Induction of reactive oxygen species without 8-hydroxydeoxyguanosine formation in DNA of initiated mouse keratinocytes treated with 12-O-tetradecanoylphorbol-13-acetate.

Evidence for the involvement of oxidative stress in 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated tumor promotion has focused on non-initiated immune cells, tumor cell lines and non-initiated epidermis treated in vivo. This paper reports the effects of TPA on 8-hydroxydeoxyguanosine (8OHdG) formation and the generation of reactive oxygen species (ROS) in cloned initiated mouse epidermal keratinocytes in order to determine whether TPA can directly damage DNA through ROS production within the keratinocytes. Using high performance liquid chromatography with electrochemical detection (HPLC-EC), TPA did not induce 8OHdG formation in DNA of initiated keratinocytes treated under a variety of conditions. The reliability of the HPLC-EC system is demonstrated by (i) the linearity of the 8OHdG standard curve; (ii) the consistency of 8OHdG measurements in calf thymus and cellular DNA; and (iii) the dose-dependent increase in 8OHdG in DNA of initiated keratinocytes treated with UVC in the presence and absence of H2O2. Though not DNA-damaging, TPA induced a 65% increase in ROS (P < 0.05) as detected by luminol-dependent chemiluminescence. These results support a mechanism for the role of oxidative stress in tumor promotion that does not involve direct DNA damage to the keratinocyte target cell. The relationship between ROS, signal transduction and tumor promotion is discussed in light of the above results which is consistent with the role of TPA-induced ROS as second messengers in signal transduction.