In vitro cytotoxic evaluation of MgO nanoparticles and their effect on the expression of ROS genes

Abstract

Water-dispersible MgO nanoparticles were tested to investigate their cytotoxic effects on oxidative stress gene expression. In this in vitro study, genes related to reactive oxygen species (ROS), glutathione S-transferase (GST) and catalase, were quantified using real-time polymerase chain reactions (molecular level) and molecular beacon technologies (cellular level). The monodispersed MgO nanoparticles, 20 nm in size, were used to treat human cancer cell lines (liver cancer epithelial cells) at different concentrations (25, 75 and 150 µg/mL) and incubation times (24, 48 and 72 h). Both the genetic and cellular cytotoxic screening methods produced consistent results, showing that GST and catalase ROS gene expression was maximized at 150 µg/mL nanoparticle treatment with 48 h incubation. However, the genotoxic effect of MgO nanoparticles was not significant compared with control experiments, which indicates its significant potential applications in nanomedicine as a diagnostic and therapeutic tool.

Figure 1

Genotoxicity of MgO nanoparticles on expression of GST in HepG2 cells. (a) RT-PCR profiles with serially diluted cDNA as a template (● 1.0 × 10⁹, ○ 1.0 × 10⁸, ▼ 1.0 × 10⁷, △ 1.0 × 10⁶, ■ 1.0 × 10⁵, □ 1.0 × 10⁴, ♦ blank) and the calibration curve of cDNA copy number with respect to the corresponding C_t values (inset). The enlarged RT-PCR profiles depend on the MgO nanoparticle incubation time of (b) 24 h, (c) 48 h and (d) 72 h, and MgO concentrations (red: control, green: 25 µg/mL, blue: 75 µg/mL, pink: 150 µg/mL).

Figure 2

Genotoxicity of MgO nanoparticles on the expression of catalase in HepG2 cells. (a) RT-PCR profiles with serially diluted cDNA as a template (● 1.0 × 10⁹, ○ 1.0 × 10⁸, ▼ 1.0 × 10⁷, △ 1.0 × 10⁶, ■ 1.0 × 10⁵, □ 1.0 × 10⁴, ♦ blank) and the calibration curve of the cDNA copy number with respect to the corresponding C_t values (Inset). The RT-PCR profiles depend on the MgO nanoparticle incubation time of (b) 24 h, (c) 48 h and (d) 72 h, and MgO concentrations (red: control, green: 25 µg/mL, blue: 75 µg/mL, pink: 150 µg/mL).

Figure 3

Gene expressions of GST and catalase of HepG2 on agarose gel. Expression of GST and catalase bands of HepG2 cell samples at 30 cycles amplification of cDNA. The gene expressions of GST (130 bp) and catalase (105 bp) showed variations in the band intensity with respect to MgO concentration and incubation time. The house keeping gene (GAPDH) for 200 bp product was also used as a reference control.

Figure 4

Confocal microscopic images of the expression of GST genes in HepG2 treated with MgO nanoparticles. Molecular beacons targeting the GST gene were delivered into live cells by a reverse permeabilization method using an activated SLO. The fluorescent live cell images at 24 h (top panel), 48 h (middle panel) and 72 h (bottom panel) were presented with excitation at 488 nm and emission detection at 530 nm. The intensity of the fluorescent signals was maximized at 48 h incubation time in proportion to MgO concentrations (scale bar size: 10 µm). The intensity of the fluorescent signals was found to be weak due to the low expression of target genes.

Figure 5

Analysis of gene expression of GST genes of each cell line by fluorometric determination. Fluorometric results showed that the gene expression of the cell line (HepG2), which depends on the MgO concentration and incubation times. Molecular beacons targeting the GST gene were delivered into live cells by a reverse permeabilization method using an activated SLO. The GST gene was expressed maximum at 48 h incubation time with proportion to MgO concentrations.