Parallel session 2

1. November 1st-2nd 2012
Human bronchial smooth muscle cells (HBSMC)
nanocytotoxic responses to quantum dots
exposure: Incidence of the lung pathology
Lyes Tabet, Melanie Welman, Lucero Castellanos and Karim Maghni
Research Center HSCM, Université de Montréal, Canada.

2. Introduction: definitions
Nanotechnology (sometimes shortened to "nanotech"): new field of research and technology
development for materials at the atomic or molecular level.
Nanoparticle: particle with at least one dimension sized from 1 to 100 nanometer (nm).
New properties at the nanometer scale
New industrial applications
Increase of industrial production
Increase risks for human to be exposed
Nanotoxicology studies to evaluate the potential risk
for human health

3. Effects of nanoprticles on the respiratory system
1/ Induction of inflammation: > 500 studies (in vitro and in vivo)
• Example of exposure to carbon nanotubes: NT1, NT2 and NT3
Bronchoalveolar lavage fluids analyses
80
200000 Neutrophils (%) TNF-α
Cells/ml
60
150000
100000 40
50000 20
2/ Change in airway reactivity: 4 studies Tabet et al., 2011
Recently, a mouse model of toluene diisocyanate-induced asthma has provided the first
evidence that nanoparticles (gold NP) can evoke airway hyperreactivity
(Hussain et al., 2010).

4. Hypothesis
The development of airway hyperreactivity in response to
nanoparticles exposure is induced by interactions of these particles
with airway smooth muscle cells. Furthermore, responses of these
cells to nanoparticles exposure will be different between allergic
asthma and normal conditions.

5. Rational
Airway smooth muscle cells is a target cell in the development of
airway hyperreactivity. However, it is still unknown whether
nanoparticles alter airway smooth muscle cells function, particularly
in allergic conditions.

6. Aim of the study
To examine in vitro in human bronchial smooth muscle cells
differences in responses to nanoparticles between normal and
asthmatic subjects. The specific mechanisms investigated were:
1 / Cytotoxic effects (viability / cell death);
2 / Oxidative / antioxidant response;
3 / Inflammatory response.

7. Quantum Dots
Quantum Dots are a spherical nanocrystals of 1 to 10 nm in diameter
The characteristics of QDs (ViveNano Co)
 Composition: Cadmium Telluride/Cadmium Sulfide
 Size: 1-10 nm

8. Biomedical applications of Quantum Dots
Biomedical applications such as nanoprobes and nanocarriers.
QDs_1
QDs_2
QDs_3
Example: QDs (with different sizes) used as nanoprobes
to target the same cancer xenograft in mice
X. Gao et al., Nat Biotechnol. 2004;22:969-976.

9. Experimental Design
• Cytotoxicity
Analyses - Mitochondrial Metabolism (reduction of MTS)
• Oxidative Stress response
- Glutathion assay (enzymatic activity)
Quantum Dots
Exposure:
0 to 1000 µg/ml
16 to 72h • Inflammatory response
- Cytokines/chemokines assays (Luminex Technology)
Characteristics of Human Bronchial Smooth Muscle Cells
Normal subject: Cells provided from the PromoCell compagny
• Male, 32 yrs, no smoker without history of asthma
Asthmatic subject: Cells provided from the Lonza compagny
• Male, 27 yrs, no smoker, diagnostic of asthma at 7yrs and treated with albuterol
Experimental protocol
For normal and asthmatic subject: 4 separate experiments with cells cultured at different passages (n=4)

10. Study of cytotoxicity: MTS assay
Principle of assay Viables cells
Mitochondrial
Succinate
Dehydrogenase
Tetrazolim salts Formazan
Cells viability
Viable Cells Dead Cells
MTS +++ MTS +

11. Results: Cells viability
Cellular Cellular
Viability (%) Viability (%)
Normal HBSMC Asthmatic HBSMC
150 150
120 120
90 90 # # #
* # #
60 * 60
* *
30 * 30 # #
* * * # #
* *
0 0
10 100 333 1000 µg/ml 10 100 333 1000 µg/ml
QDs decrease the viability of normal and asthmatic cells in a dose and time dependent manner
Cellular
Viability (%) Normal HBSMC
Asthmatic HBSMC
100
$
80 $
60
40 Normal cells are more sensitive to the cytotoxic
20 effect of QDs
0
333 1000 333 1000
QDs (µg/ml)
16h 24h
* P≤0.05 vs [10μg/ml] of QDs for normal HBSMC
# P≤0.05 vs [10μg/ml] of QDs for asthmatic HBSMC
$ P≤0.05 normal vs asthmatic HBSMC

12. Molecular mechanisms involved
in nanoparticles cytotoxicity
Oxidative stress is a major mechanism observed in cells exposed
to nanoparticles.
Oxidative stress response
Determination of the Ratio:
GSH / GSSG
Ratio ≥ 1 => Positive antioxidant cells response
Ratio < 1 => Deficient antioxidant cells response
GSH: reduced glutathion
GSSG: oxidized glutathion

13. Oxidative/antioxidative response after 24h exposure
Ratio GSH/GSSG
Normal HBSMC
4 Asthmatic HBSMC
$
3
$
2 $ * P≤0.05 vs control for normal HBSMC
$ #
# P≤0.05 vs control for asthmatic HBSMC
$ P≤0.05 normal vs asthmatic HBSMC
1 #
0
témoins
control 10 100 333 µg/ml
Ratio GSH/GSSG for QDs at [333 µg/ml]:
Ratio = 2.5 for normal HBSMC => Positive antioxidative response
Ratio = 0.5 for asthmatic HBSMC => Deficient antioxidative response

14. Molecular mechanisms involved
in nanoparticles cytotoxicity
Inflammatory response is also a major mechanism observed in
cells exposed to nanoparticles.
Oberdörster et coll. EHP 2005

15. Inflammatory response after 24h exposure
GM-CSF Eotaxin
pg/ml pg/ml Normal HBSMC
1500 120 Asthmatic HBSMC
# 100
1200
80 * P≤0.05 vs control for normal HBSMC
900 # P≤0.05 vs control for asthmatic HBSMC
60 $ P≤0.05 normal vs asthmatic HBSMC
600
40
300 20
0 0
témoins
control 100 333 µg/ml control
témoins 100 333 µg/ml
Basal Levels of GM-CSF and Eotaxin:
• Similar between normal vs asthmatic HBSMC
Levels of mediators are different according to QDs concentration
• 100 µg/ml: Eotaxin is higher in asthmatic vs normal HBSMC
• 333 µg/ml: GM-CSF is higher in asthmatic vs normal HBSMC

16. Conclusion
We found that QDs have an effects on both normal and
asthmatic human bronchial smooth muscle cells. However,
differences were observed between the two conditions, mainly:
 Nanotoxicity: more pronounced in normal cells at shorter times;
 Antioxidant capacity : Deficient in asthmatic cells;
 Inflammatory responses: Higher in asthmatic cells.
Our study suggests that QDs exposure in asthmatic subjects may
induce exacerbation of asthma. Therefore, QDs should be first
investigated for nanobiosafety before to move to biomedical
applications.

17. Acknowledgements
Dr. Karim Maghni
Dre. Melanie Welman
Mrs. Lucero Castellanos
Laboratory members
Contact:
lyes.tabet@umontreal.ca
+1 514-338-2222 # 3673