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Audrey Bruneau_In vitro immunotoxicology of quantum dots and comparison with dissolved cadmium and tellurium

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  • 1. In vitro immmunotoxicology of quantum dots and comparison with dissolved cadmium and telluriumA. BRUNEAU CACHICA a,c, F. GAGNE b, M. FORTIER a, C. GAGNON b, P.TURCOTTE b, A. TAYABALI c, T. DAVIS d, M. AUFFRET c, M. FOURNIER a a : INRS Institut Armand Frappier, 521 Boulevard des prairies, Laval, Qc, Canada b : Environnement Canada, 105 McGill, Montréal, Qc, Canada c : IUEM, LEMAR, Place Nicolas Copernic. Technopole Brest Iroise, Plouzané, France d : Canadian Space Agency, CSA, 240 Sparks street, West tower, Ottawa, On. Canada
  • 2. What’s the context ? 1- Immunotoxicity of nanoparticles and chemistry - human results 2- Immunotoxicity according to species 3- Toxicity of nanoparticles vs metals ? Discussion, Conclusion2
  • 3. Introduction, context -Emerging contaminants in wide use (15% of manufactured goods) with large economic impact ($2.6 trillion in 2014) (Biswas, 2005 ; WWICs 2007; Iavicoli et al, 2010) -Dispersed in the environment  Air, soil, water -Currently little regulation of nanoparticles  toxicity studies, principle of precaution3
  • 4. Model nanoparticle: Quantum dots (QDs) ViveNano® Core of cadmium and tellurium Core of (CdTe/ CdS): colour determination Inorganic shell: increases fluorescence and improves stability Organic shell: increases solubility and functional group conjugation (-COOH) 1-10 nm Biomolecules: - immunoglobins - oligonucleotides4
  • 5. Cd/S, Cd/Te nanoparticles, quantum dots (QDs) -Size range 5-10 nm -Stock concentration of 20 mg/ml -Use -Imaging (tumor) -Drug delivery5
  • 6. Material and methods Human Mouse Rainbow trout Blue mussel Homo Mus Oncorhyncus Mytilus edulis sapiens musculus mykiss risk analysis risk analysis risk analysis risk analysis Peripheral blood Spleen Pronephros Hemolymph Purification Nylon membrane filtration gradient Lymphocytes, macrophages, monocytes, hemocytes In vitro exposure : QDs, dissolved CdCl2, NaTeO3 and mix6
  • 7. Material and methods Biomarker analysis (flow cytometry) - Viability  propidium iodide - Phagocytosis  latex beads (1.71 µm ø) - Lymphoblastic transformation  tritiated thymidine (3H) Characterization of QD stability - sterile water - sterile sea water (mussel) - RPMI 1640 serum supplemented (human, mouse) - RPMI without bicarbonate (fish)7
  • 8. Study Goals 1- Study the immunotoxicity of nanoparticles and the chemistry of nanoparticles 2- Compare the immunotoxicity in different model organisms 3- Compare the toxicity of nanoparticles to their metallic content8
  • 9. 1-Immunotoxicity of nanoparticles and chemistry 1.1 Toxicity of QDs, blood cell viability Human monocyte/lymphocyte Autofluorescence 4% * P < 0.05 N=3 ** P< 0.019 *** P < 0.001
  • 10. 1-Immunotoxicity of nanoparticles and chemistry 1.2 Toxicity of QDs, phagocytosis Human 21-hours incubation M1 : Phagocytosis 1 bead and more M2 : Phagocytosis ≥ 3 beads * P < 0.05 N =3 ** P< 0.0110 *** P < 0.001
  • 11. 1-Immunotoxicity of nanoparticles and chemistry 1.3 Toxicity of QDs, lymphoblastic transformation Human Drastic decrease at 15 µg/ml * P < 0.05 N=3 ** P< 0.0111 *** P < 0.001
  • 12. 1-Immunotoxicity of nanoparticles and chemistry 1.4 Characterization Water Sea Water 100 100 80 80 measured measured 60 60 Predicted cadmium 40 40 20 20 concentration highly 0 0 correlated with measured 0 50 100 150 0 50 100 150 expected expected cadmium concentration in all media RPMI RPMI w/o (0.94≤R ≤ 0.99, P<0.001) 150 100 80 measured measured 100 60 40 50 20 0 0 0 50 100 150 0 50 100 150 expected expected12
  • 13. 2- Immunotoxicity according to species Immunotoxicity in model organisms IC 50 = Inhibition concentration for 50% of biological parameter QDs Human Mouse Trout Mussel Macrophage 216 > 952 > 952 435 viability Phagocytosis (≥3 425 > 952 > 952 435 beads) Lymphoblastic 29 4 20 - transformation All data expressed in µg/ml Terrestrial vertebrates were more sensitive than other13 species
  • 14. 3- Toxicity of nanoparticles vs metals ? Toxicity of QDs versus metals - Blue mussel Cd Te Cd/Te N= 9 for QDs, Cd and Cd/Te and N=16 for Te.14 * p<0.05, ** p< 0.001
  • 15. 3- Toxicity of nanoparticles vs metals ? Cd Te Cd/Te Mussel : QDs were more toxic than metals A and B: QDs (N=9) vs. dissolved Cd (N=6); C and D: QDs (N=9) vs. dissolved Te (N=6) ; * p < 0.05, ** p < 0.001, *** p15 < 0.0001
  • 16. IC 50 QDs CdCl2 NaTeO3 Mixed Viability Human 216 19 18 31 Mouse > 952 10 > 18 5 Trout 715 > 109 > 18 > 127 Mussel 582 > 109 > 18 >127 Lymphoblastic transformation Human 29 3 5 29 Mouse 4 3 1 < M1 Trout 20 10 1 3 Other models : QDs were less toxic than metals16
  • 17. Correlation between QDs vs metals Viability Mussel Trout Mice Human Cd 0.09 0.87* 0.73 0.83* Te 0.93* 0.25 0.68 0.97* Mix 0.91* 0.94* 0.77* 0.55 Immunoactivity Cd 0.97* 0.08 - 0.47 0.60 Te 0.74 -0.44 - 0.78* 0.95* Mix 0.79* 0.55 - 0.79* 0.6417
  • 18. Root 1 vs. Root 2 8 5Discriminant Mussel Rainbow trout 7 4 6Analyses 3 5 4 2 3 Root 2 1 2 Root 2 1 0 0 -1 -1 -2 -2 -3 -3 -4 -4 -5 -6 -4 -2 0 2 4 6 8 -20 -15 -10 -5 0 5 10 Root 1 Root 1 3,5 3,0 3,0 2,5 Mouse 2,5 Human 2,0 2,0 1,5 1,5 1,0 1,0 Root 2 Root 2 0,5 0,5 0,0 0,0 -0,5 -0,5 -1,0 -1,0 -1,5 -1,5 -2,0 -2,0 -2,5 -2,5 -10 -8 -6 -4 -2 0 2 4 6 -4 -3 -2 -1 0 1 2 318 Root 1 Root 1
  • 19. Discussion -Total metallic content of nanoparticles conserved in all media - Blue Mussel Toxicity QDs Metals - Other species Toxicity Metals QDs19
  • 20. - Mytilus edulis - Overall immunocompetence response patterns differed between QDs and dissolved metals - Rainbow trout - Responded differently to QDs exposure than other model organisms (Immunostimulation) - Mouse and Human - More sensitive to QDs and dissolved metals than other model organisms. - Human macrophages were the most sensitive to QDs (effect on innate immunity EC50 = 217 µg/ml) - For human: toxicity of the QDs was associated with QD components (≠mouse)20
  • 21. Cytotoxicity of the QDs could be partially due to the presence of dissolved Cd2+ and TeO3 ions in fish, mouse and humans (DA analysis) - Unique effect of QDs (distinct from metal components) observed in mussels and mice only - Rainbow trout and human cells : the immunotoxic effects of QDs were similar to those obtained with the dissolved fraction of Cd and Te mixture Mussels and mice were most able species to discriminate the effects of Cd-based NPs from the effects of dissolved Cd and Te21
  • 22. Conclusions 1- Immunotoxicity of nanoparticles and chemistry - QDs were toxic (viability, phagocytosis, lymphoblastic transformation) - Macrophages and monocytes were less sensitive to QDs and dissolved metals exposition than T lymphocytes (human, mouse and fish) 2- Immunotoxicity according to species Mouse and Human model were the most sensitive species 3- Toxicity of nanoparticles vs metals ? Cytotoxicity of the QDs could be partially due to the presence of dissolved Cd 2+ and TeO3- ions in fish, mouse and humans Not only one specie should be used as a model for the QDs risk assessments but a set of species22
  • 23. Conclusions 1- Immunotoxicity of nanoparticles and chemistry - QDs were toxic (viability, phagocytosis, lymphoblastic transformation) -Macrophages and monocytes were less sensitive to QDs and dissolved metals exposition than T lymphocytes (human, mouse and fis h) 2- Immunotoxicity according to species - Mouse and Human model were the most sensitive species 3- Toxicity of nanoparticles vs metals ? Cytotoxicity of the QDs could be partially due to the presence of dissolved Cd 2+ and TeO3- ions in fish, mouse and humans Not only one specie should be used as a model for the QDs risk assessments but a set of species23
  • 24. Conclusions 1- Immunotoxicity of nanoparticles and chemistry -QDs were toxic (viability, phagocytosis, lymphoblastic transformation) - Macrophages and monocytes were less sensitive to QDs and dissolved metals exposition than T lymphocytes (human, mouse and fis h) 2- Immunotoxicity according to species - Mouse and Human model were the most sensitive species 3- Toxicity of nanoparticles vs metals ? - Cytotoxicity of the QDs could be partially due to the presence of dissolved Cd2+ and TeO3- in fish, mouse and humans Not only one specie should be used as a model for the QDs risk assessments but a set of species24
  • 25. Conclusions 1- Immunotoxicity of nanoparticles and chemistry - QDs were toxic (viability, phagocytosis, lymphoblastic transformation) - Macrophages and monocytes were less sensitive to QDs and dissolved metals exposition than T lymphocytes (human, mouse and fis h) 2- Immunotoxicity according to species Mouse and Human model were the most sensitive species 3- Toxicity of nanoparticles vs metals ? Cytotoxicity of the QDs could be partially due to the presence of dissolved Cd 2+ and TeO3- ions in fish, mouse and humans Not only one specie should be used as a model for the QDs risk assessments but a set of species25
  • 26. Acknowledgment Funding • NSERC Canadian Research chair Associates • Centre Saint-Laurent • Aquarium de Québec All the laboratory staff26
  • 27. Thank for your attention27
  • 28. Toxicity of QDs Rainbow trout28
  • 29. Les différentes nanoparticules ? Type de Caractéristiques Forme Usage Images particule Sphère, dun Véhicule des ellipsoïde, dun Fullerène C60 Carbone molécules, tube ou dun électronique anneau Métaux purs ou Nanoparticules Agent composés Sphère inorganiques antimicrobien organiques Non solubles Molécule contenant Nanoparticules Véhicule des des microémulsions Micelle organiques molécules dans le cœur aqueux Points Solubles, Imagerie, Sphère quantiques fluorescents médecine29
  • 30. LC50 150 % normal response 100 50 0 0 10 20 30 40 50 60 70 80 90 CONCENTRATION LC5030
  • 31. Le cadmium -Industrie : production de stabilisants, de plastiques, d’alliages, de pigments, de peintures, de batteries (Huff et al., 2007) -Polluant majeur de l’environnement, 8ième place des 20 substances prioritaires (http://www.atsdr.cdc.gov, ATSDR, 2010) -Apport dans l’océan global est d’environ 8000 t/ an (1/2 = activités humaines) (Coles, 1995; Joseph, 2009) -Connu depuis les années 1950 lors d’une intoxication au cadmium, aussi appelé maladie « itai-itai » (Nogawa, 1981 ; Merrill et al, 2007) -Toxicité des ions Cd2+  induit un stress oxydatif et des métallothionéines, inactive des groupements thiols (fortes doses) (Rikans, 2000) , et cause des phénomènes apoptotiques (Stohs et al, 2000).31
  • 32. Le tellure -Principalement utilisé en optique, en électronique et pour la conception de batteries -Présent dans la croute terrestre à hauteur de 0,01 ppm, en combinaison avec des métaux (HSDB, 2010) -Peu d’études de toxicité, sur ce métalloïde, mais très pertinentes -Présent sous plusieurs formes, les oxyanions sont très toxiques TeO32- (Lawerys et al, 2007) -Toxicité chez les bactéries (Taylor et al, 1999) mais possibilité de réduction des ions tellurites - Forte toxicité : engendre des troubles digestifs , nerveux et cutanés, cause l’alopécie et l’haleine alliacée(Louria et al, 1972) -Induit la production de ROS (Chasteen et al, 2009, Jamier et al, 2009, Ogra et al., 2009) de thiols dont le glutathion (Turner et al, 2001)32
  • 33. Zolnik et al, 201033
  • 34. 3.2 Toxicité liée à la taille des nanoparticules Ultrafiltration : -Séparation des petites macromolécules (protéines, colloïdes, nanoparticules) - Poids moléculaire - La fraction qui passe la membrane = perméat - La fraction qui ne passe pas la membrane = reténa34 Document fourni par P. Turcotte
  • 35. 3- Toxicité QDs vs AgNPs IC50 des QDs et des AgNPs en fonction de différents modèles expérimentaux Viabilité QDs AgNPs Humain 216,62 µg/ml - Souris > 952,4 µg/ml 36,39 µg/ml Transformation QDs AgNPs Humain 28,70 µg/ml - Souris 4,38 µg/ml 19,06 µg/ml35
  • 36. 3.2 Les réponses cellulaires, ROS et métallothionéines Altération de la réponse mécanistique chez la souris - Production de ROS, puis inhibition - Diminution de la production de thiols * P < 0.05 ** P< 0.0136 *** P < 0.001
  • 37. 3.2 Impact sur la structure cellulaire mesurée par imagerie • Remaniement des filaments d’actine au fur et à mesure de l’augmentation de la dose de cadmium • D’autres images sont en cours d’analyse, le but est d’observer la localisation de particules.37
  • 38. 3.2 Impact sur la structure cellulaire mesurée par imagerie • Déformations de la membrane cellulaire • Perte d’intégrité cellulaire • Évacuation du contenu cellulaire • Apoptose • Nécrose38