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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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Related in: MedlinePlus

Uptake of: (1) Ag ENMs 50 nm: (A) in cytoplasmic vesicle after 2 h exposure; (B) in close proximity to chromosomes/chromatin in mitotic cells after 24 h exposure, (C) outside the cell/associated with cellular debris (48 h exposure); (2) Ag ENMs 80 nm: (A) in cytoplasmic vesicle after 24 h exposure, (B) in lysosomes after 48 h exposure; (C) in nucleus after 48 h; (3) Ag ENMs 200 nm (A) in cytoplasmic vesicles after 2 h exposure; (B) in lysosomes after 24 h exposure, (C) in vesicles localised close to the nuclear membrane (after 24 h exposure).
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Fig2: Uptake of: (1) Ag ENMs 50 nm: (A) in cytoplasmic vesicle after 2 h exposure; (B) in close proximity to chromosomes/chromatin in mitotic cells after 24 h exposure, (C) outside the cell/associated with cellular debris (48 h exposure); (2) Ag ENMs 80 nm: (A) in cytoplasmic vesicle after 24 h exposure, (B) in lysosomes after 48 h exposure; (C) in nucleus after 48 h; (3) Ag ENMs 200 nm (A) in cytoplasmic vesicles after 2 h exposure; (B) in lysosomes after 24 h exposure, (C) in vesicles localised close to the nuclear membrane (after 24 h exposure).

Mentions: The uptake and subcellular localisation of the Ag ENMs was confirmed using TEM. Firstly, the images confirmed that ENMs of all three sizes tested were taken up by A549 cells and retained their particulate nature over the exposure duration of 48 hours (Figure 2). Ag ENMs 50 nm (Figure 2.1A) were rapidly taken up by cells, and after just 2 h of exposure the ENMs were found inside the cells in dark-coloured vesicles, presumably lysosomes. Some single ENMs were localised in vesicles, very close to the cell surface. Some of those vesicles can be interpreted as endocytotic, clathrin-coated vesicles, which indicates an active mechanism of Ag ENM uptake. After 24 and 48 h exposure, Ag ENMs 50 nm were observed to form larger clusters in lysosomes (Figure 2.1B,C). The Ag ENMs 50 nm were not observed to interact directly with mitochondria in any of the tens of images assessed, although in some cases vesicles containing Ag ENMs 50 nm were found in close proximity to those organelles. Ag ENMs 50 nm were not found inside the nucleus, although in the case of mitotic cells, where the nuclear membrane was disintegrated, they were found close to the chromosomes/chromatin. Ag ENMs 80 nm (Figure 2.2A) were also found in lysosomes after 2 h of exposure, although many ENMs (more than in the case of Ag ENMs 50 nm) were found in small, presumably endocytic vesicles. After 24 h exposure, a large number of Ag ENMs 80 nm was observed in lysosomes, where they formed big clusters. After 48 h, most ENMs were located in lysosomes, although in one case ENMs were observed within the nucleus. A smaller quantity of Ag ENMs 200 nm were observed inside the cells compared to cells treated with Ag ENMs 50 and 80 nm, consistent with literature for other ENMs but also likely related to the lower numbers of ENMs exposed at constant mass. After 2 h of exposure, Ag ENMs 200 nm were found in cytoplasmic vesicles or on the cell surface. After 24 h (Figures 2.3B,C) exposure, Ag ENMs 200 nm were found in cytoplasmic vesicles. No direct interactions with other organelles were observed. However, some vesicles containing ENMs were found close to the nuclear membrane. After 48 h, much fewer ENMs 200 nm were found inside the cells compared to the smaller sized ENMs. ENMs were still localised mainly in cytoplasmic vesicles (not shown).Figure 2


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)

Uptake of: (1) Ag ENMs 50 nm: (A) in cytoplasmic vesicle after 2 h exposure; (B) in close proximity to chromosomes/chromatin in mitotic cells after 24 h exposure, (C) outside the cell/associated with cellular debris (48 h exposure); (2) Ag ENMs 80 nm: (A) in cytoplasmic vesicle after 24 h exposure, (B) in lysosomes after 48 h exposure; (C) in nucleus after 48 h; (3) Ag ENMs 200 nm (A) in cytoplasmic vesicles after 2 h exposure; (B) in lysosomes after 24 h exposure, (C) in vesicles localised close to the nuclear membrane (after 24 h exposure).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Uptake of: (1) Ag ENMs 50 nm: (A) in cytoplasmic vesicle after 2 h exposure; (B) in close proximity to chromosomes/chromatin in mitotic cells after 24 h exposure, (C) outside the cell/associated with cellular debris (48 h exposure); (2) Ag ENMs 80 nm: (A) in cytoplasmic vesicle after 24 h exposure, (B) in lysosomes after 48 h exposure; (C) in nucleus after 48 h; (3) Ag ENMs 200 nm (A) in cytoplasmic vesicles after 2 h exposure; (B) in lysosomes after 24 h exposure, (C) in vesicles localised close to the nuclear membrane (after 24 h exposure).
Mentions: The uptake and subcellular localisation of the Ag ENMs was confirmed using TEM. Firstly, the images confirmed that ENMs of all three sizes tested were taken up by A549 cells and retained their particulate nature over the exposure duration of 48 hours (Figure 2). Ag ENMs 50 nm (Figure 2.1A) were rapidly taken up by cells, and after just 2 h of exposure the ENMs were found inside the cells in dark-coloured vesicles, presumably lysosomes. Some single ENMs were localised in vesicles, very close to the cell surface. Some of those vesicles can be interpreted as endocytotic, clathrin-coated vesicles, which indicates an active mechanism of Ag ENM uptake. After 24 and 48 h exposure, Ag ENMs 50 nm were observed to form larger clusters in lysosomes (Figure 2.1B,C). The Ag ENMs 50 nm were not observed to interact directly with mitochondria in any of the tens of images assessed, although in some cases vesicles containing Ag ENMs 50 nm were found in close proximity to those organelles. Ag ENMs 50 nm were not found inside the nucleus, although in the case of mitotic cells, where the nuclear membrane was disintegrated, they were found close to the chromosomes/chromatin. Ag ENMs 80 nm (Figure 2.2A) were also found in lysosomes after 2 h of exposure, although many ENMs (more than in the case of Ag ENMs 50 nm) were found in small, presumably endocytic vesicles. After 24 h exposure, a large number of Ag ENMs 80 nm was observed in lysosomes, where they formed big clusters. After 48 h, most ENMs were located in lysosomes, although in one case ENMs were observed within the nucleus. A smaller quantity of Ag ENMs 200 nm were observed inside the cells compared to cells treated with Ag ENMs 50 and 80 nm, consistent with literature for other ENMs but also likely related to the lower numbers of ENMs exposed at constant mass. After 2 h of exposure, Ag ENMs 200 nm were found in cytoplasmic vesicles or on the cell surface. After 24 h (Figures 2.3B,C) exposure, Ag ENMs 200 nm were found in cytoplasmic vesicles. No direct interactions with other organelles were observed. However, some vesicles containing ENMs were found close to the nuclear membrane. After 48 h, much fewer ENMs 200 nm were found inside the cells compared to the smaller sized ENMs. ENMs were still localised mainly in cytoplasmic vesicles (not shown).Figure 2

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