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Transcript of Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate)...
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
AgendaInvestigate 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
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)
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)
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.
Background and Significance
Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate)
Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate)
Background and Significance
Natural Incidental Engineered
Volcanic ash Combustion Carbon
Ocean spray Cooking Quantum dots
Forest fire smoke Sandblasting Sunscreen pigments
Mineral composites Mining Silica
Cloud aerosols Welding Metals
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
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.
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
Design and Methods
Dose Response, Uptake
Imaging
Statistical Analysis
Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate)
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.
Design and Methods Uptake (ICP-MS)
Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate)
By Lisa Truong, Tanguay Lab
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.
Investigate nanomaterial biological interactions and their effects on in-vivo zebrafish (vertebrate)
Design and Methods
ImagingA 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.
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
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.
SummaryInvestigate 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