Comparative oxidative stress elicited by nanosilver in stable HSPA1A promoter-driven luciferase reporter HepG2 and A549 cells

Abstract

The use of silver nanoparticles (AgNPs) in consumer products and medical applications is growing rapidly owing to their noted antimicrobial properties. As a consequence, a rapid and reliable toxicology test that allows multiple nanosilver screening in a stress responsive pathway-based assay is needed to ensure consumer safety. Here, novel HSPA1A promoter-driven luciferase reporter gene assays based on two cell models widely used in toxicology testing, HepG2 and A549 cell lines, were developed to analyze the oxidative stress elicited by AgNPs. After AgNP exposure, the luciferase activity, cell viability, and oxidative damage in HepG2- and A549-luciferase cells were determined. The uptake and intracellular localization of AgNPs were measured by transmission electron microscopy. The cellular AgNPs were determined by inductively coupled plasma mass spectrometry. Our results showed that both reporter cells were able to take up nanoparticles and furthermore, exposure to AgNPs resulted in an obviously dose-dependent increase in transcriptional activation of the HSPA1A promoter indicated by luciferase activity. Surprisingly, luciferase activity was greater than 200× the control level in HepG2-luciferase reporter cells treated with 50 μg mL^-1 AgNPs. These results were in line with the positive responses in cytotoxicity, intracellular reactive oxygen species induction, glutathione depletion, malondialdehyde concentration, and the cellular silver content. Compared with the A549-luciferase cells, the luciferase signal in the metabolic competent HepG2-luciferase cells is obviously more sensitive and stable. We conclude that these two reporter gene cell systems, especially the HepG2-luciferase cells, demonstrated their utilities in predictive screening of the oxidative stress elicited by nanosilver.

Fig. 1. Transcriptional activation of HSPA1A promoter-driven luciferase reporter after AgNP exposure. The HepG2- and A549-luciferase cells were exposed to AgNPs (3.125–50 μg mL^–1) for 24 h. Luciferase induction was measured using the luciferase assay system. Data represent mean ± SD of three individual experiments. *P < 0.05 compared with untreated control. **P < 0.01 compared with untreated control.

Fig. 2. The cytotoxicity and oxidative damage induced by AgNPs in HepG2- and A549-luciferase cells. Cells were exposed to AgNPs (3.125–50 μg mL^–1) for 24 h. Cytotoxicity indicated as cell viability was evaluated using the CCK-8 assay (A). Oxidative damage was measured by levels of ROS (B), GSH depletion (C), and MDA concentration (D). Chemical concentrations were at the bottom. Data represent mean ± SD of three individual experiments. *P < 0.05 compared with untreated control. **P < 0.01 compared with untreated control.

Fig. 3. Intracellular localization of AgNPs in HepG2- and A549-luciferase cells was investigated by TEM. The TEM images of HepG2- (A) and A549-luciferase cells (D) were from the untreated group. The representative TEM images were for the samples from the HepG2- (B, C) and A549-luciferase cells (E, F) treated with 25 μg mL^–1 of AgNPs for 24 h.

Fig. 4. The cellular Ag dose in HepG2- and A549-luciferase cells was quantified by ICP-MS. The cells were treated with increasing concentrations of AgNPs (0, 3.125, 6.25, 12.5, 25, 50 μg mL^–1) for 24 h and the total cellular Ag content was analyzed by ICP-MS. The Ag dose was expressed as pg per cell. Results are presented as mean ± SD of two replicates.