Developing the Zebrafish Danio rerio As a Model

Developing the Zebrafish (Danio rerio) As a Model to Evaluate the Toxicity of Chlorinated and Non. Chlorinated Solvents Matthew Wenning Department of Bioresource Research Robert L. Tanguay Ph. D. and David E. Williams Ph. D. Department of Environmental and Molecular Toxicology

Overview • What are these chemicals? • Why do we care? • What are the advantages of using the zebrafish? • What happens to these chemicals? • Methods – Embryo assays and adult assay (1, 2 DAB) • Results – Embryo assays and adult assay (1, 2 DAB) • Conclusion

What Are These Chemicals? Chlorinated Solvents • Trichloroethylene (TCE) – (degreasing agent, polyvinylchloride) • Dichloroacetic Acid (DCA)* * Metabolite of parent compound shown above it

What Are These Chemicals? Non-Chlorinated Solvents • • Hexane - (extractions, adhesives) 2, 5 -Hexanedione (2, 5 -HD)* • 1, 2 -Diethylbenzene (1, 2 -DEB) - (fuel additive) • 1, 2 -Diacetylbenzene (1, 2 -DAB)* • 1, 3 -Diethylbenzene (1, 3 -DEB) • 1, 3 -Diacetylbenzene (1, 3 -DAB)* * Metabolite of parent compound shown above it

Why Do We Care? • Neurotoxic agents • Occupational • • • exposure Environmental contamination and exposure Developmental toxicity Toxicity in adults Mechanism? ? ?

What Are the Advantages of Using the Zebrafish • Embryos are transparent • Embryonic development is similar between all species, especially in early stages • They can be spawned and raised at a lower cost • Mechanism of toxicity likely conserved between zebrafish and humans

What Happens With These Chemicals? Cytochrome P 450 (CYP) Neurological effects CYP 2 E 1 Hexane 2, 5 -Hexanedione Neurological effects CYP 2 E 1 Trichloroethylene Dichloroacetic Acid

What Happens With These Chemicals? CYP 2 E 1 1, 3 -DEB No Response 1, 3 -DAB Product forms adducts with macromolecules CYP 2 E 1 1, 2 -DEB 1, 2 -DAB Molecular targets: Undefined at this point

Methods • Adult fish setup for spawning 15 -17 hours prior to exposure Spawning setup Recirculating system

Methods Embryos: • Embryos selected for exposure • Exposures started 6 -8 hours post fertilization Non-viable embryo Viable embryo

Methods Embryos: • Placed in 96 -well (200 µL) plates • One embryo per well • Exposed in 100 µL of solution at various concentrations • Broad range of concentrations evaluated – Dimethylsulfoxide (DMSO) used as a co-solvent for non-polar solvents • N = 24 for each concentration

Results Trichloroethylene Not Developmentally Toxic

Results Dichloroacetic Acid Not Highly Toxic

Results Dichloroacetic Acid Representative Images Representative images of (A) control and (B) 100 m. M DCA exposed embryos. The DCA exposed larva exhibits mild pericardial edema (*) and unlike the control animals the swim bladder (sb) is not inflated.

Results 2, 5 -Hexanedione Not Highly Toxic

Results 2, 5 -Hexanedione Representative Images Representative images of (A) control and (B) 10 m. M 2, 5 -HD exposed embryos. The 2, 5 -HD exposed larva

Results 1, 2 -Diacetylbenze Toxic But Not 1, 3 -Diacetylbenze 1, 2 DAB, but not 1, 3 DAB is developmentally toxic to zebrafish. Cumulative mortality was collected for 2 days (48 hpf). A total of 24 animals were assessed for each concentration.

Results 1, 2 -Diacetylbenzene Concentration-Dependent Response 1, 2 DAB developmental toxicity. Cumulative mortality was collected for 5 days (120 hpf). A total of 24 animals were assessed for each concentration, and the experiment was repeated 3 times.

Results 1, 2 -Diacetylbenzene Representative Images Chromogenic effect 1, 2 DAB developmental toxicity. Representative images of a control and 1, 2 DAB exposed embryos. Note the development of cardiovascular edema , curved body axis and the characteristic “blue” appearance.

Methods Adults: • Two trials testing 1, 2 -diacetylbenzene (1, 2 -DAB) • Chronic low-dose (100 ppb for 52 days) • Acute high-dose (1000 ppb days for 6 days) • 1, 2 -DAB solvated in dimethylsulfoxide (DMSO) • DMSO concentration in exposure and control groups was 0. 1%

Methods • Control and dosing solutions prepared and changed daily

Methods • Placed in 600 ml of solution • Each zebrafish placed in individual tank

Methods • Each trial contained two groups • Control and experimental groups N=4 • Transfer to clean 10 gallon tank for behavioral observation

Results Representative Images Of Adults Control (top) and 1, 2 DAB (bottom) adult fish image three days post exposure. It was a dramatic blue appearance of the 1, 2 DAB exposed fish. The appearance of the blue correlated with behavioral and motor deficits.

Conclusion • Trichloroethylene is not developmentally toxic under these conditions • Dichloroacetic acid is not developmentally toxic under these conditions • 2, 5 -Hexanedione is not developmentally toxic under these conditions • 1, 3 -Diacetylbenzene showed no toxic response • 1, 2 -Diacetylbenzene is developmentally toxic

Conclusion • Similar responses in zebrafish as in mammals with 1, 2 -DAB • Zebrafish can be used as a model to evaluate toxicity of these compounds • Future studies can be done to determine “how” the chemicals produce toxicity

Acknowledgements • Robert L. Tanguay Ph. D. • Stacey Harper Ph. D. • Ms. Jane La Du • Mr. Lijoy K. Mathew • Ms. Sumitra Sengupta • Ms. Crystal Usenko • Ms. Lisa Duong • Ms. Kate Saili • David E. Williams Ph. D. • Katherine G. Field Ph. D. • Ms. Wanda K. Crannell M. S. Tanguay Lab

Questions?

Proposed Mechanism for 1, 2 -DAB