An evaluation of the protective role of vitamin C in reactive oxygen species-induced hepatotoxicity due to hexavalent chromium in vitro and in vivo

Abstract

Backgroud: Drinking water contamination with hexavalent chromium [Cr (VI)] has become one of the most serious public health problems, thus the investigation of Cr (VI)-induced hepatotoxicity has attracted much attention in recent years.

Methods: In the present study, by determining the indices of hepatotoxicity induced by Cr (VI), the source of accumulated reactive oxygen species (ROS), and the protective effect of the antioxidant Vitamin C (Vit C), we explored the mechanisms involved in Cr (VI)-induced hepatotoxicity in vitro and in vivo.

Results: We found Cr (VI) caused hepatotoxicity characterized by the alterations of several enzymatic and cytokine markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), interleukine-1β (IL-1β), and tumor necrosis factor-α (TNF-α), etc. ROS production after Cr (VI) exposure was origins from the inhibition of electron transfer chain (ETC) and antioxidant system. Vit C inhibited ROS accumulation thus protected against Cr (VI)-induced hepatotoxicity in L-02 hepatocytes and in the rat model.

Conclusions: We concluded that ROS played a role in Cr (VI)-induced hepatotoxicity and Vit C exhibited protective effect. Our current data provides important clues for studying the mechanisms involved in Cr (VI)-induced liver injury, and may be of great help to develop therapeutic strategies for prevention and treatment of liver diseases involving ROS accumulation for occupational exposure population.

Keywords: Hepatotoxicity; Hexavalent chromium [Cr (VI)]; Mitochondrial respiratory chain complex I (MRCC I); Reactive oxygen species (ROS); Vitamin C (Vit C).

Fig. 1

Cr (VI) induced hepatotoxicity in vitro. L-02 hepatocytes were treated with Cr (VI) (8 and 16 μM) for 24 h, then the cells were collected and the indexes of hepatotoxicity were determined. a The changes of activities of enzymatic markers AST and ALT. b The production of pro-inflammatory cytokines (IL-1β, TNF-α, IFN-γ and IL-10) after Cr (VI) exposure. c Effect of Cr (VI) exposure on LTB4 level. Data represent mean ± SD. *p < 0.05, compared with the control (untreated) group

Fig. 2

Cr (VI) induced ROS accumulation in the hepatocytes. The cells were treated as described in Fig. 1. a The DCF fluorescence intensity, corresponding to the level of ROS production, was detected. b Intracellular superoxide anion. The data shows the percentage and the fluorescence intensity of positive DHE staining cells from each group. Data represent mean ± SD. *p < 0.05, compared with the control group

Fig. 3

The inhibition of ETC and antioxidant system were associated with Cr (VI)-induced ROS accumulation. The cells were treated as described in Fig. 1. a The activities of MRCC I-IV. b The mRNA and protein levels of MRCC I subunit NDUFS3. c GSH, SOD, Trx, and MDA levels. Data represent mean ± SD. *p < 0.05, compared with the control group

Fig. 4

Vit C inhibited ROS accumulation. a The hepatocytes were treated with different concentrations of Vit C (0–2430 μM) for 2 h and then analyzed for cell survival rate. (B-C) The L-02 hepatocytes were pretreated with Vit C (200 μM) for 2 h and then were exposed to Cr (VI) (8 and 16 μM) for 24 h. ROS production assay (b) and intracellular superoxide anion production assay (c) were conducted. Data represent mean ± SD. *p < 0.05, compared with the control group

Fig. 5

Vit C protected against Cr (VI)-induced hepatotoxicity in vitro. The cells were exposed to Cr (VI) (0, 8 and 16 μM) with or without the combination of 200 μM Vit C. a The changes of activities of enzymatic markers AST and ALT. b The levels of IL-1β, TNF-α and LTB4. c The levels of GSH, SOD, and Trx. d The protein expression levels of GSH, SOD, and Trx. Data represent mean ± SD. ^# p < 0.05, compared with Cr (VI) alone treatment (8 or 16 μM) group

Fig. 6

Vit C protected against Cr (VI)-induced hepatotoxicity in vivo. All rats were given the drugs by gavage at a dose of 0.5 ml/100 g body weight daily for a week (seven consecutive days). Groups are indicated by pretreatment + treatment as follows: Con, Vit C, Cr (VI) (8.84 mg/kg.bw), Vit C+ Cr (VI) (8.84 mg/kg.bw), Cr (VI) (17.68 mg/kg.bw), and Vit C+ Cr (VI) (17.68 mg/kg.bw). a Effect of Vit C pretreatment on Cr (VI)-induced alterations in rat liver histology. b The chromium contents in stool, urine, liver and plasma. c AST and ALT activities. d GSH, SOD, and MDA levels. e Free radical scavenging capacity. Data represent mean ± SD. *p < 0.05, compared with control group. ^# p < 0.05, compared with the Cr (VI) alone treatment group