toxicokinetics and biochemical toxicology of marine biotoxins barbara doerr supervisors:frank van...
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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.