Oxidative stress induced by palytoxin in human keratinocytes is mediated by a H+-dependent mitochondrial pathway

In the last decades, massive blooms of palytoxin (PLTX)-producing Ostreopsis cf. ovata have been observed along 22 Mediterranean coasts, usually associated to human respiratory and cutaneous problems. At the molecular level, 23 PLTX induces a massive intracellular Na+ influx due to the transformation of Na+/K+ ATPase in a cationic chan- 24 nel. Recently, we have demonstrated that Na+ overload is the crucial step in mediating overproduction of reactive 25 oxygen species (ROS) and cell death in human HaCaT keratinocytes, tentatively explaining PLTX-induced skin ir- 26 ritant effects. In the present study the molecular mechanisms of ROS production induced by PLTX-mediated Na+ 27 intracellular overload have been investigated. In HaCaT cells, PLTX exposure caused accumulation of superoxide 28 anion,butnotofnitricoxideorperoxynitrite/hydroxylradicals.EvenifRT-PCRandwesternblotanalysisrevealed 29 an early NOX-2 and iNOS gene and protein over-expressions, their active involvement seemed to be only partial 30 since selective inhibitors did not completely reduce O2− production. A significant role of other enzymes (COX-1, 31 COX-2,XO)wasnotevidenced.Nigericin,thatcounteractsNa+-mediatedH+-imbalance,dissipatingΔpHacross 32 mitochondrial inner membrane, and the uncouplers DNP significantly reduced O2− production. These inhibitions 33 were synergistic when co-exposed with complex-I inhibitor rotenone. These results suggest a novel mechanism 34 of O2− production induced by PLTX-mediated ionic imbalance. Indeed, the H+ intracellular overload that follows 35 PLTX-induced intracellular Na+ accumulation, could enhance ΔpH across mitochondrial inner membrane, that 36 seems to be the driving force for O2− production by reversing mitochondrial electron transport.