Parallel session 1, day 2

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. SILVER
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)
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 (latex
In 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
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9. CYTOGRAMMS OF CELLS POPULATIONS
complexity
size

10. BIOMARKER: VIABILITY
PI
PI
9

11. Sophie Gauthier Clerc copyright
BIOMARKERS: IMMUNOEFFICACITY
10

12. BIOMARKER: LYMPHOBLASTIC TRANSFORMATION
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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
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14. 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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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
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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 )
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17. 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
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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18. 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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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 WORK
AgNPs toxicity mechanism (cellular and molecular effects)
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20. Acknowledgment
Funding
 NSERC Canada Research chair
Associates
 Centre Saint-Laurent
 Aquarium de Québec
All the laboratory staff
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21. 20
O
N
U
H
A
K
Y
T
HTTP://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
18
Measured 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)