Assessment of molecular DNA damage by comet assay in HeLa cells exposed to rohituka, Aphanamixis polystachya (wall.) R.N. Parker

The stem bark extract of rohituka, Aphanamixis polystachya Wall. Parker (family Meliaceae) used traditionally to treat spleen and liver tumors was evaluated for its DNA damaging ability in the HeLa cells. The cells were treated with various concentrations of chloroform fraction of the stem bark of rohituka (APE) for 2, 4, 6 or 8 h to find out the optimum exposure time. HeLa cells embedded in agarose were microelectrophoresed in alkaline conditions to visualize the DNA damage as comets and expressed as Olive Tail Moment (OTM). Incubation of HeLa cells with APE for 6 or 8 h increased the OTM when compared with 2 or 4 h treatment and no significant difference was observed between 6 or 8 h treatment duration as a result, 6 h was considered as an optimum treatment time. The DNA repair kinetics were studied at different post-treatment times in HeLa cells exposed to 5, 10, 25, 50, 75 or 100 μg/ml APE. APE treatment caused a significant rise in the DNA damage as indicated by increased OTM when compared with the dimethyl sulfoxide treatment (p< 0.01). An approximate 7, 12, 18, 25, 34, 40-folds increase in baseline DNA damage was recorded in HeLa cells treated with 5, 10, 25, 50, 75 or 100 μg/ml APE in comparison with dimethyl sulfoxide treatment. When the APE treated cells were allowed to repair for various posttreatment times, a concentration-dependent inhibition in repair of DNA damage was observed. The repair of DNA damage was inversely related to APE concentration. A mild but non-significant repair of the damaged DNA was observed up to 2 h post-APE treatment, which remained unaltered thereafter. Exposure of HeLa cells to different concentrations of APE resulted in a concentration-dependent decline in the growth kinetics up to day 5 and 6, respectively followed by a reduction in the clonogenic potential of HeLa cells in a concentration dependent manner. Our study demonstrates that rohituka triggers DNA damage in HeLa cells at molecular level that may be the major cause of reduced clonogenicity and growth kinetics. Keywords: Aphanamixis polystachya; HeLa cells; Comet assay; DNA damage; Growth kinetics