Toxicokinetics and biochemical toxicology of marine biotoxins

Barbara Doerr

Supervisors: Frank van Pelt

John O´Halloran

Kevin James

Project Objectives

Focus on two strands

• Toxicity

- acute

- subchronic

- genotoxicity

• Metabolism

- mammalian models1

- invertebrate models1,2

• biotoxins investigated will include okadaic acid and azapiracid

1 in collaboration with CIT2 In collaboration with M. McCarthy and CIT

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

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

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

General Information

• For some biotoxins mechanisms of toxicity/molecular targets well established

- okadaic acid: Phosphatase 1 and 2A

• For others information limited

- azaspiracid

Toxicity

• Human cell lines (in vitro model)

- cell adhesion (accumulation of E-cadherin)

- disruption of cytoskelatal structures (f-actin)

Ito et al. (2000)

Toxicity

• In vivo studies (Mouse/Rat)

- inflammation

- necrosis (liver, lymphocytes)

- neurological symptoms

- oedema (lung, stomach)

- increased liver weight

- tumors as a late effect (lung)

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

Ussing chamber

Resistance over timein all 4 chambers

C D E F0

25

50

75

100t=0t=30t=60t=90t=120

Chambers

Res

ista

nce

(%

t=

0)

C = control

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

Focus of 2nd year research

• Genotoxicity

• Metabolism in vitro

Toxicity

• Genotoxicity

- micronucleus assay

• Cytotoxicity/apoptosis

- annexin staining

• Cells

- HeLa (start)

- CaCo, HepG2, lung cells

HeLa cells

CaCo cells

HepG2 cells

Human lung cells

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

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)

Toxicity

• Microscope

- staining with propidium iodide (PI)

Blank Blank

Toxicity

CdCl2 EMS

- micronuclei (MN) in presence of CdCl2 and EMS

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?

Thank you!

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.