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Genomics-based determination of nanoparticle toxicity: structure-function analysis

Genomics-based determination of nanoparticle toxicity: structure-function analysis. Alex Hadduck – Biochemistry and Biophysics Dr. Alan Bakalinsky – Food Science and Technology. Fullerene. Buckyballs -1985 discovery by Kroto, Curl, and Smalley. 1996 Nobel. Oxygen Radical Scavenger.

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Genomics-based determination of nanoparticle toxicity: structure-function analysis

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  1. Genomics-based determination of nanoparticle toxicity: structure-function analysis Alex Hadduck – Biochemistry and Biophysics Dr. Alan Bakalinsky – Food Science and Technology

  2. Fullerene • Buckyballs -1985 discovery by Kroto, Curl, and Smalley. 1996 Nobel

  3. Oxygen Radical Scavenger • Alzheimer’s • Parkinson’s • Lou Gehrig’s

  4. Drug Delivery • Osteoporosis • Tumor eradication

  5. …And Much More • Gas absorption/storage/purification • Artificial muscles • Superconductors • Combat jackets • Air pollution filters • Bridge building

  6. Toxicity of Buckyballs • Largemouth bass • DNA deformation • Eukaryotic cell growth inhibition • Antimicrobial activity • The how/why of fullerene toxicity is of great importance

  7. Experimental Overview • S. cerevisiae submitted to many conditions in order to establish toxicity parameters, mimicking possible environmental exposure. Tests monitored either cell survival or growth rate. • Once parameters have been established, gene-deletion libraries used to screen for fullerene-protective genes – over 4800 non-essential genes. • Insight into toxicity mechanisms. • Expected human (and other) orthologs.

  8. Toxicity Variables • Fullerene forms colloids – small uniform aggregates – in solution. • Aggregate size, and therefore function, very sensitive to solution chemistry. • pH, ionic strength (salts), preparation method, and organic matter (including cells) all play a role in how fullerene aggregates.

  9. Deletion Library • Mutants of a single S. cerevisiae strain, each with a unique and non-vital gene missing. • Significant increase in sensitivity in a mutant signifies missing gene plays a role in fullerene protection. • Good way to quickly get to the mechanism of toxicity

  10. 2007 – The Summer of Toxicity Parameters • Toxicity was not established early on • We struggled with finding assays that best met our needs.

  11. No visible difference between test and control

  12. If only…..

  13. New Assays and Endpoints • Without being able to reliably recreate results (either positive or negative), our first goal was to re-think how we gather our data. • New assays had to be employed – we chose to use optical density and plating • We also added another possible route of toxicity – growth rate inhibition.

  14. Plating

  15. Plating

  16. Plate Counts • Say we counted 100 cfu (colony forming units) in a plate after plating 100 microliters of a 10,000 fold dilution. • Formula: (cfu/mL plated) x dilution factor = cells/mL • So: (100/.1) x 10,000 = 1x107 cells/mL

  17. Optical Density – Growth Rate • UV spectrophotometer used to measure the light scattering of each test – a direct correlation to cell count.

  18. What We Have Learned So Far • Not toxic, but we aren’t finished • Colloidal stability of fullerene even more sensitive than we thought. • Crucial progress on proper procedures and assays for reproducible data

  19. The Future • Continue to gather data either for or against fullerene toxicity in yeast. • The hardiness of yeast may be a blessing in disguise.

  20. Thank You • Howard Hughes Medical Institute • College of Science Cripps Scholarship Fund • Dr. Alan Bakalinsky • Jeff Rowe • Vihangi Hindogalla • Dr. Qilin Li • Bin Xie

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