Marine Biotoxins: effects on bivalves and human cells Frank van Pelt 1,2 Moira McCarthy 1,3, Barbara Dörr 1,2, Ambrose Furey 1,4, Kevin James 1,3 and Bebhine Carey 1,4 and John O’Halloran 1,3. 1 Environmental Research Institute, University College Cork 2 Department of Pharmacology and Therapeutics, University College Cork 3 School of Biological, Earth and Environmental Sciences, University College Cork 4 PROTEOBIO, Department of Chemistry, Cork Institute of Technology

 Toxins produced by phytoplankton species.  Accumulate in shellfish ► foodchain  Major cause of seafood toxic syndroms in humans  Diarrhetic Shellfish Poisoning: okadaic acid (OA) dinophysistoxins (DTXs), pectenotoxins (PTXs)  Amnesic Shellfish Poisoning : domoic acid (DA) and analogues  Paralytic Shellfish Poisoning: saxitoxins (STXs), spirolides (SPXs)  Neurotoxic Shellfish Poisoning : brevetoxins (BTX)  Azaspiracid Shellfish Poisoning : azaspiracids (AZAs) and analogues Marine Biotoxins

Toxicological information on marine biotoxins is incomplete  Effects on shellfish morbidity and morality only partly investigated Environmental and economical consequences  Mammalian toxicological information, apart for acute toxicity, is limited  OA: Genotoxicity data inconclusive, proven tumour promoter.  AZAs: No data on genotoxicity, some tumours observed in longer-term toxicity study (inconclusive) Chronic human health and risk assessment Marine Biotoxins

1.OA-induced effects in shellfish  Histology  DNA fragmentation (Comet assay) 2.OA- and AZA–induced effects in human cell lines  DNA fragmentation (Comet assay)  Viability and apoptosis 3.Marine biotoxin sampling at Lough Hyne Overview

 Single Cell Gel Electrophoresis assay  extremely sensitive DNA damage assay  DNA fragmentation:  Genotoxicity  Apoptosis Comet Assay

 The blue mussel, the pacific oyster and the manila clam were exposed to okadaic acid (OA)  Acute exposure, single dose of 2µg/15l tank.  Sub-acute exposure, daily dose of either 1µg or 40µg /15l tank for 7 days.  Animals sampled at 1 day, 3 days and 7 days  Tissue damage (histology) and DNA fragmentation (Comet assay) were measured Shellfish exposure experiments

OA-induced damage in shellfish Control clam, undamaged mantle Lipofuscin granule formation, clam mantle 7 day OA exposure: 1µg/15l tank Sloughing of cells and lipofuscin formation, mussel mantle 1 day OA exposure: 40µg/15l tank

OA-induced DNA fragmentation in shellfish Acute exposure of 2µg OA/15l tank (single dose) DNA fragmentation in haemolymph and hepatopancreas cells followed over 7 days using the Comet assay * denotes significant difference from the negative controls (p < 0.05).

 Both single and repeated dosing of OA induces damage in shellfish at doses well below ‘harmful algal bloom’ levels.  Histological evidence in the mantle, haemolymph and hepatopancreas cells  DNA fragmantation in haemolymph and hepatopancreas cells  OA, even at low doses, affect shellfish health status. OA-induced damage in shellfish

Effects of OA and AZA-1 on Jurkat-T cells EMS 24 h OA 24 h AZA 1 48 h Comet analysis Viability/Apoptosis analysis

 OA induced DNA fragmentation is paralleled by decrease in cell viability and increase in apoptosis in Jurkat-T cells.  Similar results were observed with CaCo2 (intestial) and HepG2 (hepatic) cells.  AZA-1 does not cause an substantial increase in DNA fragmentation but does cause a dose-dependant loss in cell viability and increase in apoptosis  Neither OA nor AZA-1 appear to be overtly genotoxic Effects of OA and AZA-1 on Jurkat-T cells

Marine biotoxin sampling at Lough Hyne Passive sampling using Solid phase adsorption and toxin tracking (SPATT) and adsorptive resin (2 weeks submersion) Active sampling using pump and multiple filtration steps with adsorptive resin (400 l/h for 8days )

 OA, DTX1,DTX2, PTx2 and PTX2-SA were detectable following active and passive sampling (location, time, depth dependant)  13 desmethyl SPX C and Pinnatoxin G were detected in following active sampling Marine biotoxin sampling at Lough Hyne

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