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

In vitro immmunotoxicology of

quantum dots and comparison with

dissolved cadmium and tellurium

A. 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

What’s the context ? 1- Immunotoxicity of nanoparticles and chemistry - human results

2- Immunotoxicity according to species 3- Toxicity of nanoparticles vs metals ? Discussion, Conclusion

2

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 precaution

3

Model nanoparticle:

Quantum dots (QDs) ViveNano®

Core of cadmium and tellurium

4

1-10 nm

Core of (CdTe/ CdS): colour

determination

Inorganic shell: increases fluorescence and

improves stability

Organic shell: increases solubility and

functional group conjugation (-COOH)

Biomolecules:

- immunoglobins

- oligonucleotides

Cd/S, Cd/Te nanoparticles, quantum dots

(QDs)

5

-Size range 5-10 nm

-Stock concentration of 20 mg/ml

-Use

-Imaging (tumor)

-Drug delivery

Spleen Peripheral blood

Lymphocytes, macrophages, monocytes, hemocytes

In vitro exposure : QDs, dissolved CdCl2, NaTeO3 and mix

6

Nylon membrane filtration Purification

gradient

Human

Homo

sapiens

risk analysis

Mouse

Mus

musculus

risk analysis

Rainbow trout

Oncorhyncus

mykiss

risk analysis

Blue mussel

Mytilus edulis

risk analysis

Pronephros Hemolymph

Material and methods

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

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 content

8

1.1 Toxicity of QDs, blood cell viability

9

* P < 0.05

** P< 0.01

*** P < 0.001

Autofluorescence 4%

monocyte/lymphocyte

N = 3

Human

1-Immunotoxicity of nanoparticles and chemistry

1.2 Toxicity of QDs, phagocytosis

10

21-hours incubation

M1 : Phagocytosis 1 bead and

more

M2 : Phagocytosis ≥ 3 beads

* P < 0.05

** P< 0.01

*** P < 0.001

N =3

Human


1.3 Toxicity of QDs, lymphoblastic transformation

11

* P < 0.05

** P< 0.01

*** P < 0.001

N = 3

Drastic decrease at 15 µg/ml

Human


12

1.4 Characterization

Predicted cadmium

concentration highly

correlated with measured

cadmium concentration in

all media

(0.94≤R ≤ 0.99, P<0.001)

0

20

40

60

80

100

0 50 100 150

measu

red

expected

Water

0

20

40

60

80

100

0 50 100 150

measu

red

expected

Sea Water

0

50

100

150

0 50 100 150

measu

red

expected

RPMI

0

20

40

60

80

100

0 50 100 150

measu

red

expected

RPMI w/o


Immunotoxicity in model organisms

QDs Human Mouse Trout Mussel

Macrophage

viability 216 > 952 > 952 435

Phagocytosis (≥3

beads) 425 > 952 > 952 435

Lymphoblastic

transformation 29 4 20 -

IC 50 = Inhibition concentration for 50% of biological parameter

13

All data expressed in µg/ml

Terrestrial vertebrates were more sensitive than other

species

2- Immunotoxicity according to species

Toxicity of QDs versus metals - Blue mussel

14 N= 9 for QDs, Cd and Cd/Te and N=16 for Te.

* p<0.05, ** p< 0.001

Cd Te Cd/Te

3- Toxicity of nanoparticles vs metals ?

15

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, *** p

< 0.0001

Mussel : QDs were more toxic than

metals

Cd Te Cd/Te


IC 50

16

Viability

QDs CdCl2 NaTeO3 Mixed

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 metals

Correlation between QDs vs metals

17

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* 0.64

18

Root 1 vs. Root 2

-6 -4 -2 0 2 4 6 8

Root 1

-4

-3

-2

-1

0

1

2

3

4

5

Root

2

-10 -8 -6 -4 -2 0 2 4 6

Root 1

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

Root

2

Mussel

-20 -15 -10 -5 0 5 10

Root 1

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

7

8

Root

2

Mouse

Rainbow trout Discriminant

Analyses

-4 -3 -2 -1 0 1 2 3

Root 1

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

3,0

Ro

ot

2

Human

Discussion

-Total metallic content of nanoparticles conserved in all media

- Blue Mussel

- Other species

19

Toxicity QDs Metals

Toxicity Metals QDs

- 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

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 Te

21

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)


Mouse and Human model were the most sensitive species


Cytotoxicity of the QDs could be partially due to the presence of dissolved Cd2+ 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 species

22

Conclusions 1- Immunotoxicity of nanoparticles and chemistry


-Macrophages and monocytes were less sensitive to QDs and dissolved metals exposition than T lymphocytes (human, mouse and fish)


- Mouse and Human model were the most sensitive species




23


-QDs were toxic (viability, phagocytosis, lymphoblastic transformation)



- Mouse and Human model were the most sensitive species


- Cytotoxicity of the QDs could be partially due to the presence of dissolved Cd2+ and TeO3

- in fish, mouse and humans


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Mouse and Human model were the most sensitive species




25

Funding

•NSERC Canadian Research chair

Associates

•Centre Saint-Laurent

•Aquarium de Québec

All the laboratory staff

26

Acknowledgment

Thank for your attention

27

Toxicity of QDs Rainbow trout

28

Les différentes nanoparticules ?

29

Type de

particule Caractéristiques Forme Usage Images

Fullerène C60 Carbone

Sphère, d'un

ellipsoïde, d'un

tube ou d'un

anneau

Véhicule des

molécules,

électronique

Nanoparticules

inorganiques

Métaux purs ou

composés

organiques

Sphère Agent

antimicrobien

Nanoparticules

organiques

Non solubles

Molécule contenant

des microémulsions

dans le cœur

aqueux

Micelle Véhicule des

molécules

Points

quantiques

Solubles,

fluorescents Sphère

Imagerie,

médecine

LC50 CONCENTRATION

LC50

0

50

100

150

0 10 20 30 40 50 60 70 80 90

% n

orm

al re

sp

on

se

30

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

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

Zolnik et al, 2010

33

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 = reténa

Document fourni par P. Turcotte

3.2 Toxicité liée à la taille des nanoparticules

34

3- Toxicité QDs vs AgNPs

35

Viabilité QDs AgNPs

Humain 216,62 µg/ml -

Souris > 952,4 µg/ml 36,39 µg/ml

IC50 des QDs et des AgNPs en fonction de différents modèles expérimentaux

Transformation QDs AgNPs

Humain 28,70 µg/ml -

Souris 4,38 µg/ml 19,06 µg/ml

- Diminution de la production de thiols

36

3.2 Les réponses cellulaires, ROS et métallothionéines

Altération de la réponse mécanistique chez la souris - Production de ROS, puis inhibition

* P < 0.05 ** P< 0.01 *** P < 0.001

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

• Nécrose

Documents

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