High throughput screening of nanotoxicity based on oxidative stress paradigm
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1 December High throughput screening of nanotoxicity based on oxidative stress paradigm Saji George Dr Andre Nel s lab Dept. of Medicine (Division of Nanomedicine) UCLA
2 December Growing nanotech industry- need of alternate toxicity screening strategies Traditional Toxicological Approach -Based on observable experimental animals outcomes in -Time consuming -Labor and cost intensive Nature- Vol Skincare and consumer products, healthcare, electronics, photonics, biotechnology, engineering products, pharmaceuticals, drug delivery, and agriculture. US national academy of science calls for a paradigm shift in toxicity evaluation I trillion $ worth business over 1-2 yrs
3 December HTS for faster screening of toxicity of nanomaterials Cell growth and interaction with NPs Probes for assaying toxicity markers Automated imaging/readin g Advantages of HTS Automatic image analysis Assay Design Data storage and management Less labor and time consuming Data analysis Wide range of NMs properties Large batches of NMs Comprehensive array of predictive in vitro tests Cellular knowledge Easy visualization and interpretation of data Cytotoxicity assessment
4 Major pathways of toxicity Oxidative damage of proteins, DNA and membrane Inflammation:cytokines/chemokines Mitochondrial damage- Apoptosis Lysosomal damage Membrane damage December 11 28
5 December Mechanism of NP mediated toxicity- Hierarchical oxidative stress paradigm High GSH/GSSG ratio Low GSH/GSSG ratio Tier 2 Tier 3 ss ert s e vit a di xo f o l e ve L Tier 1 Cell response pathway Normal Anti-oxidant Defense Inflammation cytotoxicity Signaling pathway: Nrf-2 MAPK Mitochondrial perturbation-pt pore Genetic response: ARE AP-1 N/A Outcome PhaseII enzyme Cytokines chemokines Apoptosis Nel et al. Science 26, 311: 622-7
6 December Preliminary results (Xia et al. ACS Nano 28) B ROS D B Dead cells 1. Tier p-jnk JNK 1 1 Dead cells [Ca2+]i Lowered membrane potential % PI + Cells (M1) Φ JNK 2 p4 p46 p4 p46 % PI + Cells (M1) p-jnk 2 Fold in Fluo-4 % JC-1 low Cells 3 1 Tier 2 [Ca2+]i Actin Φ 2. HO-1 Lowered membrane potential 4 1 Tier 1 D 6 2 C Tier 2 Φ p4 p46 p4 p46 2 Superoxide 7 Actin 1 HO-1 1 H2O2 2 8 Tier 1 Tier 3 A Φ C Superoxide 1 % JC-1 low Cells Fold in DCF ROS 7 12 H2O2 Fold in Fluo-4 8 Fold in DCF 9 % MitoSOX Red+ Cells A Nanoparticle stimulated oxidant injury in human epithelial cells. % MitoSOX Red+ Cells Nanoparticle stimulated oxidant injury in murine macrophage cells Legend: At each Tier of the Hierarchical Oxidative Stress Model, nanoparticles exhibited significantly more effects than either or. Legend: At each Tier of the Hierarchical Oxidative Stress Model, exhibited significantly more effects than either or.
7 December In vitro toxicity assessment based on oxidative stress paradigm- Project outline Rat Macrophage RAW Human lung epithelial cells PS-NH2 Dimensio n Dose Tier 1 Tier 2 HO-1 expression TNF-α NQO-1 expression IL-8 Junk PhaseII enzymes Cox Duration Tier 3 Mitochondrial membrane potential Cytoplasmic Ca content PI uptake Apoptosis
8 December Tier 3 response in BEAS-2B cells subjected to nanoparticles Confocal laser scanning microscopic images of BEAS-2B cells stained with a dye-mix containing Hoechst, fluo4 and propidium iodide, after subjecting them to different treatment conditions. Confocal laser scanning microscopic images of BEAS-2B cells stained with a dye-mix containing Hoechst, JC1 and propidium iodide, after subjecting them to different treatment conditions.
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