Figure 3. Silibinin reverses CEES-induced DNA damage in skin cells.

Mouse epidermal JB6 cells (A) and fibroblasts (B) were seeded (120,000 cells/plate) and grown overnight in 60 mm petri dishes. The cells were then exposed to DMSO or 0.5 mM CEES in DMSO, or treated with either 10 µM silibinin alone or with silibinin following 30 min CEES exposure for 1 h. DNA damage in cells was measured using single cell gel electrophoresis (SCGE) or alkaline comet assay as detailed under materials and Methods. Damaged DNA seen in the form of comets [as seen in the representative pictures of epidermal cells (A) and fibroblasts (B) on top of graphical presentation of the quantified data in each panel] was scored and images were captured as described under Materials and Methods section. Data are presented as the tail extent moment (TEM; product of tail length and percentage tail DNA) and are mean ± SEM of three independent samples. *, p<0.05 as compared to CEES exposed group. DNA damage was also assessed via the detection of DNA damage markers H2A.X and p53 using western immunoblotting (C and D). Mouse epidermal JB6 cells (C) and dermal fibroblasts (D) grown over night in 100 mm plates were collected after 4 h of DMSO or 0.5 mM CEES exposure, or treatment with either 10 µM silibinin alone or with silibinin following 30 min CEES exposure, and cell lysates prepared in the lysis buffer as detailed under the Materials and Methods. The protein lysate was subjected to SDS-PAGE followed by immunoblotting using antibodies for H2A.X ser139 and p53 ser15 phosphorylations, and total levels of p53, and visualized and scanned using the Odyssey™ Infrared Imager. Protein loading was checked by stripping and re-probing the membranes with β-actin antibody and the results obtained were quantified by densitometric analysis of the immunoblots.