Toxicity of palytoxin and Ostreopsis extracts on intestinal, liver, and lung human cells

Transcription

1 v1 Toxicity of palytoxin and Ostreopsis extracts on intestinal, liver, and lung human cells 1 Ferron PJ, 2 Solino L., 1 Lanceleur R., 2 Diogene J., 1 Fessard V. 1 : French Agency for Food and Safety, Toxicology contaminant Unit 2 : Institute of Agrifood Research and Technology, Spain, Department of aquaculture GDR Phycotox 216 Villefranche sur mer

2 Diapositive 1 v1??? mettre le logo da l'irta v.fessard; 14/3/216

3 Ostreopsis Benthic dinoflagelates 9 species for Ostreopsis genus Commonly found in tropical waters of the Pacific Expansion of Ostreopsis ovata into Mediterranean sea Credit : Smithsonian Institution Inhalation or skin exposure Ostreopsis Human toxicity Credit : Smithsonian Institution Accumulation in food chain Oral exposure 2

4 Palytoxin and Ovatoxins Molecular Mass = 268,13 Da! Very large non polymeric natural molecules Palytoxins: several analogues Ovatoxins are palytoxin like toxins produced by Ostreopsis ovata Palytoxins and ovatoxins target Na+/K+ ATPase 3

5 Culture of Ostreopsis ovata Monoclonal strain of Ostreopsis ovata from the agricultural Institute of Catalogna ID : IRTA SMM 11 1 Isolated in Erbro strait, in South Catalogna Identified as a producer of ovatoxins Conditions de culture : Diluted Gaillard Medium 36% Salinity 24 C 1 µmol photons.m².s 1 cycle 12h/12h 3 weeks of culture 4

6 Purification and fraction Ostreopsis culture Ostreopsis ovata crude extract OSTR Concentration Dissolved in methanol and sonicated Sedimentation and filtration Liquid chromatography UPLC Water / Acetonitrile Evaporation and suspension in MeOH Ostreopsis ovata crude extract OSTR Purified Ovatoxins OVA 5

7 Toxicity of Palytoxin, OSTR and OVA Palytoxin target organs Aerosol exposure, the main way of exposure in mediteranean Lung Liver Organ highly exposed through blood filtration and involved in detoxication Food exposure, mostly reported in Pacific areas Intestine 6

8 Evaluation of OSTR and OVA toxicity A549 : human pulmonary cell line Isolated from Adenocarcinoma Similar as pulmonary alveolar cells : human hepatocyte cell line Isolated from Adenocarcinoma Similar as hepatocytes and biliary cells Caco 2 : human enterocyte cell line Isolated from Adenocarcinoma Similar as enterocyte 7

9 Lysosomal toxicity Based on the neutral red uptake assay PLTX OSTR26 OVA A Caco % viability 75 5 % viability 75 5 % viability PLTX [µg/ml]; OSTR/OVA [µgalg.eq/ml] PLTX [µg/ml]; OSTR/OVA [µgalg.eq/ml] PLTX [µg/ml]; OSTR/OVA [µgalg.eq/ml] Palytoxin [µg/ml] OSTR26 [µgalg.eq/ml] A549 Caco-2 IC5 95% CI IC5 95%CI IC5 95%CI 9 7,3-11,1 15,1 12,8-17,8 NE 139,6 9,3-215,7 357,9 244,8-523,2 NE OVA present no toxic effects 8

10 Lysosomal toxicity Based on the neutral red uptake assay PLTX OSTR26 OVA A Caco % viability 75 5 % viability 75 5 % viability PLTX [µg/ml]; OSTR/OVA [µgalg.eq/ml] PLTX [µg/ml]; OSTR/OVA [µgalg.eq/ml] PLTX [µg/ml]; OSTR/OVA [µgalg.eq/ml] Palytoxin [µg/ml] OSTR26 [µgalg.eq/ml] A549 Caco-2 IC5 95% CI IC5 95%CI IC5 95%CI 9 7,3-11,1 15,1 12,8-17,8 NE 139,6 9,3-215,7 357,9 244,8-523,2 NE OVA present no toxic effects Intestinal cells are not affected by PLTX and OSTR 9

11 Lysosomal toxicity Based on the neutral red uptake assay PLTX OSTR26 OVA A Caco % viability 75 5 % viability 75 5 % viability PLTX [µg/ml]; OSTR/OVA [µgalg.eq/ml] PLTX [µg/ml]; OSTR/OVA [µgalg.eq/ml] PLTX [µg/ml]; OSTR/OVA [µgalg.eq/ml] Palytoxin [µg/ml] OSTR26 [µgalg.eq/ml] A549 Caco-2 IC5 95% CI IC5 95%CI IC5 95%CI 9 7,3-11,1 15,1 12,8-17,8 NE 139,6 9,3-215,7 357,9 244,8-523,2 NE OVA present no toxic effects Intestinal cells are not affected by PLTX and OSTR Pulmonary cells are more sensitive than hepatocytes 1

12 Cellular inflammation Release of Interleukin 8 Chemokine involved in polynuclear leukocytes recruitment Positive control PLTX OSTR26 OVA26 15 A549 6 Caco-2 6 IL-8 in pg/ml IL-8 in pg/ml 4 2 IL-8 in pg/ml PLTX [µg/ml]; OSTR/OVA [µgalg/ml] PLTX [µg/ml]; OSTR/OVA [µgalg/ml] PLTX [µg/ml]; OSTR/OVA [µgalg/ml] Low effect on pulmonary cells 11

13 Cellular inflammation Release of Interleukin 8 Chemokine involved in polynuclear leukocytes recruitment Positive control PLTX OSTR26 OVA26 15 A549 6 Caco-2 6 IL-8 in pg/ml IL-8 in pg/ml 4 2 IL-8 in pg/ml PLTX [µg/ml]; OSTR/OVA [µgalg/ml] PLTX [µg/ml]; OSTR/OVA [µgalg/ml] PLTX [µg/ml]; OSTR/OVA [µgalg/ml] Low effect on pulmonary cells OVA has no effect on enterocytes and hepatocytes 12

14 Cellular inflammation Release of Interleukin 8 Chemokine involved in polynuclear leukocytes recruitment Positive control PLTX OSTR26 OVA26 15 A549 6 Caco-2 6 IL-8 in pg/ml IL-8 in pg/ml 4 2 IL-8 in pg/ml PLTX [µg/ml]; OSTR/OVA [µgalg/ml] PLTX [µg/ml]; OSTR/OVA [µgalg/ml] PLTX [µg/ml]; OSTR/OVA [µgalg/ml] Low effect on pulmonary cells OVA has no effect on enterocytes and hepatocytes OSTR induced high response on intestinal cells 13

15 Profile of cellular toxicity A549 Caco 2 PLTX OSTR OVA Immunostaining for automated microscopy Cell Count Apoptosis Genotoxicity Inflammation Oxydative stress DAPI Active Caspase3 γh2ax NF kb NRF2 [OA] 15 nm 14

16 High content screening Cellomique : Discipline basée sur l'analyse quantitative de donnée sur des cellules par microcopie Trois grandes composantes: acquisition d'images, analyse d'images, gestion des données 15

17 Profile of cellular toxicity Palytoxin Viabilité % Caspase 3 Fold H2AX Fold NFkB NRF2 15 A Caco PLTX [µg/ml] PLTX [µg/ml] PLTX [µg/ml]. High toxicity on pulmonary cells 16

18 Profile of cellular toxicity Palytoxin Viabilité % Caspase 3 Fold H2AX Fold NFkB NRF2 15 A Caco PLTX [µg/ml] PLTX [µg/ml] PLTX [µg/ml]. High toxicity on pulmonary cells Enterocytes are not sensitive to PLTX 17

19 Profile of cellular toxicity Palytoxin Viabilité % Caspase 3 Fold H2AX Fold NFkB NRF2 15 A Caco PLTX [µg/ml] PLTX [µg/ml] PLTX [µg/ml]. High toxicity on pulmonary cells Enterocytes are not sensitive to PLTX PLTX induce apoptosis, genotoxicity and oxydative stress in pulmonary cells and hepatocytes 18

20 Profile of cellular toxicity OSTR Viabilité % Caspase 3 Fold H2AX Fold NFkB NRF2 15 A Caco OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] High toxicity on pulmonary cells 19

21 Profile of cellular toxicity OSTR Viabilité % Caspase 3 Fold H2AX Fold NFkB NRF2 15 A Caco OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] High toxicity on pulmonary cells Low effect on enterocytes 2

22 Profile of cellular toxicity OSTR Viabilité % Caspase 3 Fold H2AX Fold NFkB NRF2 15 A Caco OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] High toxicity on pulmonary cells Low effect on enterocytes OSTR induced apoptosison pulmonary cells and hepatocytes 21

23 Profile of cellular toxicity OSTR Viabilité % Caspase 3 Fold H2AX Fold NFkB NRF2 15 A Caco OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] High toxicity on pulmonary cells Low effect on enterocytes OSTR induced apoptosison pulmonary cells and hepatocytes Oxydative stress on pulmonary cells 22

24 Profile of cellular toxicity OSTR Viabilité % Caspase 3 Fold H2AX Fold NFkB NRF2 15 A Caco OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] OSTR [µg Alg/eq. ml] High toxicity on pulmonary cells Low effect on enterocytes OSTR induced apoptosison pulmonary cells and hepatocytes Oxydative stress on pulmonary cells OSTR induced genotoxicity on hepatocytes 23

25 Analysis of Ostreopsis crude extract Hybrid linear ion trap LTQ Orbitrap XL Fourier transform mass spectrometer (FTMS) Toxic compound IRTA-SMM-11-1 (OSTR) ( Microalgae Eq/mL) Concentration (ng/ml) % Putative palytoxin ovatoxin a ovatoxin b ovatoxin c ovatoxin d ovatoxin e ovatoxin g total

26 Analysis of Ostreopsis crude extract Hybrid linear ion trap LTQ Orbitrap XL Fourier transform mass spectrometer (FTMS) Toxic compound IRTA-SMM-11-1 (OSTR) ( Microalgae Eq/mL) Concentration (ng/ml) % Putative palytoxin ovatoxin a ovatoxin b ovatoxin c ovatoxin d ovatoxin e ovatoxin g total

27 Analyzed of ovatoxins fraction OVTX-a (ng/ml) OVTX- b (ng/ml) *OVTX-like components (ng/ml) Total (ng/ml) OVA OSTR : 186 ng/ml of OVTX A OVA : 24 ng/ml of OVTX A 74 more OVTX A in OSTR than in OVA! 26

28 Summary of toxicity profile Cell lines Toxins EC5 NRU IL 8 secretion Viability (based on cell Caspase 3 activation H2AX phosphorylatio NFkB translocation NRF2 translocation A549 12,51 pg/µl Palytoxin Caco 2 NE + NE NE NE NE NE 15,53 pg/µl / +/ A µalgeq/ml NE NE + OSTR26 OVA26 Caco 2 NE + NE NE NE + NE 39, NE NE µalgeq/ml + A549 NE + NE NE NE NE + Caco 2 NE NE NE NE NE NE NE NE +/ NE NE NE NE NE NE = No Effect 27

29 Conclusion Palytoxin is highly toxic on human pulmonary and hepatocytes cell lines Strain IRTA SMM 11 1 of Ostreopsis ovata is a good producer of ovatoxin A and B Toxicity Lung Liver Intestine Palytoxin OVTX-A / OVTX-B Apoptose YES YES Genotoxicity YES Only liver cells Oxydative stress YES Only lung cells Inflammation YES Only intestinal cells 28

30 Perspectives More purified materials Investigate new cellular markers qpcr and tissue specific cytokines 29