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 Mediterranean coasts, usually associated to human respiratory and cutaneous problems. At the molecular level, PLTX induces a massive intracellular Na+ influx due to the transformation of Na+/K+ ATPase in a cationic channel. Recently, we have demonstrated that Na+ overload is the crucial step in mediating overproduction of reactive oxygen species (ROS) and cell death in human HaCaT keratinocytes, tentatively explaining PLTX-induced skin irritant effects. In the present study the molecular mechanisms of ROS production induced by PLTX-mediated Na+ intracellular overload have been investigated. In HaCaT cells, PLTX exposure caused accumulation of superoxide anion, but not of nitric oxide or peroxynitrite/hydroxyl radicals. Even if RT-PCR and western blot analysis revealed an early NOX-2 and iNOS gene and protein over-expressions, their active involvement seemed to be only partial since selective inhibitors did not completely reduce O2− production. A significant role of other enzymes (COX-1, COX-2, XO) was not evidenced. Nigericin, that counteracts Na+-mediated H+-imbalance, dissipating ΔpH across mitochondrial inner membrane, and the uncouplers DNP significantly reduced O2− production. These inhibitions were synergistic when co-exposed with complex-I inhibitor rotenone. These results suggest a novel mechanism of O2− production induced by PLTX-mediated ionic imbalance. Indeed, the H+ intracellular overload that follows PLTX-induced intracellular Na+ accumulation, could enhance ΔpH across mitochondrial inner membrane, that seems to be the driving force for O2− production by reversing mitochondrial electron transport.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (258 K)Download as PowerPoint slideHighlights► PLTX induces superoxide (O2−) production by reversing mitochondrial transport chain. ► The mechanism of O2− production is dependent on PLTX-induced ionic imbalance. ► The results led to the proposal of a novel mechanism of O2− production. ► Enhanced Na+, by increasing H+ level and blocking mitochondrial chain, induces O2−.