Audrey Bruneau_Susceptibility of the immune system in three animal models exposed to silver nanoparticles (AgNPs)
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Audrey Bruneau_Susceptibility of the immune system in three animal models exposed to silver nanoparticles (AgNPs)

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Audrey Bruneau_Susceptibility of the immune system in three animal models exposed to silver nanoparticles (AgNPs) Presentation Transcript

  • 1. SUSCEPTIBILITY OF THE IMMUNE SYSTEM OF THREE ANIMAL MODELS EXPOSED TO SILVER NANOPARTICLES Bruneau A. a,c, Fortier M.a, Gagné F. b, C. Gagnonb, P. Turcotte b,Tayabali A.d, Auffret M. c, Fournier M. a a : INRS Institut Armand Frappier, 531 Boulevard des prairies, Laval, Qc, Canada. b : Environment Canada, 105 Mc Gill, Montréal, Qc, Canada. c : IUEM, Lemar, Place Nicolas Copernic. Technopole Brest Iroise, Plouzané, France.d : HECSB, Health Canada, Rm 201A, Environmental Health Centre, 50 Colombine Driveway, Ottawa, Canada
  • 2. INTRODUCTION Silver nanoparticles (AgNPs) are mainly employed for their antimicrobial properties.  Textile  Medical plastic  Food packaging Silver nanoparticles are the main particles of interest Risk: metal silver represent an environmental hazard = toxic, persistent and bioaccumulative (under at least some circumstances) (Luoma et al, 2009) Need more regulation to define a status 1
  • 3. SILVERo Control infections (Jain et al., 2009; Pradeep et al., 2009)o700 000 Kg of silver enter in aquatic media per year (Purcelle & Peters,1998)oSilver toxicity is mainly due to ionic form Ag+ (Edwards-Jones, 2009; Liu & Hurt, 2010)oArgyria cause (skin pigmentation) (Hollinger, 1996; Hammond et al, 2004)oIn vitro silver induce viability variation, decrease in cellular proliferation,oxydative burst, and cellular damages (Liedberg & Lundeberg 1989; McCauley, Linares et al.1989; Kuroyanagi, Kim et al. 1991; Zapata, R et al. 1993; Hollinger 1996) 2
  • 4. OBJECTIVES Validation of nanoparticles studied in the project Evaluation of immune system performance of different animal models Calculation of IC 50 for animal models in order to evaluate more sensitive species Comparison of different immune parameters to identify the most representative 3
  • 5. SILVER NANOPARTICLES Characteristics: - Metallic contents > 97% silver - Sodium polyacrylate coating - COOH groups at surface - Stock concentration 1.5 mg/ml 10 nm Core: Silver Polymer coating (polyacrylate sodium)5 4
  • 6. MATERIALS AND METHODS Blue mussel Rainbow trout Mouse Mytilus edulis Onchorynchus Mus musculus mykiss Hemolymph Pronephros Spleen Purification Purification Lymphocytes, macrophages and hemocytes 5
  • 7. MATERIALS AND METHODS Immune cells 21 h Viability (propidium iodide) and Phagocytosis (latexIn vitro exposure to silver beads) nanoparticles ( 0 to 71 µg/ml) 48 H Viability and lymphoblastic and 72h transformation Flow cytometry (viability and phagocytosis) and radioactivity count (lymphoblastic transformation) Imagery (Transmission Electron Microscopy) immune cell structure 6
  • 8. TRANSMISSION ELECTRON MICROSCOPY 10 nm NPs Fixation Embed in paraffin × 12 000 × 30 000 Section and photograph 7
  • 9. CYTOGRAMMS OF CELLS POPULATIONS complexity size
  • 10. BIOMARKER: VIABILITY PI PI 9
  • 11. Sophie Gauthier Clerc copyright BIOMARKERS: IMMUNOEFFICACITY10
  • 12. BIOMARKER: LYMPHOBLASTIC TRANSFORMATION 11
  • 13. RESULTS - IC 50 Lymphoblastic Species Viability Immunoactivity Immunoefficacity transformation Mouse 36 ˃ 71 ˃ 71 11 Trout 55 ˃ 71 ˃ 71 7 Mussel ˃ 71 21 12 × Mouse ˃ Trout ˃ Mussel 12
  • 14. RESULTS - IC 50o Mouse macrophage and lymphocyte viability is more sensitive than that of fish cells.o Phagocytosis of mussels hemocytes (IC 50 = 21µg / ml) is inhibited while that of trout and mouse models is not.o The lymphocytes of rainbow trout are more sensitive than those of mouse to silver nanoparticles 13
  • 15. RESULTS - IC 50 NANO VS. METALS Immune cells Mouse AgNPs AgNO3 In vitro exposure to Macrophages viability 36 1.6 dissolved silver (AgNO3) in same Lymphocytes viability 25 1.7 concentrations of Immunoactivity ˃71 1.7 AgNPs Immunoefficacity ˃71 1 Lymphoblastic Biomarkers 11 0.9 transformation analyses IC 50 14
  • 16. IMAGERY : IMPACT ON CELL STRUCTURE Mouse Control 37.5 µg/ml ×1500 and ×5000- Vacuole formation- Nuclear fragmentation ×1200, ×2000,- Piknosis ×2500- Lysis 17 µg/ml (×1500 ) 15
  • 17. DISCUSSIONo AgNPs disrupt immune performance o Dose-dependant toxicity (Maurer-Jones et al., 2010) o Effects varied in different animal models: immunostimulation or immunodepression (Iavicoli et al., 2010)  difference in immune system (Nappi et al., 2000) o Gradient of species Immune parameters Gradient of species from more to less sensitive Viability Mouse > Trout > Mussel Phagocytosis Mussel > Mouse Lymphoblastic transformation Trout ≥ Mouse 16
  • 18. DISCUSSIONo Phagocytic cells are les sensitives than lymphocyteso NPs were internalized in cells o Yue et al., 2009 macrophages of miceo Apoptosis and necrosis in immune cells for high concentrations of AgNPs o Nel et al., 2006 , Teodoro et al., 2011 decrease in ATP production  Apoptosis initiationo Dissolved silver is more toxic than silver nanoparticle 17
  • 19. CONCLUSION- Cellular toxicity- Toxicity is variable according to the kind of cells and model animal- Nanoparticles of silver are toxic, at low doses in certain cases- Phagocytosis is less sensitive than lymphoblastic transformation- Mouse is the most representative specie FUTURE WORKAgNPs toxicity mechanism (cellular and molecular effects) 18
  • 20. Acknowledgment Funding  NSERC Canada Research chair Associates  Centre Saint-Laurent  Aquarium de Québec All the laboratory staff 19
  • 21. 20 O N U H A K Y THTTP://WWW.COLDTRUTH.COM/2009/ENVIRONMENTAL-HEALTH/NANOTECHNOLOGY-ENVIRONMENTAL-HEALTH/FEDS-QUESTION-SAFETY-OF-NANOSILVER-USED-IN-ODOR-EATING-CLOTHING-FAVORED-BY-ASTRONAUTS-HIKERS-AND-BABIES/
  • 22. BIOMARKERS ANALYSIS
  • 23. MATERIALS AND METHODS Biomarker analysis (flow cytometry) - Viability  propidium iodide - Phagocytosis  latex beads (1.71 µm ø) - Lymphoblastic transformation  tritiated thymidine (3H) Imagery (Electronic microscopy) - immune cells structure 7
  • 24. PI PI
  • 25. ICP-MS ANALYSES 20 18Measured concentration(ppb) 16 14 12 Water 10 Sea water 8 RPMI wo No stability in 6 RPMI sea water 4 2 160 Measured concentration(ppb) 0 140 0 20 40 60 80 120 Theorical concentration (µg/ml) 100 Water 80 Sea water 60 RPMI wo 40 RPMI 20 0 0 20 40 60 80 100 Theorical concentration (µg/ml)