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Is the toxic potential of nanosilver dependent on its size?

Huk, Anna, Izak-Nau, Emilia, Reidy, Bogumila, Boyles, Matthew, Duschl, Albert, Lynch, Iseult, Dušinska, Maria·2014·Particle and Fibre Toxicology
DOI 10.1186/s12989-014-0065-1PMC PMC4274708
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Figure 1 TEM characterization of pristine Ag ENMs : (A) Ag ENMs 200 nm , (B) Ag ENMs 80 nm, ( C) Ag ENMs 50 nm; scale bar : 1 μm.
Figure 1 TEM characterization of pristine Ag ENMs : (A) Ag ENMs 200 nm , (B) Ag ENMs 80 nm, ( C) Ag ENMs 50 nm; scale bar : 1 μm.CC BY · PubMed Central
Figure 2 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).
Figure 2 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).CC BY · PubMed Central
Figure 3 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.
Figure 3 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.CC BY · PubMed Central
Figure 4 Induction of IL -8 and MCP- 1 in A549 cells exposed to Ag ENMs 50, 80 and 200 nm. Cells were treated with 6 concentrations (μg/cm2) of Ag ENMs for 24 h. The data are expressed as mean ± SD of at least three independent experiments. *statistically significant (p < 0.05) difference from the unexposed control. Horizontal line represents expressed level of IL8 (81.8 ± 31.5 pg/ml) and MCP-1 (143.3 ± 26.68 pg/ml) in untreated cells. TNFα (20 ng/ml) as a positive control (IL-8) gave 386.5 ± 87.92 pg/ml and (MCP-1) gave 528.3 ± 134.52 pg/ml.
Figure 4 Induction of IL -8 and MCP- 1 in A549 cells exposed to Ag ENMs 50, 80 and 200 nm. Cells were treated with 6 concentrations (μg/cm2) of Ag ENMs for 24 h. The data are expressed as mean ± SD of at least three independent experiments. *statistically significant (p < 0.05) difference from the unexposed control. Horizontal line represents expressed level of IL8 (81.8 ± 31.5 pg/ml) and MCP-1 (143.3 ± 26.68 pg/ml) in untreated cells. TNFα (20 ng/ml) as a positive control (IL-8) gave 386.5 ± 87.92 pg/ml and (MCP-1) gave 528.3 ± 134.52 pg/ml.CC BY · PubMed Central
Figure 5 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/ cm 2 ) 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.
Figure 5 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/ cm 2 ) 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.CC BY · PubMed Central
Figure 6 Effect of 24 h treatment with 50, 80 and 200 nm Ag ENMs (0.21 -15.6 μg/ cm 2 ) on induction of hprt gene 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%).
Figure 6 Effect of 24 h treatment with 50, 80 and 200 nm Ag ENMs (0.21 -15.6 μg/ cm 2 ) on induction of hprt gene 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%).CC BY · PubMed Central

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Authors
Huk, A., Izak-Nau, E., Reidy, B., Boyles, M., Duschl, A., Lynch, I., & Dušinska, M.
Venue
Particle and Fibre Toxicology
Publisher
Springer Science and Business Media LLC
Published
2014-12
Volume
11
Issue
1
ISSN
1743-8977
DOI
10.1186/s12989-014-0065-1
PMC
PMC4274708
Cited by
73
References
2
Language
en
Licence
creativecommons.org/licenses/by/4.0 (unspecified)

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