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Is the toxic potential of nanosilver dependent on its size?

Huk A, Izak-Nau E, Reidy B, Boyles M, Duschl A, Lynch I, Dušinska M - Part Fibre Toxicol (2014)

Bottom Line: However, re-calculation of Ag ENMs concentrations from mass unit to surface area and number of ENMs per cm2 highlighted that 200 nm Ag ENMs, are the most toxic.Strong cytotoxic and genotoxic effects were observed in cells exposed to Ag ENMs 50 nm, but Ag ENMs 200 nm had the most mutagenic potential.Additionally, we showed that expression of concentrations of ENMs in mass units is not representative.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Nanosilver is one of the most commonly used engineered nanomaterials (ENMs). In our study we focused on assessing the size-dependence of the toxicity of nanosilver (Ag ENMs), utilising materials of three sizes (50, 80 and 200 nm) synthesized by the same method, with the same chemical composition, charge and coating.

Methods: Uptake and localisation (by Transmission Electron Microscopy), cell proliferation (Relative growth activity) and cytotoxic effects (Plating efficiency), inflammatory response (induction of IL-8 and MCP-1 by Enzyme linked immune sorbent assay), DNA damage (strand breaks and oxidised DNA lesions by the Comet assay) were all assessed in human lung carcinoma epithelial cells (A549), and the mutagenic potential of ENMs (Mammalian hprt gene mutation test) was assessed in V79-4 cells as per the OECD protocol. Detailed physico-chemical characterization of the ENMs was performed in water and in biological media as a prerequisite to assessment of their impacts on cells. To study the relationship between the surface area of the ENMs and the number of ENMs with the biological response observed, Ag ENMs concentrations were recalculated from μg/cm2 to ENMs cm2/cm2 and ENMs/cm2.

Results: Studied Ag ENMs are cytotoxic and cytostatic, and induced strand breaks, DNA oxidation, inflammation and gene mutations. Results expressed in mass unit [μg/cm2] suggested that the toxicity of Ag ENMs is size dependent with 50 nm being most toxic. However, re-calculation of Ag ENMs concentrations from mass unit to surface area and number of ENMs per cm2 highlighted that 200 nm Ag ENMs, are the most toxic. Results from hprt gene mutation assay showed that Ag ENMs 200 nm are the most mutagenic irrespective of the concentration unit expressed.

Conclusion: We found that the toxicity of Ag ENMs is not always size dependent. Strong cytotoxic and genotoxic effects were observed in cells exposed to Ag ENMs 50 nm, but Ag ENMs 200 nm had the most mutagenic potential. Additionally, we showed that expression of concentrations of ENMs in mass units is not representative. Number of ENMs or surface area of ENMs (per cm2) seem more precise units with which to compare the toxicity of different ENMs.

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Cytotoxic effects of 50, 80 and 200 nm Ag ENMs on A549 cells measured as Relative growth activity (RGA) and Plating efficiency (PE). Relative growth activity (Upper figures): Cells were treated with 5 concentrations (μg/cm2) of Ag ENMs for 2, 24 and 48 h and cell number was counted immediately following staining. Plating efficiency (Lower figures): Cells were treated with 5 concentrations (μg/cm2) of Ag ENMs for 2, 24 and 48 h. Immediately after the exposure 100 cells per dish was inoculated and the number of cell clones was calculated after 10 days of incubation. Note that ENMs that had been internalised during the initial exposure remained in the cells and were diluted only by cell division over the 10 days. Columns represent cytotoxicity relative to 100% of control. The data are expressed as mean ± SD of three independent experiments. *a statistically significant (p < 0.05) difference from the unexposed (control) cells.
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Fig3: Cytotoxic effects of 50, 80 and 200 nm Ag ENMs on A549 cells measured as Relative growth activity (RGA) and Plating efficiency (PE). Relative growth activity (Upper figures): Cells were treated with 5 concentrations (μg/cm2) of Ag ENMs for 2, 24 and 48 h and cell number was counted immediately following staining. Plating efficiency (Lower figures): Cells were treated with 5 concentrations (μg/cm2) of Ag ENMs for 2, 24 and 48 h. Immediately after the exposure 100 cells per dish was inoculated and the number of cell clones was calculated after 10 days of incubation. Note that ENMs that had been internalised during the initial exposure remained in the cells and were diluted only by cell division over the 10 days. Columns represent cytotoxicity relative to 100% of control. The data are expressed as mean ± SD of three independent experiments. *a statistically significant (p < 0.05) difference from the unexposed (control) cells.

Mentions: The effect of Ag ENMs with 3 different sizes and varying concentrations on cellular toxicity and proliferation was examined after 2, 24, 48 h exposure using PE and RGA. Data are presented with respect to the control cells that had no Ag ENM treatment (negative control). A clear concentration response was observed for all tested ENMs. On a mass basis, Ag ENMs 50 nm were considered the most cytotoxic of the tested materials (Figure 3), already evident during the 2 h exposure period in both PE and RGA tests. Conversely, expressing the data as ENMs/cm2 or ENMs cm2/cm2, we observed the reverse trend, where from the three tested materials, Ag ENMs 200 nm gave the highest toxic response in A549 cells (Additional file 4: Figures S5 and S6). To highlight this ambiguity in dosimetry, an IC50 was calculated for each system, and is summarized in Table 2. There were no statistically significant differences between IC50 values calculated in mass units [μg/cm2]. However, IC50 values (RGA) expressed as number of ENMs [ENMs*1011/cm2] or surface area of ENMs [ENMs cm2/cm2] were found to be several-fold less for Ag ENMs 200 nm compared with Ag ENMs 50 and 80 nm. Thus, a smaller number of the larger Ag ENMs can induce 50% impedance of cell proliferation compared with ENMs with sizes 50 and 80 nm.Figure 3


Is the toxic potential of nanosilver dependent on its size?

Huk A, Izak-Nau E, Reidy B, Boyles M, Duschl A, Lynch I, Dušinska M - Part Fibre Toxicol (2014)

Cytotoxic effects of 50, 80 and 200 nm Ag ENMs on A549 cells measured as Relative growth activity (RGA) and Plating efficiency (PE). Relative growth activity (Upper figures): Cells were treated with 5 concentrations (μg/cm2) of Ag ENMs for 2, 24 and 48 h and cell number was counted immediately following staining. Plating efficiency (Lower figures): Cells were treated with 5 concentrations (μg/cm2) of Ag ENMs for 2, 24 and 48 h. Immediately after the exposure 100 cells per dish was inoculated and the number of cell clones was calculated after 10 days of incubation. Note that ENMs that had been internalised during the initial exposure remained in the cells and were diluted only by cell division over the 10 days. Columns represent cytotoxicity relative to 100% of control. The data are expressed as mean ± SD of three independent experiments. *a statistically significant (p < 0.05) difference from the unexposed (control) cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4274708&req=5

Fig3: Cytotoxic effects of 50, 80 and 200 nm Ag ENMs on A549 cells measured as Relative growth activity (RGA) and Plating efficiency (PE). Relative growth activity (Upper figures): Cells were treated with 5 concentrations (μg/cm2) of Ag ENMs for 2, 24 and 48 h and cell number was counted immediately following staining. Plating efficiency (Lower figures): Cells were treated with 5 concentrations (μg/cm2) of Ag ENMs for 2, 24 and 48 h. Immediately after the exposure 100 cells per dish was inoculated and the number of cell clones was calculated after 10 days of incubation. Note that ENMs that had been internalised during the initial exposure remained in the cells and were diluted only by cell division over the 10 days. Columns represent cytotoxicity relative to 100% of control. The data are expressed as mean ± SD of three independent experiments. *a statistically significant (p < 0.05) difference from the unexposed (control) cells.
Mentions: The effect of Ag ENMs with 3 different sizes and varying concentrations on cellular toxicity and proliferation was examined after 2, 24, 48 h exposure using PE and RGA. Data are presented with respect to the control cells that had no Ag ENM treatment (negative control). A clear concentration response was observed for all tested ENMs. On a mass basis, Ag ENMs 50 nm were considered the most cytotoxic of the tested materials (Figure 3), already evident during the 2 h exposure period in both PE and RGA tests. Conversely, expressing the data as ENMs/cm2 or ENMs cm2/cm2, we observed the reverse trend, where from the three tested materials, Ag ENMs 200 nm gave the highest toxic response in A549 cells (Additional file 4: Figures S5 and S6). To highlight this ambiguity in dosimetry, an IC50 was calculated for each system, and is summarized in Table 2. There were no statistically significant differences between IC50 values calculated in mass units [μg/cm2]. However, IC50 values (RGA) expressed as number of ENMs [ENMs*1011/cm2] or surface area of ENMs [ENMs cm2/cm2] were found to be several-fold less for Ag ENMs 200 nm compared with Ag ENMs 50 and 80 nm. Thus, a smaller number of the larger Ag ENMs can induce 50% impedance of cell proliferation compared with ENMs with sizes 50 and 80 nm.Figure 3

Bottom Line: However, re-calculation of Ag ENMs concentrations from mass unit to surface area and number of ENMs per cm2 highlighted that 200 nm Ag ENMs, are the most toxic.Strong cytotoxic and genotoxic effects were observed in cells exposed to Ag ENMs 50 nm, but Ag ENMs 200 nm had the most mutagenic potential.Additionally, we showed that expression of concentrations of ENMs in mass units is not representative.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Nanosilver is one of the most commonly used engineered nanomaterials (ENMs). In our study we focused on assessing the size-dependence of the toxicity of nanosilver (Ag ENMs), utilising materials of three sizes (50, 80 and 200 nm) synthesized by the same method, with the same chemical composition, charge and coating.

Methods: Uptake and localisation (by Transmission Electron Microscopy), cell proliferation (Relative growth activity) and cytotoxic effects (Plating efficiency), inflammatory response (induction of IL-8 and MCP-1 by Enzyme linked immune sorbent assay), DNA damage (strand breaks and oxidised DNA lesions by the Comet assay) were all assessed in human lung carcinoma epithelial cells (A549), and the mutagenic potential of ENMs (Mammalian hprt gene mutation test) was assessed in V79-4 cells as per the OECD protocol. Detailed physico-chemical characterization of the ENMs was performed in water and in biological media as a prerequisite to assessment of their impacts on cells. To study the relationship between the surface area of the ENMs and the number of ENMs with the biological response observed, Ag ENMs concentrations were recalculated from μg/cm2 to ENMs cm2/cm2 and ENMs/cm2.

Results: Studied Ag ENMs are cytotoxic and cytostatic, and induced strand breaks, DNA oxidation, inflammation and gene mutations. Results expressed in mass unit [μg/cm2] suggested that the toxicity of Ag ENMs is size dependent with 50 nm being most toxic. However, re-calculation of Ag ENMs concentrations from mass unit to surface area and number of ENMs per cm2 highlighted that 200 nm Ag ENMs, are the most toxic. Results from hprt gene mutation assay showed that Ag ENMs 200 nm are the most mutagenic irrespective of the concentration unit expressed.

Conclusion: We found that the toxicity of Ag ENMs is not always size dependent. Strong cytotoxic and genotoxic effects were observed in cells exposed to Ag ENMs 50 nm, but Ag ENMs 200 nm had the most mutagenic potential. Additionally, we showed that expression of concentrations of ENMs in mass units is not representative. Number of ENMs or surface area of ENMs (per cm2) seem more precise units with which to compare the toxicity of different ENMs.

Show MeSH
Related in: MedlinePlus