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 Alzheimer’s Parkinson’s Lou Gehrig’s

Drug Delivery Osteoporosis Tumor eradication

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

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

importance

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.

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.

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

2007 – The Summer of Toxicity Parameters

Toxicity was not established early on We struggled with finding assays that best

met our needs.

No visible difference between test and control

If only…..

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.

Plating

Plating

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

Optical Density – Growth Rate UV spectrophotometer

used to measure the light scattering of each test – a direct correlation to cell count.

BY4742 Growth 1

-2.5

-2

-1.5

-1

-0.5

0

0 2 4 6

Time (hours)

LN A600

Control

29 mg/L Fullerene

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

The Future Continue to gather data either for or against

fullerene toxicity in yeast. The hardiness of yeast may be a blessing in

disguise.

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