Arachidonic acid cascade inhibitors modulate phorbol ester-induced oxidative stress in female ICR mouse skin: differential roles of 5-lipoxygenase and cyclooxygenase-2 in leukocyte infiltration and activation.

We recently developed a short-term assay for skin H2O2 generation induced by double 12-O-tetradecanoylphorbol-13-acetate (TPA) applications for mechanistic study on skin epidermal carcinogenesis. In the present study, we investigated the individual roles of arachidonic acid metabolism in H2O2 generation in mouse skin inflammation. The experiments using inhibitors of arachidonic acid (AA) metabolism showed that corticosteroid and a lipoxygenase (LO) inhibitor expectedly suppressed double TPA application-induced H2O2 generation through the interference of chemotactic action but not by direct decomposition or scavenging. We also demonstrated that the treatment of AA (1 mumol) and 5-LO metabolites including leukotriene B4 (LTB4) partly mimicked, though soybean LO-derived lipid hydroperoxide and prostaglandins did not, the priming effect evaluated by edema formation and leukocyte infiltration. We also confirmed that inflammatory leukocytes accumulated by LTB4 generated a significant amount of H2O2 by TPA stimulation. These results suggested that 5-LO metabolites of AA are the potential key molecules in the TPA-induced priming event. Interestingly, the cyclooxygenase (COX-) 2-selective inhibitor nimesulide (NS) and celecoxib (CXB) showed different responses than those of other inhibitors. These agents showed no specific potential to inhibit the priming event but significantly suppressed H2O2 generation, lipid peroxidation, and hyperplasia in mouse skin. From the results based on an in vitro leukocyte differentiation model, we speculated that the antioxidant effect of the COX-2 inhibitors might be partly associated with both counteraction of proinflammatory cytokine-enhanced ROS generation and inhibition of CD11b, an important molecule for cell adhesion, expression. Indeed, the topical application of NS attenuated the number of infiltrated leukocytes induced by TPA in mouse skin. Thus, these gathered data indicated the differential roles of 5-LO and COX-2 in leukocyte adhesion, infiltration, and H2O2 generation.