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 MaghniResearch Center HSCM, Université de Montréal, Canada.
Introduction: definitionsNanotechnology (sometimes shortened to "nanotech"): new field of research and technologydevelopment 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
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., 2011Recently, a mouse model of toluene diisocyanate-induced asthma has provided the firstevidence that nanoparticles (gold NP) can evoke airway hyperreactivity (Hussain et al., 2010).
HypothesisThe development of airway hyperreactivity in response tonanoparticles exposure is induced by interactions of these particleswith airway smooth muscle cells. Furthermore, responses of thesecells to nanoparticles exposure will be different between allergicasthma and normal conditions.
RationalAirway smooth muscle cells is a target cell in the development ofairway hyperreactivity. However, it is still unknown whethernanoparticles alter airway smooth muscle cells function, particularlyin allergic conditions.
Aim of the studyTo examine in vitro in human bronchial smooth muscle cellsdifferences in responses to nanoparticles between normal andasthmatic subjects. The specific mechanisms investigated were:1 / Cytotoxic effects (viability / cell death);2 / Oxidative / antioxidant response;3 / Inflammatory response.
Quantum DotsQuantum Dots are a spherical nanocrystals of 1 to 10 nm in diameterThe characteristics of QDs (ViveNano Co) Composition: Cadmium Telluride/Cadmium Sulfide Size: 1-10 nm
Biomedical applications of Quantum DotsBiomedical 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 miceX. Gao et al., Nat Biotechnol. 2004;22:969-976.
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)
Study of cytotoxicity: MTS assayPrinciple of assay Viables cells Mitochondrial Succinate Dehydrogenase Tetrazolim salts FormazanCells viability Viable Cells Dead Cells MTS +++ MTS +
Results: Cells viability Cellular CellularViability (%) Viability (%) Normal HBSMC Asthmatic HBSMC150 150120 120 90 90 # # # * # # 60 * 60 * * 30 * 30 # # * * * # # * * 0 0 10 100 333 1000 µg/ml 10 100 333 1000 µg/mlQDs 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
Molecular mechanisms involved in nanoparticles cytotoxicityOxidative stress is a major mechanism observed in cells exposedto nanoparticles.Oxidative stress responseDetermination of the Ratio: GSH / GSSG Ratio ≥ 1 => Positive antioxidant cells response Ratio < 1 => Deficient antioxidant cells responseGSH: reduced glutathionGSSG: oxidized glutathion
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
Molecular mechanisms involved in nanoparticles cytotoxicityInflammatory response is also a major mechanism observed incells exposed to nanoparticles. Oberdörster et coll. EHP 2005
Inflammatory response after 24h exposure GM-CSF Eotaxin pg/ml pg/ml Normal HBSMC1500 120 Asthmatic HBSMC # 1001200 80 * P≤0.05 vs control for normal HBSMC900 # P≤0.05 vs control for asthmatic HBSMC 60 $ P≤0.05 normal vs asthmatic HBSMC600 40300 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
Conclusion We found that QDs have an effects on both normal andasthmatic 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 mayinduce exacerbation of asthma. Therefore, QDs should be firstinvestigated for nanobiosafety before to move to biomedicalapplications.
Acknowledgements Dr. Karim Maghni Dre. Melanie Welman Mrs. Lucero Castellanos Laboratory members Contact: firstname.lastname@example.org +1 514-338-2222 # 3673