Fig 1.
Characterization of AgNPs using transmission electron microscopy (TEM).
(A) A representative microscopy image shows shape of AgNPs. (B) The histogram illustrates the range of particle size distribution with the mean size of 18.3 nm obtained from TEM measurements of 200 particles.
Table 1.
The summary characteristics of AgNPs.
Fig 2.
The TEM examination of hFOB 1.19.
(A) The ultrastructural features of a control cell; mitochondria (M), nucleus (N), Golgi complex (Ga); (B-C) Cells exposed to AgNPs 30 μg/mL for 24 h. cell. (B) AgNPs are found within the cell (arrows). Micrographs show vacuoles (V) with randomly oriented AgNPs of quite regular morphology (arrows). The well-developed Golgi complex (Ga) is located in the cytoplasm in perinuclear areas. (C) Cells exposed to AgNPs 30 μg/mL for 24 h show swelling of the endoplasmic reticulum. Ribosomes may have dissociated from the endoplasmic reticulum (RER) and the cytoplasm is filled with free ribosomes. The nucleus (N) presents a normal aspect.
Fig 3.
The ultrastructural alterations of hFOB 1.19 cells after exposure to 30 or 60 μg/mL AgNPs for 48 h.
(A) Cells show ultrastructural changes and formation of multiple blebs. Enlarged nucleus (N), pushing the cytoplasm towards the periphery. (B) The cell exposed to 30 μg/mL AgNPs for 48 h contains double-membraned autophagic vacuoles—autophagolysosomes with engulfed organelles displaying degenerative changes (arrowheads). Autophagosome (asterisk) and lysosome (arrow) are indicated. Note condensed mitochondria and disorganization of the inner membrane system. (C) Apoptosis in cells exposed to 60 μg/mL AgNPs for 48 h. A decrease in cell volume and chromatin condensation show induction of apoptosis in the osteoblast on a right side of figure. Note a loss of microvilli and formation protrusions from the surface of plasma membrane—known as blebs. The autolytic vacuoles are visible in the cytoplasm. Features of necrotic lysis, such as disintegration of cytoplasmic membrane, electron-lucent nuclear chromatin, heavily vacuolized cytoplasm (arrows) and lack of organelles are prominent.
Fig 4.
Evaluation of cytotoxicity of AgNPs and Ag released in cell medium from 30 and 60 μg/mL AgNPs on hFOB 1.19 cells.
Results are expressed as % LDH release by cells into the culture medium and presented as mean ± standard deviation of 3 independent experiment. ***p<0.001 exposed cells v/s control.
Fig 5.
Pretreatment of hFOB 1.19 with L-NIL attenuated AgNPs-induced cell death.
A representative dot plot of flow cytometry. A representative dot plot of flow cytometry. AgNPs induced apoptosis and necrosis in hFOB 1.19 cells an effect attenuated by L-NIL. Viable cells are shown in the lower left field (low Annexin V and PI staining; AV- PI-). The lower right field (AV+ PI-) represents the apoptotic cells, and the higher right field (AV+ PI+) indicates late apoptotic/necrotic cells. The higher left field (AV- PI+) shows the dead cells. L-NIL significantly attenuated number of apoptotic and dead cells.
Fig 6.
AgNPs-induced apoptosis in hFOB 1.19 cells and protective effect of L-NIL.
(A) AgNPs-induced apoptosis, (B) Bax, Bcl-2 protein and (C) Bax, Bcl-2 mRNA levels. L-NIL significantly attenuated: (A) number of apoptotic cells, (B) Bax protein, (C) mRNA level and significantly increased: (B) Bcl-2 protein, (C) mRNA level. Data are expressed as means ± SD of 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 exposed cells v/s control or as indicated.
Fig 7.
AgNPs-induced necrosis in hFOB 1.19 cells and protective effect of L-NIL.
Nec-1 failed to protect cells from AgNPs-induced cell death. Data are expressed as means ± SD of 3 independent experiments. *p<0.05; ***p<0.001 exposed cells v/s control or as indicated.
Fig 8.
AgNPs-induced expression of iNOS in hFOB 1.19 cells.
(A) AgNPs increased generation of NO and its inhibition by L-NIL. (B) Immunoblot of iNOS and (C) mRNA levels. Data are expressed as means ± SD of 4 independent experiments. **p<0.01; ***p<0.001 exposed cells v/s control (con) or as indicated.
Table 2.
Pearson’s correlation coefficient between AgNPs and iNOS.
Fig 9.
AgNPs-induced generation of nitrotyrosine in hFOB 1.19 and its inhibition by L-NIL.
(A) A bar graph showing data as measured by ELISA; (B) Representative immunoblots. Data are expressed as means ± SD of 3 independent experiments. *p<0.05; **p<0.01 exposed cells v/s control or as indicated.
Fig 10.
Transfection of hFOB 1.19 with iNOS siRNA prevents AgNPs-induced cell death.
The hFOB 1.19 cells were transfected with either 50 nM iNOS or control siRNA for 24 h, then cells were exposed for 48 h to 60 μg/mL AgNPs (the highest working concentration). (A) A representative immunoblot demonstrates efficient of transfection of iNOS siRNA. iNOS siRNA prevents AgNPs-induced increase of (B) NO, (C) NT, (D) apoptosis level and attenuated AgNPs-induced (E) cell death. Data are expressed as means ± SD of 3 independent experiments. **p<0.01; ***p<0.001 exposed cells v/s control or as indicated.