RETRACTED: Combined Effect of Silica Nanoparticles and Benzo[a]pyrene on Cell Cycle Arrest Induction and Apoptosis in Human Umbilical Vein Endothelial Cells

Abstract

Particulate matter (PM) such as ultrafine particulate matter (UFP) and the organic compound pollutants such as polycyclic aromatic hydrocarbon (PAH) are widespread in the environment. UFP and PAH are present in the air, and their presence may enhance their individual adverse effects on human health. However, the mechanism and effect of their combined interactions on human cells are not well understood. We investigated the combined toxicity of silica nanoparticles (SiNPs) (UFP) and Benzo[a]pyrene (B[a]P) (PAH) on human endothelial cells. Human umbilical vascular endothelial cells (HUVECs) were exposed to SiNPs or B[a]P, or a combination of SiNPs and B[a]P. The toxicity was investigated by assessing cellular oxidative stress, DNA damage, cell cycle arrest, and apoptosis. Our results show that SiNPs were able to induce reactive oxygen species generation (ROS). B[a]P, when acting alone, had no toxicity effect. However, a co-exposure of SiNPs and B[a]P synergistically induced DNA damage, oxidative stress, cell cycle arrest at the G2/M check point, and apoptosis. The co-exposure induced G2/M arrest through the upregulation of Chk1 and downregulation of Cdc25C, cyclin B1. The co-exposure also upregulated bax, caspase-3, and caspase-9, the proapoptic proteins, while down-regulating bcl-2, which is an antiapoptotic protein. These results show that interactions between SiNPs and B[a]P synergistically potentiated toxicological effects on HUVECs. This information should help further our understanding of the combined toxicity of PAH and UFP.

Keywords: Benzo[a]pyrene; HUVECs; co-exposure toxicity; silica nanoparticles.

Figure 1

Characterization of SNPs. (A) TEM images show spherical SNPs with good monodispersity in distilled water; (B) Size distribution of SNPs showing a normal distribution curve, mean = 62.720 ± 10.917.

Figure 2

Effects of SiNPs and/or B[a]P on HUVECs’ viability. (A) Cell viability of various concentrations of SiNPs; (B) Cell viability of various concentrations of B[a]P; (C) Cell viability of HUVECs treated with DMSO (1%), SiNPs (10 μg/mL), B[a]P (1 μM), and their mixture (10 μg/mL + 1 μM); (D) Profile plot shows a synergy interaction between SiNPs and B[a]P (F = 6.476, p = 0.021). * p < 0.05, ** p < 0.01 for treated group compared to control, while # p < 0.05 for combined groups compared to single treated groups.

Figure 3

Morphological changes in HUVECs observed under an electron microscope after 24 h of exposure to B[a]P and/ SiNPs. (A) Control group; (B) HUVECs exposed to DMSO (0.1%); (C) HUVECs exposed to B[a]P (1 μM); (D) HUVECs exposed to SiNP (10 μg/mL); (E) HUVECs exposed to B[a]P + SiNPs (10 μg/mL + 1 μM).

Figure 4

Intracellular ROS generated by treated HUVECs. (A) ROS level; (B) Interaction plots showing a synergy interaction between SiNPs and B[a]P (F = 7.301, p = 0.027). * p < 0.05, ** p < 0.01 for the treated group compared to the control, while # p < 0.05 for combined groups compared to single treated groups.

Figure 5

HUVEC oxidative stress caused by SiNPs and B[a]P co-exposure. (A) Malondialdehyde content increased; (B) Profile plots shows that SiNPs and B[a]P synergistically increased the malondialdehyde content (F = 5.084, p = 0.026); (C) Decreased superoxide dismutase activity; (D) Profile plots shows that the superoxide dismutase activity decrease was additive (F = 3.506, p = 0.143); (E) Decrease in glutathione peroxidase activity; (F) Profile plot shows a synergy interaction in the decrease of glutathione peroxidase activity (F = 11.174, p = 0.006). ** p < 0.01 for the treated group compared to the control, while # p < 0.05 for the combined group compared to single treated groups.

Figure 5

HUVEC oxidative stress caused by SiNPs and B[a]P co-exposure. (A) Malondialdehyde content increased; (B) Profile plots shows that SiNPs and B[a]P synergistically increased the malondialdehyde content (F = 5.084, p = 0.026); (C) Decreased superoxide dismutase activity; (D) Profile plots shows that the superoxide dismutase activity decrease was additive (F = 3.506, p = 0.143); (E) Decrease in glutathione peroxidase activity; (F) Profile plot shows a synergy interaction in the decrease of glutathione peroxidase activity (F = 11.174, p = 0.006). ** p < 0.01 for the treated group compared to the control, while # p < 0.05 for the combined group compared to single treated groups.

Figure 6

DNA damage in HUVECs induced by SiNPs and/or B[a]P exposure. (A–E) show the representative fluorescence images of PI-stained nuclei of the control and cells treated with DMSO, B[a]P, SiNPs, and B[a]P + SiNPs, respectively; (F) Shows the DNA damage rate, tail DNA percentage (%), tail length, and OTM; (G) Interaction plot illustrates the synergistic effect of SiNPs and B[a]P on the DNA damage of HUVECs. * p < 0.05, ** p < 0.01 for the treated group compared to the control, while # p < 0.05 for the combined group compared to single treated groups.

Figure 7

Cell cycle phase distribution in various treatment groups. (A) Control; (B) DMSO (0.1%); (C) B[a]P (1 μM); (D) SiNPs (10 μg/mL); (E) B[a]P + SiNPs (1 μM + 10 μg/mL) treatment; (F) Percentages are mean ± SD of each cell cycle phase for triplicate experiments; (G) Factorial analysis plots show a synergy interaction between SiNPs and B[a]P (F = 27.637, p = 0.001). ** p < 0.01 for the treated group compared to the control, while # p < 0.05 for the combined group compared to single treated groups; (H) Western blot results show a significant increase in Chk1 expression and decrease in Cdc25C, Cyclin B1, and Cdc2 expression; (I) the graph shows a significant change in the protein expression of the co-exposed group compared to the other groups.

Figure 8

Apoptosis of HUVECs induced by SiNPs and/or B[a]P. (A) Control; (B) DMSO; (C) B[a]P; (D) SiNPs; (E) B[a]P + SiNPs; (F) The percentage of apoptotic cells; (G) Synergistic interaction between SiNPs and B[a]P illustrated by interaction plots (F = 23.838, p = 0.001. * p < 0.05, ** p < 0.01 for the treated group compared to the control, while # p < 0.05 for the combined group compared to single treated groups; (H) Western blot results show a decrease in Bcl-2 expression and increase in Bax, Caspase 9, and Caspase 3 expression in the co-exposed group; (I) the graph shows a significant change in the protein expression of the co-exposed group compared to the other groups.