P21-64 Characterization of the toxic effects by the marine toxin ovatoxin-a on human skin keratinocytes

Ovatoxin-a (OVTX-a) is the major palytoxin (PLTX) analogue identified in the benthic dinoflagellate Ostreopsis cf. ovata from the Mediterranean area. Humans can be exposed to OVTX-a mainly through inhalation of marine aerosol and/or skin contact with seawater during dinoflagellates’ blooms, with possible threat to public health. Despite the hazard posed by PLTX has been extensively characterized, very few data are currently available for OVTX-a. Hence, this study was aimed at assessing the cutaneous in vitro effects of OVTX-a using spontaneously immortalized HaCaT keratinocytes. The effects of OVTX-a (1x10-16–1x10-7 M) in HaCaT cells were compared to those of the reference toxin (PLTX), in terms of cell viability, cell necrosis, reactive oxygen species (ROS) production and mitochondrial depolarization. After 4 h exposure, OVTX-a induced a concentration-dependent cell viability reduction (EC50=8.3x10-9 M), with one order of magnitude lower potency than that of PLTX (EC50=3.7x10-10 M). Accordingly, OVTX-a induced a concentration-dependent increase of cell necrosis with a potency lower than that of PLTX. Moreover, despite OVTX-a increased ROS production similarly to PLTX, it caused a lower mitochondrial depolarization in keratinocytes with respect to the reference toxin. Then, to investigate the possible mechanisms involved in OVTX-a cytotoxicity, the same cellular parameters were assessed in presence of ouabain (OUA, 1.0x10-5 M) as inhibitor of Na+/K+ ATPase, the molecular target of PLTX, or diphenyliodonium chloride (DPI, 5.0x10-6), a non-specific inhibitor of flavoprotein-based enzymes, known to be involved in PLTX-induced oxidative stress. On the whole, results suggested that OVTX-a and PLTX share the same molecular target and mechanism of cytotoxicity. In conclusion, this study provided a contribution in the characterization of the toxic effects of OVTX-a in skin keratinocytes. Although less potent than PLTX, the OVTX-a cytotoxic effects at nanomolar concentrations after a short exposure time rise some concern for humans exposed to this toxin during Ostreopsis blooms.