Post on 11-May-2015
description
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-Immunotoxicity of nanoparticles and chemistry
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
1-Immunotoxicity of nanoparticles and chemistry
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
1-Immunotoxicity of nanoparticles and chemistry
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
3- Toxicity of nanoparticles vs metals ?
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)
2- Immunotoxicity according to species
Mouse and Human model were the most sensitive species
3- Toxicity of nanoparticles vs metals ?
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
- 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)
2- Immunotoxicity according to species
- Mouse and Human model were the most sensitive species
3- Toxicity of nanoparticles vs metals ?
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
23
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)
2- Immunotoxicity according to species
- Mouse and Human model were the most sensitive species
3- Toxicity of nanoparticles vs metals ?
- Cytotoxicity of the QDs could be partially due to the presence of dissolved Cd2+ and TeO3
- 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
24
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)
2- Immunotoxicity according to species
Mouse and Human model were the most sensitive species
3- Toxicity of nanoparticles vs metals ?
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
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