1. Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) May 2010 Joe Fisher, PhD Student in Public Health GRA –Tanguay Lab, Environmental and Molecular Toxicology H576: R03 Small Grant Proposal Oregon State University

2. Agenda Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) Specific Aims 1. Identify distribution pathways of nanomaterials in zebrafish (biological transport). 2. Detect the distribution of nanomaterials in zebrafish (biological fate). 3. Correlate nanomaterial exposed zebrafish to biological fate and transport. Background and Significance Nanomaterials are becoming more prevalent in society Preliminary Studies ONAMI: OHSU, OSU, PNNL, PSU, UO PhD Committee Design and Methods Dose Response, Uptake Imaging Statistical Analysis

3. Specific Aims Identify distribution pathways of nanomaterials in zebrafish (biological transport). Detect the distribution of nanomaterials in zebrafish (biological fate). Correlate nanomaterial exposed zebrafish to biological fate and transport. Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate)

4. Background and Significance Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) The zebrafish, Danio rerio, is a tropical freshwater fish belonging to the minnow family (Cyprinidae) of order Cypriniformes. It is a popular aquarium fish, and is an important vertebrate model organism in scientific research. Single cell dividing into two cells.24 hours post fertilization (hpf)

5. Background and Significance Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) The zebrafish, Danio rerio, is a tropical freshwater fish belonging to the minnow family (Cyprinidae) of order Cypriniformes. It is a popular aquarium fish, and is an important vertebrate model organism in scientific research.

6. Background and Significance Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate)

7. Background and Significance NaturalIncidentalEngineered Volcanic ashCombustionCarbon Ocean sprayCooking Quantum dots Forest fire smoke Sandblasting Sunscreen pigments Mineral compositesMiningSilica Cloud aerosolsWeldingMetals

8. Background and Significance Nanomaterials are becoming more prevalent in society Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) Over 1000 consumer products worldwide … Photo by David Hawxhurst, Woodrow Wilson Center

9. Unknown Insufficient Data Nanomaterial Human Health Risks and Risk Assessment Preliminary Studies Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) Health Risk = f(hazard, exposure). Leads to mortality, morbidity, and disability.

10. Preliminary Studies ONAMI: OHSU, OSU, PNNL, PSU, UO PhD Committee Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) Major Professor: Anna Harding Committee Members: Robert Tanguay Daniel Sudakin Ellen Smit John Bolte

11. Design and Methods Dose Response, Uptake Imaging Statistical Analysis Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate)

12. Design and Methods Dose Response Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) By Lisa Truong, Tanguay Lab Embryo Each embryo is dechorionated at 6hpf, loaded into 96 well plates, and exposed to 100 uL at 8 hpf. Mortality is recorded at 120 hpf.

13. Design and Methods Uptake (ICP-MS) Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) By Lisa Truong, Tanguay Lab

14. Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) Design and Methods Imaging Ten dechorionated embryos are exposed to fluorescent nanoparticles in a glass vial with 1000 uL of exposure medium. After the exposure period, an embryo is washed in fish water and moved to a glass bottom dish. Low melt agarose is poured over the embryo. The embryo is anesthetized with tricain. Water is added to the dish. The glass bottom dish is moved to the microscope for imaging.

15. Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) Design and Methods Imaging A laser scanning confocal microscope image from the z-stack shows florescent quantum dots in the internal part of the embryo. The z-stack focus the image at each layer from the bottom of the embryo to the top. Each image is a slice progressing from the bottom to the top.

16. Design and Methods Imaging Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) Light Microscopy: Laser Scanning – 405 to 800 nm 200 nm spatial resolution, ~ 3 nm spectral resolution PNNL – Zeiss LSM 710 NLO and other Electron Microscopy: PNNL – A variety available including cryo preparation UO – A variety available including cryo preparation Note: Primary imaging method is high resolution 3D light microscopy

17. Design and Methods Statistical Analysis Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) Biological Transport: Imaging – Cardiovascular system, Central nervous system, Cellular transport, Kidney, and Liver by fluorescence in XYZ space and time series. Biological Fate: Embryos evaluated Imaging – target organs, and location by fluorescence Dose absorbed by ICP-MS (whole embryo, target organs) Data are expressed as mean ±SE. Statistical significance is determined by Student's t-test. Biological Exposures: Embryos evaluated every 24 hpf for mortality and developmental progression. 120 hpf for mortality, morbidity, and behavior. Scored binary with 24 embryos/treatment, 80% power, and 0.05 significance and compared using one-way ANOVA.

18. Summary Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate) Specific Aims 1. Identify distribution pathways of nanomaterials in zebrafish (biological transport). 2. Detect the distribution of nanomaterials in zebrafish (biological fate). 3. Correlate nanomaterial exposed zebrafish to biological fate and transport. Background and Significance Nanomaterials are becoming more prevalent in society Preliminary Studies ONAMI: OHSU, OSU, PNNL, PSU, UO PhD Committee Design and Methods Dose Response, Uptake Imaging Statistical Analysis