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