The protective activity of alpha-hederine against H2O2 genotoxicity in HepG2 cells by alkaline comet assay.

ABSTRACT

This study was designed to evaluate the protective effect of alpha-hederine (alpha-hed) against H2O2-mediated DNA damage on HepG2 cell line by the alkaline comet assay. For the protective effect of alpha-hed study, cells were treated according to three protocols pre-treatment, simultaneous treatment and post-treatment. The effect of alpha-hed on catalase activity was evaluated after treating the cells with 3.36 mg/ml of 3-amino-1,2,4-triazole (AMT) singly or in combination with alpha-hed (1.5 or 3 microg/ml) and H2O2 (8.8 microM) during 1 h. The catalase activity was also biochemically measured after treating cells with alpha-hed at 1.5, 3, or 15 microg/ml during 1 h. Additionally, the influence of alpha-hed on membrane RedOx potential, pool of reduced glutathione and total protein content was evaluated by flow cytometry. In the pre-treatment, the two concentrations of alpha-hed (1.5 and 3 microg/ml) decreased the lesions induced by H2O2 (8.8 microM) significantly. This decrease was about 57.2% and 66.1%, respectively. Similar results were observed when cells were treated with alpha-hed and H2O2 simultaneously. The decrease of H2O2-induced lesions was about 78.2% and 83.2% (alpha-hed 1.5 and 3 microg/ml, respectively). In the post-treatment protocol, this decrease was not significant. The combination of AMT and H2O2 induced more DNA damage than H2O2 alone (tail moment (TM) means was 31.4% and 21.8%, respectively). When alpha-hed was added to this mixture, TM means were reduced significantly (17.4% for alpha-hed 1. 5 microg/ml and 15.5% for alpha-hed 3 microg/ml). Up to 6.9 microg/ml, alpha-hed enhanced catalase activity (60.5%), followed by a decrease of the activity. Total protein content and membrane RedOx potential were slightly increased up to 11 microg/ml (14% and 3.6%, respectively) followed by a drop and a plateau. Pool of reduced glutathione remained unchanged up to 10 microg/ml then dropped and reached a plateau. In conclusion, alpha-hed could exert its protective effect against H2O2-mediated DNA damage by scavenging free radicals or by enhancing the catalase activity.