Generation of Reactive Oxygen Species and Subsequent DNA Fragmentation in Bovine Cultured Somatic Cells

The present study was conducted to examine the reactive oxygen species (ROS) generation levels and subsequent DNA damage in the bovine cultured somatic cells. Bovine ear skin cells were classified by serum starvation, confluence and cycling cells. Cells were stained in 10 ${\mu}M$ dichlorohydrofluorescein diacetate ($H_2DCFDA$) or 10 ${\mu}M$ hydroxyphenyl fluorescein (HPF) dye to measure the $H_2O_2$ or $^{\cdot}OH$ radical levels. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each cell. $H_2O_2$ and $^{\cdot}OH$ radical levels of cultured somatic cells were high in confluence group ($7.1{\pm}0.7$ and $8.4{\pm}0.4$ pixels/cell, respectively) and significantly low in serum starvation group ($4.9{\pm}0.4$ and $7.0{\pm}0.4$ pixels/cell, respectively, p<0.05). Comet tail lengths of serum starvation ($148.3{\pm}5.7$ ${\mu}M$) and confluence ($151.1{\pm}5.0$ ${\mu}M$) groups were found to be significantly (p<0.05) increased in comparison to that of cycling group ($137.1{\pm}7.5$ ${\mu}M$). These results suggest that the culture type of donor cells can affect the ROS generation, which leads the DNA fragmentation of the cells.