Microsomal glutathione transferase 1 protects against toxicity induced by silica nanoparticles but not by zinc oxide nanoparticles

Abstract

Microsomal glutathione transferase 1 (MGST1) is an antioxidant enzyme located predominantly in the mitochondrial outer membrane and endoplasmic reticulum and has been shown to protect cells from lipid peroxidation induced by a variety of cytostatic drugs and pro-oxidant stimuli. We hypothesized that MGST1 may also protect against nanomaterial-induced cytotoxicity through a specific effect on lipid peroxidation. We evaluated the induction of cytotoxicity and oxidative stress by TiO(2), CeO(2), SiO(2), and ZnO in the human MCF-7 cell line with or without overexpression of MGST1. SiO(2) and ZnO nanoparticles caused dose- and time-dependent toxicity, whereas no obvious cytotoxic effects were induced by nanoparticles of TiO(2) and CeO(2). We also noted pronounced cytotoxicity for three out of four additional SiO(2) nanoparticles tested. Overexpression of MGST1 reversed the cytotoxicity of the main SiO(2) nanoparticles tested and for one of the supplementary SiO(2) nanoparticles but did not protect cells against ZnO-induced cytotoxic effects. The data point toward a role of lipid peroxidation in SiO(2) nanoparticle-induced cell death. For ZnO nanoparticles, rapid dissolution was observed, and the subsequent interaction of Zn(2+) with cellular targets is likely to contribute to the cytotoxic effects. A direct inhibition of MGST1 by Zn(2+) could provide a possible explanation for the lack of protection against ZnO nanoparticles in this model. Our data also showed that SiO(2) nanoparticle-induced cytotoxicity is mitigated in the presence of serum, potentially through masking of reactive surface groups by serum proteins, whereas ZnO nanoparticles were cytotoxic both in the presence and in the absence of serum.

Figure 1

TEM images of the metal oxide nanoparticles TiO₂, CeO₂, ZnO, and SiO₂. Scale bar: 20 nm.

Figure 2

Cytotoxicity induced by metal oxide nanoparticles in MCF-7 cells. Dose-dependent cytotoxicity induced by TiO₂, CeO₂, ZnO, and SiO₂, in the presence (A) or absence (B) of serum at 24 h, assessed as metabolic activity by MTT assay. The results are expressed by mean values ± SD (n = 3–4); *<0.05, **<0.01, ***<0.001 (for SiO₂ compared to control, i.e., no treatment); ##<0.01, ###<0.001 (for ZnO compared control).

Figure 3

Particle uptake following exposure of MCF-7 cells. Uptake of TiO₂ and CeO₂ nanoparticles was evident, while no evidence of cellular uptake of intact ZnO nanoparticles was observed, likely due to rapid dissolution. SiO₂ nanoparticles appeared to interact with the plasma membrane, but uptake was difficult to ascertain due to the similarity of the particles to other cellular structures in terms of size, shape, and electron density. Cells were incubated with particles (50 μg/mL) for 2 h. Scale bars for control cells are 5 and 2 μm (left and right, respectively), 2 μm for TiO₂, 5 μm for CeO₂, and 500 nm for ZnO as well as SiO₂.

Figure 4

MGST1 protects against cytotoxicity induced by CuOOH. (A) Western blot confirmation of MGST1 overexpression (17 kDa) using a specific antirat MGST1 antibody. β-Actin (42 kDa) was used as loading control. Cytotoxicity induced by CuOOH, measured as (B) metabolic activity using the MTT assay (50 μM CuOOH, 1 h incubation), (C) LDH release (50 μM CuOOH, 1 h incubation), and (D) colony formation (CFE) assay (5 μM CuOOH, 3 h incubation and further incubation for 7 days), is prevented by MGST1.

Figure 5

MGST1 protects against SiO₂ nanoparticle-induced cytotoxicity but not ZnO nanoparticle-induced cytotoxicity. MGST1 protection against nanoparticle-induced cytotoxicity at 24 h was assessed using MTT assay for assessment of metabolic activity (A,B), LDH assay to monitor cell membrane damage (C,D), and CFE assay to monitor the late effects of particle exposure (24 h exposure, followed by a further 7 day incubation) (E,F). MGST1 overexpressing cells are indicated by filled squares and solid line, antisense transfected cells by triangles and dashed line, and MCF-7 wild-type cells by diamonds and dotted line. The results are expressed as mean values ± SD (n = 3–4); *<0.05, **<0.01, ***<0.01.

Figure 6

MGST1 protects against SiO₂ nanoparticle-induced but not ZnO nanoparticle-induced mitochondrial ROS production (A,B) and lipid peroxidation (C,D). Cells were incubated with 20 μg/mL nanoparticles for 2 h in the absence of serum. MitoSOX was used for measurement of mitochondria-specific superoxide generation and C₁₁-BODIPY^581/591 was used as a surrogate marker for lipid peroxidation; ***<0.01.

Figure 7

MGST1 protects against SiO₂ nanoparticle-induced mitochondrial depolarization (A) and oxidative DNA damage (B,C). Cells were incubated with 20 μg/mL SiO₂ nanoparticles for 2 h in the absence of serum. Fpg-comet assay was used to determine oxidative DNA damage (% of DNA content in the comet tail), and the TMRE assay was used for measurement of the dissipation of the mitochondrial transmembrane potential. Representative photographs depicting comet tails (seen in WT cells and antisense transfected cells) are shown. The data are expressed as mean values ± SD (n = 3); *<0.05, **<0.01.

Figure 8

Intracellular glutathione (GSH) and toxicity of SiO₂ nanoparticles. ThioGlo-1 assay was used to determine GSH levels. (A) Exposure to SiO₂ nanoparticles (20 μg/mL, 2 h in the absence of serum) (A) results in a depletion of GSH, and this is reversed in MGST1 overexpressing cells. (B) Buthionine-[S,R]-sulfoximine (BSO)-induced depletion of intracellular GSH after 24 h. (C,D) Toxicity of SiO₂ nanoparticles (50 μg/mL, 24 h) is not affected by co-treatment with BSO (50 μM) (C) or N-acetylcysteine (NAC, 1 mM), a precursor of GSH.