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
Mussel HL
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Control T
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s
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s0
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* *
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ail D
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ail D
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Oyster HL
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1-day
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ail D
NA
Oyster HP
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**
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ail D
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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
% t
ail D
NA
0
20
40
60
% o
f to
tal P
op
ula
tio
n
0
25
50
75
100
% ta
il DNA
0
20
40
60
% o
f tot
al po
pulat
ion
0
25
50
75
100
ViableEarly ApoptosisLate Apoptosis/necrosis
EMS 24 h
OA24 h
AZA 148 h
% t
ail D
NA
0
20
40
60
% o
f to
tal p
op
ula
tio
n
0
20
40
60
80
100
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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Aug 4 SB Aug 4 NB Aug 4 WT Aug 17 SB
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DTX-2 accumulation in diaion and amberlite
ng/g Diaion ng/g Amberlite
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