Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

Abstract

The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

Keywords: aniline; apoptosis; hepatocytes; reactive oxygen species.

Figure 1

ROS levels of hepatocytes exposed to aniline. ROS levels gradually increased with concentration of aniline, in a dose-response relationship (A–D). A. 0 μg/mL; B. 2.50 μg/mL; C. 5.0 μg/mL; D. 10.0 μg/mL; E. 10.0 μg/mL + 20 mmol/L NAC; F. Positive control (H₂O₂).

Figure 2

Changes in GSH, SOD, CAT and MDA levels after exposure of hepatocytes to aniline for 24 h. A. 0 μg/mL; B. 2.50 μg/mL; C. 5.0 μg/mL; D. 10.0 μg/mL; E. 10.0 μg/mL + 20 mmol/L NAC. Each data set mean value is a composite of three independent experiments with SD shown. * compared with control, p < 0.05; Δ compared with 10.0 μg/mL group, p < 0.05. (a) GSH; (b) SOD; (c) CAT; (d) MDA.

Figure 3

Mitochondrial membrane potential of hepatocytes exposed to aniline for 24 h. Data represented are mean ± SD of three identical experiments made in triplicate. A. 0 μg/mL; B. 2.50 μg/mL; C. 5.0 μg/mL; D.10.0 μg/mL; E. 10.0 μg/mL + 20 mmol/L NAC. * Compared with control, p < 0.05; Δ compared with 10.0 μg/mL group, p < 0.05.

Figure 4

The degree of DNA damage of hepatocytes exposed to aniline for 24 h. (a) 0 μg/mL; (b) 2.50 μg/mL; (c) 5.0 μg/mL; (d) 10.0 μg/mL; (e) 10.0 μg/mL + 20 mmol/L NAC; (f) positive control (H₂O₂). (Original magnification 100×).

Figure 5

Viability of hepatocytes exposed to aniline. Hepatocytes were cultured in the absence or presence of 1.25, 2.5, 5.0 or 10.0 μg/mL aniline for 24 h. Cell viability was determined based on the MTT assay. Data represented are mean ± SD of three identical experiments made in triplicate.

Figure 6

Apoptosis of hepatocytes exposed to 0, 2.50, 5.0 or 10.0 μg/mL aniline for 24 h. Cells were treated with aniline, then stained with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide using flow cytometry. (a) 0 μg/mL; (b) 2.50 μg/mL; (c) 5.0 μg/mL; (d) 10.0 μg/mL; (e) 10.0 μg/mL + 20 mmol/L NAC; (f) Apoptotic cells (%). Data represented are mean ± SD of three identical experiments made in triplicate. * Compared with control, p < 0.05; Δ compared with 10.0 μg/mL group, p < 0.05.