Parallel session 1

1. 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

2. 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

3. 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

4. Model nanoparticle:
Quantum dots (QDs) ViveNano®
Core of cadmium and tellurium
Core of (CdTe/ CdS): colour
determination
Inorganic shell: increases fluorescence and
improves stability
Organic shell: increases solubility and
functional group conjugation (-COOH)
1-10 nm
Biomolecules:
- immunoglobins
- oligonucleotides
4

5. Cd/S, Cd/Te nanoparticles, quantum dots
(QDs)
-Size range 5-10 nm
-Stock concentration of 20 mg/ml
-Use
-Imaging (tumor)
-Drug delivery
5

6. Material and methods
Human Mouse Rainbow trout Blue mussel
Homo Mus Oncorhyncus Mytilus edulis
sapiens musculus mykiss risk analysis
risk analysis risk analysis risk analysis
Peripheral blood Spleen Pronephros Hemolymph
Purification Nylon membrane filtration
gradient
Lymphocytes, macrophages, monocytes, hemocytes
In vitro exposure : QDs, dissolved CdCl2, NaTeO3 and mix
6

7. 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

8. 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

9. 1-Immunotoxicity of nanoparticles and chemistry
1.1 Toxicity of QDs, blood cell viability
Human monocyte/lymphocyte
Autofluorescence 4%
* P < 0.05
N=3 ** P< 0.01
9 *** P < 0.001

10. 1-Immunotoxicity of nanoparticles and chemistry
1.2 Toxicity of QDs, phagocytosis
Human
21-hours incubation
M1 : Phagocytosis 1 bead and
more
M2 : Phagocytosis ≥ 3 beads
* P < 0.05
N =3 ** P< 0.01
10 *** P < 0.001

11. 1-Immunotoxicity of nanoparticles and chemistry
1.3 Toxicity of QDs, lymphoblastic transformation
Human
Drastic decrease at 15 µg/ml
* P < 0.05
N=3 ** P< 0.01
11 *** P < 0.001

12. 1-Immunotoxicity of nanoparticles and chemistry
1.4 Characterization
Water Sea Water
100 100
80 80
measured
measured
60 60 Predicted cadmium
40 40
20 20 concentration highly
0 0 correlated with measured
0 50 100 150 0 50 100 150
expected expected cadmium concentration in
all media
RPMI RPMI w/o (0.94≤R ≤ 0.99, P<0.001)
150 100
80
measured
measured
100
60
40
50
20
0 0
0 50 100 150 0 50 100 150
expected expected
12

13. 2- Immunotoxicity according to species
Immunotoxicity in model organisms
IC 50 = Inhibition concentration for 50% of biological parameter
QDs Human Mouse Trout Mussel
Macrophage
216 > 952 > 952 435
viability
Phagocytosis (≥3
425 > 952 > 952 435
beads)
Lymphoblastic
29 4 20 -
transformation
All data expressed in µg/ml
Terrestrial vertebrates were more sensitive than other
13 species

14. 3- Toxicity of nanoparticles vs metals ?
Toxicity of QDs versus metals
- Blue mussel
Cd Te Cd/Te
N= 9 for QDs, Cd and Cd/Te and N=16 for Te.
14 * p<0.05, ** p< 0.001

15. 3- Toxicity of nanoparticles vs metals ?
Cd Te Cd/Te
Mussel : QDs were more toxic than
metals
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
15 < 0.0001

16. IC 50
QDs CdCl2 NaTeO3 Mixed
Viability
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
16

17. Correlation between QDs vs metals
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
17

18. Root 1 vs. Root 2 8
5
Discriminant Mussel Rainbow trout
7
4
6
Analyses
3 5
4
2
3
Root 2
1 2
Root 2
1
0
0
-1 -1
-2
-2
-3
-3
-4
-4 -5
-6 -4 -2 0 2 4 6 8 -20 -15 -10 -5 0 5 10
Root 1 Root 1
3,5
3,0
3,0
2,5
Mouse 2,5
Human
2,0
2,0
1,5
1,5
1,0
1,0
Root 2
Root 2
0,5
0,5
0,0
0,0
-0,5
-0,5
-1,0
-1,0
-1,5
-1,5
-2,0
-2,0
-2,5 -2,5
-10 -8 -6 -4 -2 0 2 4 6 -4 -3 -2 -1 0 1 2 3
18 Root 1 Root 1

19. Discussion
-Total metallic content of nanoparticles conserved in all
media
- Blue Mussel
Toxicity
QDs Metals
- Other species
Toxicity
Metals QDs
19

20. - 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

21. 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

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 Cd 2+ 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

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 fis h)
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 Cd 2+ 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

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 fis h)
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

25. 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 fis h)
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 Cd 2+ 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

26. Acknowledgment
Funding
• NSERC Canadian Research chair
Associates
• Centre Saint-Laurent
• Aquarium de Québec
All the laboratory staff
26

27. Thank for your attention
27

28. Toxicity of QDs
Rainbow trout
28

29. Les différentes nanoparticules ?
Type de
Caractéristiques Forme Usage Images
particule
Sphère, d'un
Véhicule des
ellipsoïde, d'un
Fullerène C60 Carbone molécules,
tube ou d'un
électronique
anneau
Métaux purs ou
Nanoparticules Agent
composés Sphère
inorganiques antimicrobien
organiques
Non solubles
Molécule contenant
Nanoparticules Véhicule des
des microémulsions Micelle
organiques molécules
dans le cœur
aqueux
Points Solubles, Imagerie,
Sphère
quantiques fluorescents médecine
29

30. LC50
150
% normal response
100
50
0
0 10 20 30 40 50 60 70 80 90
CONCENTRATION LC50
30

31. 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

32. 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

33. Zolnik et al, 2010
33

34. 3.2 Toxicité liée à la taille des nanoparticules
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
34 Document fourni par P. Turcotte

35. 3- Toxicité QDs vs AgNPs
IC50 des QDs et des AgNPs en fonction de différents modèles expérimentaux
Viabilité QDs AgNPs
Humain 216,62 µg/ml -
Souris > 952,4 µg/ml 36,39 µg/ml
Transformation QDs AgNPs
Humain 28,70 µg/ml -
Souris 4,38 µg/ml 19,06 µg/ml
35

36. 3.2 Les réponses cellulaires, ROS et métallothionéines
Altération de la réponse
mécanistique chez la souris
- Production de ROS, puis
inhibition
- Diminution de la
production de thiols
* P < 0.05
** P< 0.01
36 *** P < 0.001

37. 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
38