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:

lyes.tabet@umontreal.ca

+1 514-338-2222 # 3673