Vitamin D and melatonin protect the cell's viability and ameliorate the CCl4 induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines

Abstract

Carbon tetrachloride (CCl4) is widely used to induce liver toxicity in in vitro/in vivo models. Lipid peroxidation (LPO) begins with toxicity and affects cell viability. Recently, the beneficial effects of melatonin and Vitamin D on cell proliferation in human normal and cancer cells were found. This study was planned to evaluate antioxidant and cytoprotective activity of melatonin and Vitamin D in CCl4 induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines. Based on the cytotoxicity assay, melatonin and Vitamin D were evaluated for cytotoprotective potential against CCl4 induced toxicity in HepG2 and Hep3B liver cell lines by monitoring cell viability, LPO and glutathione (GSH) level. Different dosages of CCl4 (0.1, 0.2, 0.3 and 0.4 % v/v) were applied to HepG2 and Hep3B cells in order to determine the most toxic dosage of it in a time dependent manner. The same experiments were repeated with exogenously applied melatonin (MEL) and Vitamin D to groups treated with/without CCL4. Cell viability was determined with MTT measurements at the 2nd, 24th and 48th h. GSH content and Malondialdehyde levels were measured from the cell lysates. As a result, both melatonin and Vitamin D administration during CCl4 exposure protected liver cells from CCl4 induced cell damage. Increase in LPO and decrease in GSH were found in the CCl4 groups of both cells. Contrary to these results administration of MEL and Vitamin D on cells exhibited results similar to the control groups. Therefore, melatonin and Vitamin D might be a promising therapeutic agent in several toxic hepatic diseases.

Keywords: CCl4; Cell proliferation; Hep3B; HepG2; Liver cytotoxicity; Melatonin; Vitamin D.

Fig. 1

Cell death (%) was determined at the 2nd, 24th and 48th h by the MTT assay. CCl₄ exposure induced prominent cell death in liver cell lines in a dose and time dependent manner. Data are from six independent experiments for each condition. a 0.4 % exposure of CCl₄ affected HepG2 cell viability the most. b Reduction of Hep3B cell number was prominent at 0.4 % CCl₄ dosage. Data are presented as mean ± SEM. *p < 0.05 versus control group

Fig. 2

Cell number (%) was determined by MTT assay following 2, 24, and 48 h of exposure of MEL and Vitamin D alone or with CCl₄. The percentages of the cell number were shown on the bars of the graphic. Decrease in cell number as a result of CCl₄ exposure in HepG2 cells was assessed. Supplementation of MEL and Vitamin D to CCl₄ almost protected the cell viability in a time dependent manner. Increase in cell number of Vitamin D alone and with CCl₄ groups compared to the control group at 24 h was prominent. Data are presented as mean ± SEM. *p < 0.05 versus control group

Fig. 3

The cell number (%) of MEL and Vitamin D on CCl₄ exposure in Hep3B liver cells by MTT assay on the 2nd, 24th and 48th h of exposure. The percentages of the cell number in MEL and Vitamin D treatment alone were similar to the control group. Hep3B cell number was diminished after CCl₄ exposure. MEL and Vitamin D treatment with CCl₄ increased cell proliferation in Hep3B liver cells in a time dependent manner. Data are presented as mean ± SEM. *p < 0.05 versus control group

Fig. 4

The discrepancy between groups was evaluated. a MDA values of HEP3B cells are shown. b The variability of MDA content was determined in HepG2 cells. c GSH content of HEP3B cells are shown. d The date of GSH in HepG2 cells showed parallel results with the data of HEP3B cells. Data are presented as mean ± SEM. *p < 0.05 versus CCl₄ value was accepted as statistically significant (versus CCL₄)