1. Toxicokinetics and biochemical toxicology of marine biotoxins Barbara Doerr Supervisors:Frank van Pelt John O´Halloran Kevin James

2. Project Objectives Focus on two strands Toxicity - acute - subchronic - genotoxicity Metabolism - mammalian models 1 - invertebrate models 1,2 biotoxins investigated will include okadaic acid and azapiracid 1 in collaboration with CIT 2 In collaboration with M. McCarthy and CIT

3. General Information Approx. 4000 known phytoplankton species - 60-80 potentially toxin-producing Toxin first assimilated by bivalves & other shellfish Accumulation/transfer throughout the foodweb Significant environmental impact - morbidity/mortalities to birds and marine mammals - major cause of seafood toxic syndroms in humans

4. General Information Main biotoxins associated with seafood toxic syndroms - saxiotoxin - domoic acid - okadaic acid - azaspiracid Main vectors of algal toxins to humans - filter-feeding bivalves (mussels, clams, scallops, oysters) - herbivourous finfish Alexandrium spp. Dinophysis spp. Karenia brevis Azadinium spinosum

5. General Information Symptoms (acute toxicity) Common symptoms - nausea, vomiting, severe diarrhoea, stomach cramps Toxin specific symptoms - paralysis, respiratory difficults - headache, confusion, disorientation Occur rapid (30min to 18h) Full recovery of clinical symptoms in most cases after a few days

6. General Information For some biotoxins mechanisms of toxicity/molecular targets well established - okadaic acid: Phosphatase 1 and 2A For others information limited - azaspiracid

7. Toxicity Human cell lines (in vitro model) - cell adhesion (accumulation of E-cadherin) - disruption of cytoskelatal structures (f-actin) Ito et al. (2000)

8. Toxicity In vivo studies (Mouse/Rat) - inflammation - necrosis (liver, lymphocytes) - neurological symptoms - oedema (lung, stomach) - increased liver weight - tumors as a late effect (lung)

9. Toxicity Starting point: electrophysiological investigation in target tissue (Ussing chamber) Measuring a) fluid/electrolytes - ion-transport across tissues b) permeability/tissue integrity - resistence - flux Methodology established & optimised using OA, subsequently other biotoxins would have been investigated

10. Ussing chamber C = control

11. Ussing chamber Cell monolayers (2d approach) - more sensitive - less interference - CaCo cells - treatment as for whole tissue Conclusion - method has been established - results not comparable

12. Focus of 2nd year research Genotoxicity Metabolism in vitro

13. Toxicity Genotoxicity - micronucleus assay Cytotoxicity/apoptosis - annexin staining Cells - HeLa (start) - CaCo, HepG2, lung cells HeLa cells CaCo cells HepG2 cells Human lung cells

14. Toxicity In vitro micronucleus assay - cytogenic damage (mammalian cells) - micronuclei a) chromosome loss b) chromosome/chromatid fragments - detection a) microscope b) flow cytometer www.nature.com

15. Toxicity Microscope - visual detection of micronuclei Flow cytometer - higher sample number - higher sensistivity Annexin staining (flow cytometer) - appoptosis/viability of cells www.flow.csc.mrc.ac.uk Dopp et al. (1994)

16. Toxicity Microscope - staining with propidium iodide (PI) Blank

17. Toxicity CdCl 2 EMS - micronuclei (MN) in presence of CdCl 2 and EMS

18. Metabolism Distribution and metabolism of biotoxins poorly described Do they influence type and extend of toxicity? Most biotoxins are lipophilic e.g. azaspiracid - distribution, metabolism, elimination unknown - indications that compunds are persistent - further/different metabolism in mammalians/humen - which enzymes are involved? Long term effects?

19. Thank you!

20. References Alfonso, A., Y. Roman, et al. (2005). "Azaspiracid-4 inhibits Ca2+ entry by stored operated channels in human T lymphocytes." Biochem Pharmacol 69(11): 1627- 1636. Clark L.L. (2009). "A guide to Ussing chamber studies in mouse intestine.“ Am J Physiol Gastrointest Liver Physiol 296: 1151-1166 Ito, E., M. Satake, et al. (2002). "Chronic effects in mice caused by oral administration of sublethal doses of azaspiracid, a new marine toxin isolated from mussels." Toxicon 40(2): 193-203. Ito, E., M. Satake, et al. (2000). "Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland." Toxicon 38(7): 917-930. James K.J., Carey B., O´Halloran J., van pelt F., Skrabácová Z. (2009), Shellfish Toxicity – Epidemiology and Human Health Implications of Marine Algal Toxins. Epidemiology and Infection James, K. J., M. J. Fidalgo Saez, et al. (2004). "Azaspiracid poisoning, the food-borne illness associated with shellfish consumption." Food Addit Contam 21(9): 879-892. Nzoughet, K. J., J. T. Hamilton, et al. (2008). "Azaspiracid: first evidence of protein binding in shellfish." Toxicon 51(7): 1255-1263. Roman, Y., A. Alfonso, et al. (2002). "Azaspiracid-1, a potent, nonapoptotic new phycotoxin with several cell targets." Cell Signal 14(8): 703-716. Roman, Y., A. Alfonso, et al. (2004). "Effects of Azaspiracids 2 and 3 on intracellular cAMP, [Ca2+], and pH." Chem Res Toxicol 17(10): 1338-1349. Ronzitti, G., P. Hess, et al. (2007). "Azaspiracid-1 alters the E-cadherin pool in epithelial cells." Toxicol Sci 95(2): 427-435. Rossini, G. P. (2005). "Functional assays in marine biotoxin detection." Toxicology 207(3): 451-462. Twiner, M. J., N. Rehmann, et al. (2008). "Azaspiracid shellfish poisoning: a review on the chemistry, ecology, and toxicology with an emphasis on human health impacts." Mar Drugs 6(2): 39-72. Ueoka, R., A. Ito, et al. (2009). "Isolation of azaspiracid-2 from a marine sponge Echinoclathria sp. as a potent cytotoxin." Toxicon 53(6): 680-684. Vilarino, N., K. C. Nicolaou, et al. (2007). "Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells." Biochem Pharmacol 74(2): 327-335.