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Parallel session 2
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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.
November 1st-2nd 2012
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
Increase of industrial production
Increase risks for human to be exposed
Nanotoxicology studies to evaluate the potential risk
for human health
New industrial applications
Effects of nanoprticles on the respiratory system
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).
Tabet et al., 2011
1/ Induction of inflammation: > 500 studies (in vitro and in vivo)
• Example of exposure to carbon nanotubes: NT1, NT2 and NT3
2/ Change in airway reactivity: 4 studies
Cells/ml Neutrophils (%) TNF-α 200000
150000
100000
50000
80
60
40
20
Bronchoalveolar lavage fluids analyses
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.
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.
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.
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
Biomedical applications of Quantum Dots
Example: QDs (with different sizes) used as nanoprobes
to target the same cancer xenograft in mice
Biomedical applications such as nanoprobes and nanocarriers.
X. Gao et al., Nat Biotechnol. 2004;22:969-976.
QDs_1
QDs_2
QDs_3
• Cytotoxicity
- Mitochondrial Metabolism (reduction of MTS)
• Oxidative Stress response - Glutathion assay (enzymatic activity)
• 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 Design
Exposure:
16 to 72h
Quantum Dots
0 to 1000 µg/ml
Analyses
Experimental protocol
For normal and asthmatic subject: 4 separate experiments with cells cultured at different passages (n=4)
Study of cytotoxicity: MTS assay
Mitochondrial
Succinate
Dehydrogenase
Tetrazolim salts Formazan
Viables cells
MTS +
Viable Cells Dead Cells
MTS +++
Principle of assay
Cells viability
Results: Cells viability
QDs decrease the viability of normal and asthmatic cells in a dose and time dependent manner
* 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
0
30
60
90
120
150
10 100 333 1000 µg/ml
Cellular Viability (%) Asthmatic HBSMC
# #
# #
#
# #
# #
0
30
60
90
120
150
10 100 333 1000 µg/ml
Cellular Viability (%)
Normal HBSMC
*
* * *
*
* *
* * *
Normal cells are more sensitive to the cytotoxic
effect of QDs
QDs (µg/ml) 16h 24h
$ $
Cellular
Viability (%)
0
20
40
60
80
100
333 1000 333 1000
Normal HBSMC
Asthmatic HBSMC
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
Molecular mechanisms involved
in nanoparticles cytotoxicity
Oxidative/antioxidative response after 24h exposure
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
* P≤0.05 vs control for normal HBSMC
# P≤0.05 vs control for asthmatic HBSMC
$ P≤0.05 normal vs asthmatic HBSMC
0
1
2
3
4
témoins 10 100 333 µg/ml
Ratio GSH/GSSG
#
$ #
$
$
$
control
Normal HBSMC
Asthmatic HBSMC
Oberdörster et coll. EHP 2005
Inflammatory response is also a major mechanism observed in
cells exposed to nanoparticles.
Molecular mechanisms involved
in nanoparticles cytotoxicity
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
Inflammatory response after 24h exposure
0
20
40
60
80
100
120
témoins 100 333 µg/ml
pg/ml Eotaxin
0
300
600
900
1200
1500
témoins 100 333 µg/ml
pg/ml GM-CSF
#
* P≤0.05 vs control for normal HBSMC
# P≤0.05 vs control for asthmatic HBSMC
$ P≤0.05 normal vs asthmatic HBSMC
control control
Normal HBSMC
Asthmatic HBSMC
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.
Dr. Karim Maghni
Dre. Melanie Welman
Mrs. Lucero Castellanos
Laboratory members
Acknowledgements
Contact:
+1 514-338-2222 # 3673