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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

Effects on immune cells (RAW 264.7 murine macrophages).Comparative evaluation of cell viability by resazurin assay (A and B) and NRU assay (C and D) after exposure to ZnO (NM-110 and NM-111). Cells were grown for 24h in complete growth medium and then exposed for 24h, 48h and 72h to different concentrations of ZnO NMs. At the end of the incubation, cell viability was assessed using two different assays (resazurin and NRU). Data are means ± SD of 10 independent determinations in two separate experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post-hoc test. *p< 0.05, **p< 0.01, ***p< 0.001.
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pone.0127174.g005: Effects on immune cells (RAW 264.7 murine macrophages).Comparative evaluation of cell viability by resazurin assay (A and B) and NRU assay (C and D) after exposure to ZnO (NM-110 and NM-111). Cells were grown for 24h in complete growth medium and then exposed for 24h, 48h and 72h to different concentrations of ZnO NMs. At the end of the incubation, cell viability was assessed using two different assays (resazurin and NRU). Data are means ± SD of 10 independent determinations in two separate experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post-hoc test. *p< 0.05, **p< 0.01, ***p< 0.001.

Mentions: A higher toxicity of ZnO (NM-110 and NM-111) was observed in macrophages. The exposure of HMDM to ZnO NMs for 24h induced a dose-dependent decrease in cell viability (Fig 4). However, the IC50 values were always >100 μg/ml for both NMs. We also detected increased levels of TNF-α in the HMDM culture medium (Fig. A in S2 File) after exposure to concentrations between 5 and 50 μg/ml of NM-110 and between 1 and 25 μg/ml of NM-111. The loss of cell viability at higher doses could prevent the cytokine secretion. The RAW 264.7 cells (Fig 5) showed a marked loss of viability after exposure to ZnO NMs for 24 to 72h, which was dose- but not time-dependent. The IC50 values were between 10–25 μg/ml. We observed a complete loss of viability at concentrations ≥64 μg/ml for NM-110 and ≥32 μg/ml for NM-111. Similar results (IC50 = 15–25 μg/ml) were registered also in MH-S cells (Fig 6) with a complete loss of viability observed (resazurin assay) already after 24h of exposure to concentrations ≥32 μg/ml of both NM-110 (uncoated) and NM-111 (coated). The negative correlation between the time-points and the NM concentration observed in some cases could be explained by differences in the doses delivered to the cells in function of time or perhaps by the proliferation rate of the cells at the time-points evaluated. In other cases (i.e., for NM-111) the correlations were contradictory between the two cytotoxicity assays (NRU and resazurin). Thus, it seems a peculiar effect of the assay, highlighting the importance of applying multiple assays for testing of NMs.


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)

Effects on immune cells (RAW 264.7 murine macrophages).Comparative evaluation of cell viability by resazurin assay (A and B) and NRU assay (C and D) after exposure to ZnO (NM-110 and NM-111). Cells were grown for 24h in complete growth medium and then exposed for 24h, 48h and 72h to different concentrations of ZnO NMs. At the end of the incubation, cell viability was assessed using two different assays (resazurin and NRU). Data are means ± SD of 10 independent determinations in two separate experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post-hoc test. *p< 0.05, **p< 0.01, ***p< 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127174.g005: Effects on immune cells (RAW 264.7 murine macrophages).Comparative evaluation of cell viability by resazurin assay (A and B) and NRU assay (C and D) after exposure to ZnO (NM-110 and NM-111). Cells were grown for 24h in complete growth medium and then exposed for 24h, 48h and 72h to different concentrations of ZnO NMs. At the end of the incubation, cell viability was assessed using two different assays (resazurin and NRU). Data are means ± SD of 10 independent determinations in two separate experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post-hoc test. *p< 0.05, **p< 0.01, ***p< 0.001.
Mentions: A higher toxicity of ZnO (NM-110 and NM-111) was observed in macrophages. The exposure of HMDM to ZnO NMs for 24h induced a dose-dependent decrease in cell viability (Fig 4). However, the IC50 values were always >100 μg/ml for both NMs. We also detected increased levels of TNF-α in the HMDM culture medium (Fig. A in S2 File) after exposure to concentrations between 5 and 50 μg/ml of NM-110 and between 1 and 25 μg/ml of NM-111. The loss of cell viability at higher doses could prevent the cytokine secretion. The RAW 264.7 cells (Fig 5) showed a marked loss of viability after exposure to ZnO NMs for 24 to 72h, which was dose- but not time-dependent. The IC50 values were between 10–25 μg/ml. We observed a complete loss of viability at concentrations ≥64 μg/ml for NM-110 and ≥32 μg/ml for NM-111. Similar results (IC50 = 15–25 μg/ml) were registered also in MH-S cells (Fig 6) with a complete loss of viability observed (resazurin assay) already after 24h of exposure to concentrations ≥32 μg/ml of both NM-110 (uncoated) and NM-111 (coated). The negative correlation between the time-points and the NM concentration observed in some cases could be explained by differences in the doses delivered to the cells in function of time or perhaps by the proliferation rate of the cells at the time-points evaluated. In other cases (i.e., for NM-111) the correlations were contradictory between the two cytotoxicity assays (NRU and resazurin). Thus, it seems a peculiar effect of the assay, highlighting the importance of applying multiple assays for testing of NMs.

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