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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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Effect of 24 h treatment with 50, 80 and 200 nm Ag ENMs (0.21-15.6 μg/cm2) on induction ofhprtgene mutations in V79-4 cells (bar graphs, left hand scale). The mutant frequencies (x106) are expressed as the mean ± SD of two independent experiments, with two independent harvests per experiment. Horizontal line shows hprt gene mutant frequency in untreated cells (8.47 ± 3.83). MMS (0.03 μM, 30 min), a positive control gave 80.03 ± 22.13 hprt gene mutations. Significant difference from unexposed control (*p < 0.05, **p < 0.01, ***p < 0,001). Cytotoxic effects – Plating efficiency (PE) (horizontal marker line, right hand scale) is expressed as the mean of two independent experiments. Results represent cytotoxicity relative to 100% of the negative control. Cytotoxicity of MMS has not been observed (PE = 97.99 ± 6.14%).
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Fig6: Effect of 24 h treatment with 50, 80 and 200 nm Ag ENMs (0.21-15.6 μg/cm2) on induction ofhprtgene mutations in V79-4 cells (bar graphs, left hand scale). The mutant frequencies (x106) are expressed as the mean ± SD of two independent experiments, with two independent harvests per experiment. Horizontal line shows hprt gene mutant frequency in untreated cells (8.47 ± 3.83). MMS (0.03 μM, 30 min), a positive control gave 80.03 ± 22.13 hprt gene mutations. Significant difference from unexposed control (*p < 0.05, **p < 0.01, ***p < 0,001). Cytotoxic effects – Plating efficiency (PE) (horizontal marker line, right hand scale) is expressed as the mean of two independent experiments. Results represent cytotoxicity relative to 100% of the negative control. Cytotoxicity of MMS has not been observed (PE = 97.99 ± 6.14%).

Mentions: The mutagenic potential of 50, 80, 200 nm Ag ENMs was examined using the hprt gene mutation assay according to OECD guideline 476. Two independent experiments, each with two mutation harvests, were performed (Figure 6A). Ag ENM 200 nm were observed to be the most mutagenic (p < 0.001), at all concentrations tested. The data are presented in mass units; however, this pattern holds true when plotted as number of ENMs, or by surface area. We observed the highest frequency of hprt mutants in the first experiment; mutant frequency was 9.6 ± 5.25 times higher than the negative control. The frequency of induced mutants in several groups was comparable with the mutant frequency found with the positive control (MMS). For cells treated with Ag ENMs 50 and 80 nm there was a trend towards higher mutant frequency of 80 nm ENMs compared with 50 nm; however, this was not significant.Figure 6


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)

Effect of 24 h treatment with 50, 80 and 200 nm Ag ENMs (0.21-15.6 μg/cm2) on induction ofhprtgene mutations in V79-4 cells (bar graphs, left hand scale). The mutant frequencies (x106) are expressed as the mean ± SD of two independent experiments, with two independent harvests per experiment. Horizontal line shows hprt gene mutant frequency in untreated cells (8.47 ± 3.83). MMS (0.03 μM, 30 min), a positive control gave 80.03 ± 22.13 hprt gene mutations. Significant difference from unexposed control (*p < 0.05, **p < 0.01, ***p < 0,001). Cytotoxic effects – Plating efficiency (PE) (horizontal marker line, right hand scale) is expressed as the mean of two independent experiments. Results represent cytotoxicity relative to 100% of the negative control. Cytotoxicity of MMS has not been observed (PE = 97.99 ± 6.14%).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Effect of 24 h treatment with 50, 80 and 200 nm Ag ENMs (0.21-15.6 μg/cm2) on induction ofhprtgene mutations in V79-4 cells (bar graphs, left hand scale). The mutant frequencies (x106) are expressed as the mean ± SD of two independent experiments, with two independent harvests per experiment. Horizontal line shows hprt gene mutant frequency in untreated cells (8.47 ± 3.83). MMS (0.03 μM, 30 min), a positive control gave 80.03 ± 22.13 hprt gene mutations. Significant difference from unexposed control (*p < 0.05, **p < 0.01, ***p < 0,001). Cytotoxic effects – Plating efficiency (PE) (horizontal marker line, right hand scale) is expressed as the mean of two independent experiments. Results represent cytotoxicity relative to 100% of the negative control. Cytotoxicity of MMS has not been observed (PE = 97.99 ± 6.14%).
Mentions: The mutagenic potential of 50, 80, 200 nm Ag ENMs was examined using the hprt gene mutation assay according to OECD guideline 476. Two independent experiments, each with two mutation harvests, were performed (Figure 6A). Ag ENM 200 nm were observed to be the most mutagenic (p < 0.001), at all concentrations tested. The data are presented in mass units; however, this pattern holds true when plotted as number of ENMs, or by surface area. We observed the highest frequency of hprt mutants in the first experiment; mutant frequency was 9.6 ± 5.25 times higher than the negative control. The frequency of induced mutants in several groups was comparable with the mutant frequency found with the positive control (MMS). For cells treated with Ag ENMs 50 and 80 nm there was a trend towards higher mutant frequency of 80 nm ENMs compared with 50 nm; however, this was not significant.Figure 6

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