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

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

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

2

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

4

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

TRANSMISSION ELECTRON MICROSCOPY

× 12 000 × 30 000

NPs

Fixation

Embed in paraffin

Section and photograph

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10 nm

CYTOGRAMMS OF CELLS POPULATIONS

8

size

co

mp

lexity

BIOMARKER: VIABILITY

9

PI

PI

BIOMARKERS: IMMUNOEFFICACITY

10

Sophie

Gauth

ier

Cle

rc c

opyr

ight

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

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

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 )

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

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

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20

BIOMARKERS ANALYSIS

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

PI

PI

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