An update on the in vitro toxicity and detection of ovatoxin-a

In the last two decades, Ostreopsis cf. ovata blooms have been recorded with increasing frequency and intensity along the Mediterranean coasts where they have been shown to produce palytoxin (PLTX)-like compounds, principally ovatoxin-a (OVTX-a). Simultaneously, episodes of adverse effects in humans (e.g. cough, fever, dyspnea, rhinorrhea and dermatitis) after inhalation and/or skin contact with marine aerosols or direct contact to seawater were also reported. However, to date no cases of human poisonings linked to OVTXs-contaminated seafood have been documented in this area. Considering the increasing sanitary problems ascribable to OVTXs during Ostreopsis blooms, development of rapid detection methods for risk assessment is of particular importance. Due to the lack of sufficient toxin for in vivo studies, OVTX-a was evaluated for its potential toxicity in vitro using skin HaCaT keratinocytes, one of the most sensitive cell models to the reference toxin PLTX. OVTX-a was about 100-fold less potent than PLTX in reducing HaCaT cell viability (EC50=1.1x10-9 M vs 1.8x10-11 M, MTT test), consistent with their binding affinities to these cells (Kd =1.2x10-9 vs 2.7x10-11 M, saturation experiments on intact cells). Accordingly, OVTX-a hemolytic activity was lower than that of PLTX (EC50=3.4x10-8 M vs 5.9x10-9 M). Although OVTX-a cytotoxicity appears to be lower than that of the reference compound PLTX, its significant effect at nanomolar concentrations raises significant concerns for human health. A sandwich immunoassay (ELISA) previously developed and characterized for PLTX, was assessed for its suitability to quantify OVTX-a. The assay was able to detect OVTX-a in a sensitive and accurate manner (Bias: 0.3%), in both O. cf. ovata extracts (LOQ = 9.6 ng/ml) and contaminated mussels (LOQ = 11 mg/kg). As such, it is a suitable screening method for OVTX-a detection within monitoring programs. The anti-PLTX antibodies used in the ELISA also allowed OVTX-a immunolocalization in O. cf. ovata samples. As expected, toxins were not detectable in the dinoflagellte Coolia monotis, frequently found associated with Ostreopsis blooms. To increase the sensitivity of the ELISA, an ultrasensitive electrochemiluminescence-based sensor for PLTXs detection was developed, taking advantage of the specificity provided by anti-PLTX antibodies, the good conductive properties of carbon nanotubes, and the excellent sensitivity achieved by a luminescence based transducer.