Regulation of apoptosis through bcl-2/bax proteins expression and DNA damage by nano-sized gadolinium oxide

Abstract

Gadolinium oxide (Gd₂O₃) nanoparticles (GNPs) are applied in industrial products, for example, additives, optical glass, and catalysis. There are various suggestions of metal nanoparticles paradigm but the underlying basic mechanism about the toxicity of metal nanoparticles, for example GNPs, remains unclear. This experiment was done to measure the effective toxicity of GNPs (10, 25, 50, and 100 µg/mL) over 24 and 48 h and to evaluate toxicity mechanism in human neuronal (SH-SY5Y) cells. GNPs produced reactive oxygen species (ROS), as evaluated by 2', 7'-dichlorodihydrofluorescein diacetate. Due to incorporation into cells, GNPs generated ROS in a concentration- and time-dependent manner. To determine the toxicity of GNP mechanism related to ROS, we also found chromosome condensation and dysfunction of mitochondrial membrane potential (MMP) after exposure of GNPs. Furthermore, the increased cell apoptosis rate and DNA fragmentation were closely related to the increased dose and exposure duration of GNPs in SH-SY5Y cells. The reduction in MMP with a simultaneous increase in the expression of bax/bcl2 gene ratio indicated that mitochondria-mediated pathway involved in GNPs induced apoptosis. Thus, our finding has provided valuable insights into the probable mechanism of apoptosis caused by GNPs at in vitro level.

Keywords: DNA fragmentation; GNPs; ROS; SH-SY5Y cells; apoptosis.

Figure 1

Characterization of GNPs (A). TEM image (B). Size distribution (%) of GNPs generated by TEM image. Abbreviations: GNPs, gadolinium oxide nanoparticles; TEM, transmission electron microscopy.

Figure 2

Morphology of SH-SY5Y cells. (A) Control cells (B) after exposure to 100 µg/mL GNPs for 24 h, (C) after exposure to 100 µg/mL GNPs for 48 h. Arrow (→) indicates damaged SH-SY5Y cells. Scale bar is 50 µm. Abbreviation: GNPs, gadolinium oxide nanoparticles.

Figure 3

Cell toxicity by MTT assay (A, B), LDH leakage (C, D) in SH-SY5Y cells exposed to gadolinium oxide nanoparticles for 24 and 48 h. Data are represented as mean ± SE. *P<0.01 vs control. Abbreviations: LDH, lactate dehydrogenase; MTT, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide; SE, standard error.

Figure 4

ROS production induced by GNPs. (A) The fluorescence image of SH-SY5Y cells treated with 10–100 µg/mL of GNPs for 24–48 h and stained with DCFHDA. (B) % ROS production due to GNPs in cells. SH-SY5Y cells were pretreated with or without NAC (1.5 mM) for 1 h and then exposed to GNPs (100 µg/mL) for 24 and 48 h. Images were snapped in phase contrast cum fluorescence microscope (Nikon, model 80i). Each value represents the mean ± SE of three experiments. *P<0.01 vs control. Values with alphabet superscript differ significantly (P<0.01) between exposed concentrations of GNPs (100 µg/mL) and GNPs (100 µg/mL) + NAC groups in same durations. Abbreviations: DCFHDA, dichloro-dihydro-fluorescein diacetate; GNPs, gadolinium oxide nanoparticles; NAC, N-acetyl-cysteine; ROS, reactive oxygen species; SE, standard error.

Figure 5

Gadolinium oxide nanoparticles-induced dose- and time-dependent mitochondrial membrane potential in SH-SY5Y cells.

Figure 6

GNPs induced oxidative stress biomarkers (A). GSH (B). LPO (C). SOD (D). CAT in SH-SY5Y cells. Each value represents the mean ± SE of three experiments. *P<0.01 vs control. Abbreviations: CAT, catalase; GSH, glutathione; LPO, lipid peroxide; MDA, malondialdehyde; SE, standard error; SOD, superoxide dismutase.

Figure 6

GNPs induced oxidative stress biomarkers (A). GSH (B). LPO (C). SOD (D). CAT in SH-SY5Y cells. Each value represents the mean ± SE of three experiments. *P<0.01 vs control. Abbreviations: CAT, catalase; GSH, glutathione; LPO, lipid peroxide; MDA, malondialdehyde; SE, standard error; SOD, superoxide dismutase.

Figure 7

Chromosomal condensation and and induction of caspase-3 activity in SH-SY5Y cells after exposure to GNPs. Notes: (A) Chromosomal condensation and (B) induction of caspase-3 activity in SH-SY5Y cells after exposure to GNPs for 24 and 48 h. Each value represents the mean ± SE of three experiments. *P<0.01 vs control. Arrow (→) indicates fragmented chromosome. Abbreviations: GNPs, gadolinium oxide nanoparticles; SE, standard error.

Figure 8

DNA strand breakage in SH-SY5Y cells because of GNPs (A). % tail DNA (B). Olive tail moment (C). Control cell (D). Exposed cell to GNPs (50 µg/mL) for 48 h. Each value represents the mean ± SE of three experiments. *P<0.01 vs control. Scale bar is 50 µm. Abbreviations: GNPs, gadolinium oxide nanoparticles; OTM, Olive tail moment; SE, standard error.

Figure 9

Western blot analysis of protein involved in apoptosis because of GNPs for 48 h exposure. (A) Bax, Bcl2. β-actin was used as an internal control to normalize result. (B) Relative quantification of protein expression levels. (C) Quantitative real-time polymerase chain reaction analysis of mRNA levels of apoptotic genes in SH-SY5Y cells exposed to GNPs. Results represent average ± SE of triplicate experiments. *P<0.01 vs control. Abbreviations: GNPs, gadolinium oxide nanoparticles; SE, standard error.