Marine Biotoxins: effects on bivalves and

human cells

Frank van Pelt1,2 Moira McCarthy1,3, Barbara Dörr1,2, Ambrose Furey1,4, Kevin James1,3 and Bebhine Carey1,4 and John O’Halloran1,3.

1Environmental Research Institute, University College Cork

2Department of Pharmacology and Therapeutics, University College Cork

3School of Biological, Earth and Environmental Sciences, University College Cork

4PROTEOBIO, Department of Chemistry, Cork Institute of Technology

PROT E OB IOPROT EOB IOMASS SPECTROMETRY CENTRE FOR PROTEOMICS AND BIOTOXIN RESEARCH

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

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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

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ViableEarly ApoptosisLate Apoptosis/necrosis

EMS 24 h

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AZA 148 h

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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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DTX-2 accumulation in diaion and amberlite

ng/g Diaion ng/g Amberlite

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