Products of Lipid Peroxidation as a Factor in the Toxic Effect of Silver Nanoparticles

Abstract

In our previous study we have shown that nanoparticles have different effects depending on the energy metabolism of the cell, which is an important factor in the context of oncology and diabetes. Here we assess the influence of AgNPs on cellular lipid components in varying glucose concentrations. To assess the effect of silver nanoparticles on cell lipids, we measured cell viability, the fluidity of the cell membranes, the content of amino groups in proteins, the level of lipid peroxidation products, the concentration of 4-hydroxynonenal (4-HNE), and the concentration of lipid peroxides. The obtained results show differences in the formation of lipid peroxidation products in cells exposed to oxidative stress induced by nanoparticles. In addition, we have shown that the metabolic state of the cell is a factor significantly affecting this process.

Keywords: lipid peroxidation; oxidative stress; silver nanoparticles.

Figure 1

The effect of hydrogen peroxide on the survival of Hep G2 cells (1 × 10³ cells per well). The cells were cultured in a medium with two variants of glucose concentration: 5.5 mmol/dm³ and 25 mmol/dm³ for 24 h, treated with H₂O₂ for 10 min, the measurements were taken after another 24 h. IC 50 parameters were compared with Student’s t-test, n = 6, α = 0.05.

Figure 2

Generation of lipid peroxides in Hep G2 cell line grown in 5.5 (A,B) and 25 mmol/dm³ (C,D) glucose media, exposed to silver nanoparticles for 4 (A,C) and 24 h (B,D). Cells were seeded in density of 5 × 10⁵ per well and lipid peroxides were analyzed with flow cytometry (BODIPY® 581/591 C11). * denotes statistically significant difference compared to control (untreated cells). ANOVA followed by Dunnett’s post hoc test, n = 6, α = 0.05.

Figure 3

Measurement of the SSC-A parameter in Hep G2 cells (5 × 10⁵ cells per well) grown in 5.5 mmol/ dm³, and 25 mmol/ dm³ glucose media after 4 and h incubation with 12.5, 25, and 50 μg/cm³ silver nanoparticles concentrations. Comparisons between cells cultured in media with different glucose concentration was determined by comparison of areas under the curves with Student’s t-test.

Figure 4

Alterations in the fluidity of the membrane in cells exposed to different silver nanoparticle concentrations were estimated by the ratio of fluorescence intensity of pyrene excimer and monomer. Cells (1 × 10⁴ per well) were cultured in 5.5 mmol/dm³ and 25 mmol/dm³ glucose concentration medium and incubated with silver nanoparticles for 4 (A) and 24 (B) h. AgNPs were used in concentrations: 0.39, 0.78, 1.56, 3.125, 6.25, and 12.5 µg/cm³, concentrations were logarithmized for graphing purposes. Comparison of cells grown in medium with different glucose levels was performed by comparing areas under the curves with Student’s t-test, n = 3, α = 0.05. Differences between varying concentrations of silver nanoparticles in the same medium were tested with anova followed by Dunnett’s post hoc test and marked with *.

Figure 5

HNE content of cells cultured at different glucose concentrations (ELISA). The cells were subjected to a 4 h (A) and 24 h (B) treatment with silver nanoparticles (12.5 µg/cm³), untreated cells performed as the control; * denotes a statistically significant difference with respect to the control, Student’s t-test, n = 3, α = 0.05.

Figure 6

Logarithm of the fluorescence intensity ratio of protein-bound fluorescamine for cells exposed and nonexposed to nanoparticles (control). Fluorescence was normalized with respect to the protein content. Statistical analysis was performed using Student’s t-test, n = 3. The concentration of silver nanoparticles was 25 μg/cm³. Cells were collected after 4 h incubation (A) and after 24 h incubation, (B) with silver nanoparticles. * denotes a statisticcally significant difference.