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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
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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
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o 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)
SILVER
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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
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SILVER NANOPARTICLES
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Characteristics: - Metallic contents > 97% silver - Sodium polyacrylate coating - COOH groups at surface - Stock concentration 1.5 mg/ml
Core: Silver
Polymer coating (polyacrylate sodium)
10 nm
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Blue mussel
Mytilus edulis
Rainbow trout
Onchorynchus
mykiss
Mouse
Mus musculus
Hemolymph Pronephros Spleen
Purification Purification
Lymphocytes, macrophages and hemocytes
MATERIALS AND METHODS
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Immune cells
In vitro exposure to silver nanoparticles
( 0 to 71 µg/ml)
21 h
48 H
and 72h
Viability (propidium iodide) and Phagocytosis (latex
beads)
Viability and lymphoblastic transformation
Flow cytometry (viability and phagocytosis) and radioactivity count (lymphoblastic transformation)
MATERIALS AND METHODS
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Imagery (Transmission Electron Microscopy) immune cell structure
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TRANSMISSION ELECTRON MICROSCOPY
× 12 000 × 30 000
NPs
Fixation
Embed in paraffin
Section and photograph
7
10 nm
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CYTOGRAMMS OF CELLS POPULATIONS
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size
co
mp
lexity
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BIOMARKER: VIABILITY
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PI
PI
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BIOMARKERS: IMMUNOEFFICACITY
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Sophie
Gauth
ier
Cle
rc c
opyr
ight
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BIOMARKER: LYMPHOBLASTIC TRANSFORMATION
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RESULTS - IC 50
Species Viability Immunoactivity Immunoefficacity Lymphoblastic transformation
Mouse 36 ˃ 71 ˃ 71 11
Trout 55 ˃ 71 ˃ 71 7
Mussel ˃ 71 21 12 ×
12
Mouse ˃ Trout ˃ Mussel
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RESULTS - IC 50
o 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
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RESULTS - IC 50 NANO VS. METALS
AgNPs AgNO3
Macrophages viability 36 1.6
Lymphocytes viability 25 1.7
Immunoactivity ˃71 1.7
Immunoefficacity ˃71 1
Lymphoblastic
transformation 11 0.9
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Immune cells
In vitro exposure to dissolved silver
(AgNO3) in same concentrations of
AgNPs
Biomarkers analyses
IC 50
Mouse
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IMAGERY : IMPACT ON CELL STRUCTURE
×1500 and ×5000
37.5 µg/ml
- Vacuole formation - Nuclear fragmentation - Piknosis - Lysis
Control
×1200, ×2000, ×2500
Mouse
15
17 µg/ml (×1500 )
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DISCUSSION
o 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
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Immune parameters Gradient of species from more to
less sensitive
Viability Mouse > Trout > Mussel
Phagocytosis Mussel > Mouse
Lymphoblastic transformation Trout ≥ Mouse
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DISCUSSION
o Phagocytic cells are les sensitives than lymphocytes
o NPs were internalized in cells
o Yue et al., 2009 macrophages of mice
o 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 initiation
o Dissolved silver is more toxic than silver nanoparticle
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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 WORK
AgNPs toxicity mechanism (cellular and molecular effects)
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Funding
NSERC Canada Research chair
Associates
Centre Saint-Laurent
Aquarium de Québec
All the laboratory staff
Acknowledgment
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THANK
YOU
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TH.C
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20
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BIOMARKERS ANALYSIS
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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
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PI
PI
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ICP-MS ANALYSES
0
2
4
6
8
10
12
14
16
18
20
0 20 40 60 80
Mea
su
red
co
ncen
tra
tio
n(p
pb
)
Theorical concentration (µg/ml)
Water
Sea water
RPMI wo
RPMI
0
20
40
60
80
100
120
140
160
0 20 40 60 80 100
Mea
su
red
co
ncen
tra
tio
n(p
pb
)
Theorical concentration (µg/ml)
Water
Sea water
RPMI wo
RPMI
No stability in
sea water