J Vet Sci.  2014 Dec;15(4):485-493. 10.4142/jvs.2014.15.4.485.

N-acetylcysteine protects against cadmium-induced oxidative stress in rat hepatocytes

• Wang J
• Zhu H
• Liu X
• Liu Z

• KMID: 2070234
• DOI: http://doi.org/10.4142/jvs.2014.15.4.485

Abstract

Cadmium (Cd) is a well-known hepatotoxic environmental pollutant. We used rat hepatocytes as a model to study oxidative damage induced by Cd, effects on the antioxidant systems, and the role of N-acetylcysteine (NAC) in protecting cells against Cd toxicity. Hepatocytes were incubated for 12 and 24 h with Cd (2.5, 5, 10 microM). Results showed that Cd can induce cytotoxicity: 10 microM resulted in 36.2% mortality after 12 h and 47.8% after 24 h. Lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase activities increased. Additionally, reactive oxygen species (ROS) generation increased in Cd-treated hepatocytes along with malondialdehyde levels. Glutathione concentrations significantly decreased after treatment with Cd for 12 h but increased after 24 h of Cd exposure. In contrast, glutathione peroxidase activity significantly increased after treatment with Cd for 12 h but decreased after 24 h. superoxide dismutase and catalase activities increased at 12 h and 24 h. glutathione S-transferase and glutathione reductase activities decreased, but not significantly. Rat hepatocytes incubated with NAC and Cd simultaneously had significantly increased viability and decreased Cd-induced ROS generation. Our results suggested that Cd induces ROS generation that leads to oxidative stress. Moreover, NAC protects rat hepatocytes from cytotoxicity associated with Cd.

Keyword

cadmium; hepatocytes; oxidative stress; rat

MeSH Terms

Acetylcysteine/*metabolism
Animals
Antioxidants/*metabolism
Cadmium/*toxicity
Cell Survival/drug effects
Cells, Cultured
Environmental Pollutants/*toxicity
Hepatocytes/drug effects/metabolism
*Oxidative Stress
Rats
Rats, Sprague-Dawley
Reactive Oxygen Species/*metabolism
Acetylcysteine
Antioxidants
Cadmium
Environmental Pollutants
Reactive Oxygen Species

Figure

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  Fig. 1 Effect of cadmium (Cd) on the viability of rat hepatocytes. The cells were incubated with 0, 2.5, 5, and 10 µM Cd for 12 and 24 h. Viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay. Each experiment was repeated six times and data are expressed as the mean ± SD (**p < 0.01).

• 

  Fig. 2 Effects of Cd on (A) lactate dehydrogenase (LDH) activity, (B) aspartate aminotransferase (AST) activity, and (C) alanine aminotransferase (ALT) activity in rat hepatocytes. Cells were incubated with 0, 2.5, 5, and 10 µM Cd for 12 and 24 h. Each experiment was repeated six times and data are expressed as the mean ± SD (**p < 0.01).

• 

  Fig. 3 Effect of Cd on reactive oxygen species (ROS) production. Hepatocytes were treated with 5 µM of Cd for 45 min or 1.5, 6, 12, and 24 h. The cells were then stained with DCFH2-DA. Intracellular ROS levels were measured by flow cytometry as described in the Materials and Methods section. Results are expressed as a representative histogram (A, control; B, 5 µM Cd; respectively 1.5 h) and mean fluorescence obtained from the histogram statistics (C). Each bar represents the mean ± SD (n = 3). **p < 0.01 and *p < 0.05 compared to the control. DCF: dichlorofluorescein, FITC: fluorescein isothiocyanate.

• 

  Fig. 4 Effects of Cd on (A) glutathione (GSH) and (B) malondialdehyde (MDA) levels in rat hepatocytes. Cells were treated with 0, 2.5, 5, and 10 µM Cd for 12 and 24 h. Each experiment was repeated six times and data are expressed as the mean ± SD (*p < 0.05 and **p < 0.01).

• 

  Fig. 5 Effects of Cd on (A) glutathione peroxidase (GPx), (B) glutathione S-transferase (GST), (C) glutathione reductase (GR), (D) superoxide dismutase (SOD), and (E) catalase (CAT) activities. Hepatocytes were treated with 2.5, 5, and 10 µM Cd. GPx, GST, GR, SOD, and CAT activities were measured after 12 and 24 h. Each experiment was repeated six times and data are expressed as the mean ± SD (*p < 0.05 and **p < 0.01).

• 

  Fig. 6 Effect of N-acetylcysteine (NAC) on Cd-induced cytotoxicity in hepatocytes. Cells were incubated with NAC (1 or 2 mM) and Cd (2.5, 5, or 10 µM) simultaneously for 24 h. An MTT assay was then performed to evaluate cytotoxicity. Each experiment was repeated six times and data are expressed as the mean ± SD (**p < 0.01, #p < 0.05, and ##p < 0.01 compared to cells treated with Cd alone).

• 

  Fig. 7 Inhibitory effect of NAC on Cd-induced ROS generation (as monitored by DCF fluorescence). The cells were incubated with NAC (2 mM) and Cd (5 µM) at the same time for 1.5 h. Bars represent the mean ± SD (n = 3). **p < 0.01 and #p < 0.05 compared to cells treated with Cd alone.

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