellular systems calculated at different time points (24 to 72h or 10 days). The heat map shows a higher toxicity of ZnO NMs, followed by SiO2 and TiO2. The highest sensitivity after short term exposure to NMs was noted in the case of murine alveolar macrophages (MH-S) while Calu-3 were the most resistant cells. The long term exposure (10 days) of NIH3T3 and mES cells to NMs induced also significant cytotoxic effect.
Mentions: The cytotoxicity results obtained after the exposure of these cell systems to all six NMs allowed us to calculate the IC50 for each NM and to compare the results obtained at different time points, between different cellular systems and with different assays (Fig 3). In addition, a hazard ranking of these NMs could be established. The concentrations tested in these studies were generally between 1 and 100 μg/ml, with lower and higher limits at 0.125 and 200 μg/ml, respectively. Therefore, the concentrations to induce maximum effect (100% cell death) could not always be established, as the administration of too high doses of NMs could in some cases cause interferences with the assays. Furthermore, we have used the IC50 index in order to establish a hazard ranking of NMs. For this reason we have divided the results in six categories of toxicity, depending on the calculated IC50 value: <10, 10.1–30.0, 30.1–50.0, 50.1–70.0, 70.1–100 and >100 μg/ml. These IC50 values were calculated from the cell viability data by GraphPad Prism Software. The biological assays applied in this study allowed also the evaluation of different cytotoxicity mechanisms by assessing the damage of cell membrane (i.e. lactate dehydrogenase (LDH) assay), lysosomal integrity (Neutral Red Uptake) or the cellular metabolism (i.e. resazurin assay, water soluble tetrazolium—WST-1 and WST-8 assays). In addition to these colorimetric assays, we have evaluated the cytotoxicity and cytostatic effects of NMs by performing the colony forming efficiency (CFE) assay in Caco-2 cells after long term/repeated exposure. The transepithelial electrical resistance (TEER) of Calu-3 and Caco-2 cells was measured in order to evaluate epithelial barrier damages caused by exposure to NMs. The potential toxicity of all six NMs on embryonic tissues was also evaluated, by performing the Embryonic Stem Cell Test (EST). Prior to the toxicity testing, the evaluation of lipopolysaccharides (LPS) contamination using the conventional Limulus Amebocyte Lysate (LAL) chromogenic assay was performed for all NMs; the results showed that the LPS values for all samples were below the maximum admissible limit of 0.5 EU/ml [24, 25]. We also evaluated the possible interferences between the NMs and the assays reagents or the readouts and we did, indeed, observe some interference especially in the case of TiO2 NMs. However, this was found to not affect the outcome of the toxicity assays performed.

Bottom Line: Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (NM-200) and pyrogenic (NM-203).Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'.A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103).

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.

No MeSH data available.


Related in: MedlinePlus