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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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Level of DNA damage – strand breaks (A, B) and oxidised DNA lesions expressed as NET FPG (C, D) in A549 cells exposed to different concentrations of Ag ENMs (μg/cm2) with sizes 50, 80, 200 nm for 2 h (A, C) and 24 h (B, D). NET FPG was estimated as FPG-sensitive sites minus strand breaks, representing altered purines. Horizontal lines represent level of strands breaks (SBs)/oxidised bases (NET FPG) in untreated cells (A: 1.36 ± 0.84; B: 0.76 ± 0.5; C: 1.99 ± 1.7; D: 3.3 ± 1.56% tail DNA). The data are expressed as mean ± SD of three independent experiments. *significant (p < 0.05) difference from the unexposed control. Hydrogen peroxide (50 μM, 5 min in PBS), a positive control (SBs) gave 38.72 ± 6.1% tail DNA. Photosensitiser Ro19-8022 (1 μM in PBS, 5 min, on ice), a positive control for oxidised DNA lesions (NET FPG) gave 33.2 ± 10.96% tail DNA.
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Fig5: Level of DNA damage – strand breaks (A, B) and oxidised DNA lesions expressed as NET FPG (C, D) in A549 cells exposed to different concentrations of Ag ENMs (μg/cm2) with sizes 50, 80, 200 nm for 2 h (A, C) and 24 h (B, D). NET FPG was estimated as FPG-sensitive sites minus strand breaks, representing altered purines. Horizontal lines represent level of strands breaks (SBs)/oxidised bases (NET FPG) in untreated cells (A: 1.36 ± 0.84; B: 0.76 ± 0.5; C: 1.99 ± 1.7; D: 3.3 ± 1.56% tail DNA). The data are expressed as mean ± SD of three independent experiments. *significant (p < 0.05) difference from the unexposed control. Hydrogen peroxide (50 μM, 5 min in PBS), a positive control (SBs) gave 38.72 ± 6.1% tail DNA. Photosensitiser Ro19-8022 (1 μM in PBS, 5 min, on ice), a positive control for oxidised DNA lesions (NET FPG) gave 33.2 ± 10.96% tail DNA.

Mentions: The standard alkaline Comet assay was employed for detection of single and double strand breaks, and a modified version with FPG was used to detect oxidized DNA lesions (Figure 5). A significant concentration response was observed at all time points and for all ENMs tested. The strongest effect was observed in cells treated with 50 nm Ag ENMs at all tested concentrations, and our data demonstrated that the genotoxic potential of Ag ENMs is both concentration and size dependent. No statistically significant difference was found between the level of DNA damage induced by Ag ENMs 80 and 200 nm at any exposure time (Figure 5A and B or Figure 5C and D). An increased level of oxidised DNA lesions was also observed in all treated groups (Figure 5C and D), but again the strongest effect was demonstrated in cells exposed to the smallest ENMs at the shortest exposure time. A significant level of DNA oxidation was already seen in cells exposed at the lowest concentration (1.1 μg/cm2) of Ag ENM 50 nm. The high genotoxic response to ENMs 50 nm can be coupled with the higher number of ENMs compared to the number of 80 nm and 200 nm ENMs. Re-calculating the data from mass units [μg/cm2] to number of ENMs [ENMs*1011/cm2] or surface area of ENMs [ENMs cm2/cm2] showed that Ag 50 nm ENMs are the most genotoxic only at short time exposures – 2 h, but for longer treatment times the highest DNA damaging effect was observed with 200 nm Ag ENMs. Supplementary figure (Additional file 4: Figure S7 and S8).Figure 5


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)

Level of DNA damage – strand breaks (A, B) and oxidised DNA lesions expressed as NET FPG (C, D) in A549 cells exposed to different concentrations of Ag ENMs (μg/cm2) with sizes 50, 80, 200 nm for 2 h (A, C) and 24 h (B, D). NET FPG was estimated as FPG-sensitive sites minus strand breaks, representing altered purines. Horizontal lines represent level of strands breaks (SBs)/oxidised bases (NET FPG) in untreated cells (A: 1.36 ± 0.84; B: 0.76 ± 0.5; C: 1.99 ± 1.7; D: 3.3 ± 1.56% tail DNA). The data are expressed as mean ± SD of three independent experiments. *significant (p < 0.05) difference from the unexposed control. Hydrogen peroxide (50 μM, 5 min in PBS), a positive control (SBs) gave 38.72 ± 6.1% tail DNA. Photosensitiser Ro19-8022 (1 μM in PBS, 5 min, on ice), a positive control for oxidised DNA lesions (NET FPG) gave 33.2 ± 10.96% tail DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Level of DNA damage – strand breaks (A, B) and oxidised DNA lesions expressed as NET FPG (C, D) in A549 cells exposed to different concentrations of Ag ENMs (μg/cm2) with sizes 50, 80, 200 nm for 2 h (A, C) and 24 h (B, D). NET FPG was estimated as FPG-sensitive sites minus strand breaks, representing altered purines. Horizontal lines represent level of strands breaks (SBs)/oxidised bases (NET FPG) in untreated cells (A: 1.36 ± 0.84; B: 0.76 ± 0.5; C: 1.99 ± 1.7; D: 3.3 ± 1.56% tail DNA). The data are expressed as mean ± SD of three independent experiments. *significant (p < 0.05) difference from the unexposed control. Hydrogen peroxide (50 μM, 5 min in PBS), a positive control (SBs) gave 38.72 ± 6.1% tail DNA. Photosensitiser Ro19-8022 (1 μM in PBS, 5 min, on ice), a positive control for oxidised DNA lesions (NET FPG) gave 33.2 ± 10.96% tail DNA.
Mentions: The standard alkaline Comet assay was employed for detection of single and double strand breaks, and a modified version with FPG was used to detect oxidized DNA lesions (Figure 5). A significant concentration response was observed at all time points and for all ENMs tested. The strongest effect was observed in cells treated with 50 nm Ag ENMs at all tested concentrations, and our data demonstrated that the genotoxic potential of Ag ENMs is both concentration and size dependent. No statistically significant difference was found between the level of DNA damage induced by Ag ENMs 80 and 200 nm at any exposure time (Figure 5A and B or Figure 5C and D). An increased level of oxidised DNA lesions was also observed in all treated groups (Figure 5C and D), but again the strongest effect was demonstrated in cells exposed to the smallest ENMs at the shortest exposure time. A significant level of DNA oxidation was already seen in cells exposed at the lowest concentration (1.1 μg/cm2) of Ag ENM 50 nm. The high genotoxic response to ENMs 50 nm can be coupled with the higher number of ENMs compared to the number of 80 nm and 200 nm ENMs. Re-calculating the data from mass units [μg/cm2] to number of ENMs [ENMs*1011/cm2] or surface area of ENMs [ENMs cm2/cm2] showed that Ag 50 nm ENMs are the most genotoxic only at short time exposures – 2 h, but for longer treatment times the highest DNA damaging effect was observed with 200 nm Ag ENMs. Supplementary figure (Additional file 4: Figure S7 and S8).Figure 5

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