A study of the mechanism of in vitro cytotoxicity of metal oxide nanoparticles using catfish primary hepatocytes and human HepG2 cells

Abstract

Nanoparticles (NPs), including nanometal oxides, are being used in diverse applications such as medicine, clothing, cosmetics and food. In order to promote the safe development of nanotechnology, it is essential to assess the potential adverse health consequences associated with human exposure. The liver is a target site for NP toxicity, due to NP accumulation within it after ingestion, inhalation or absorption. The toxicity of nano-ZnO, TiO(2), CuO and Co(3)O(4) was investigated using a primary culture of channel catfish hepatocytes and human HepG2 cells as in vitro model systems for assessing the impact of metal oxide NPs on human and environmental health. Some mechanisms of nanotoxicity were determined by using phase contrast inverted microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, reactive oxygen species (ROS) assays, and flow cytometric assays. Nano-CuO and ZnO showed significant toxicity in both HepG2 cells and catfish primary hepatocytes. The results demonstrate that HepG2 cells are more sensitive than catfish primary hepatocytes to the toxicity of metal oxide NPs. The overall ranking of the toxicity of metal oxides to the test cells is as follows: TiO(2)<Co(3)O(4)<ZnO<CuO. The toxicity is due not only to ROS-induced cell death, but also to damages to cell and mitochondrial membranes.

Fig. 1

Phase contrast microscopy light micrographs of HepG2 control cells (A); HepG2 cells treated with 50 mg/l ZnO NPs (B) and 50 mg/l TiO₂ NPs (C) for 48 h; HepG2 cells treated with 25 mg/l CuO NPs (D) and 25 mg/l Co₃O₄ NPs (E) for 48 h.

Fig. 2

Phase contrast microscopy light micrographs of the catfish control primary hepatocytes (A); catfish primary hepatocytes treated with 200 mg/l ZnO NPs (B) and 200 mg/l TiO₂ NPs (C) for 48 h; the catfish primary hepatocytes treated with 200 mg/l CuO NPs (D) and 200 mg/l Co₃O₄ NPs (E) for 48 h.

Fig. 3

MTT activities of HepG2 cells (A) and primary hepatocytes (B) treated with metal oxide NPs of various concentrations for 48 h (replicate number ≥4). Figure only reflects the data points of effective concentrations. Histograms labeled with different letters represent significant differences (p < 0.05).

Fig. 4

TEM images of the control cells of catfish primary hepatocytes, (A, 8,000X); the primary hepatocytes treated with ZnO (B, 20,000X) and TiO₂ (C, 60,000X) at the concentration of 200 mg/l for 48 h, respectively; HepG2 cells treated with ZnO (D, 10,000X) and TiO₂ (E, 15,000X) at the concentration of 200 mg/l for 48 h, respectively; and the control HepG2 cells (F, 60,000X). The arrows indicate where the nanoparticles are located.

Fig. 5

The accumulation of intracellular ROS in HepG2 cells induced by metal oxide NPs of various concentrations for 24 h (A), 48h (B) and the catfish primary hepatocytes treated with metal oxide NPs of various concentrations for 24 h (C), 48 h (D).

Fig. 5

The accumulation of intracellular ROS in HepG2 cells induced by metal oxide NPs of various concentrations for 24 h (A), 48h (B) and the catfish primary hepatocytes treated with metal oxide NPs of various concentrations for 24 h (C), 48 h (D).

Fig. 6

Bivariate plots of ZnO-induced effects on the primary hepatocyte cells as determined by flow cytometric assay. Control cells (A); the cells treated with ZnO for 48 h at the concentration of 25 mg/l (B); the concentration of 50 mg/l (C); the concentration of 100 mg/l (D); the concentration of 200 mg/l (E). In each plot, Upper Left (UL) depicts non-viable cells or PI positive cells, Upper Right (UR) depicts apoptotic and necrotic cells or annevin V and PI positive cells, Lower Left (LL) depicts live cells or annexin V and PI negative, Lower Right (LR) depicts annexin V positive cells or early apoptotic cells.