1. 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

2. 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
Conclusion

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

4. 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

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

6. 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

7. What Happens With These Chemicals? Cytochrome P450 (CYP)
CYP 2E1
Hexane
2,5-Hexanedione
Neurological
effects
Trichloroethylene
Dichloroacetic Acid
CYP 2E1
Neurological
effects

8. What Happens With These Chemicals?
1,3-DAB
1,3-DEB
CYP 2E1
No Response
Product forms adducts with macromolecules
Molecular targets:
Undefined at this point
1,2-DAB
1,2-DEB
CYP 2E1

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

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

11. 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

12. Results
Trichloroethylene Not Developmentally Toxic

13. Results
Dichloroacetic Acid Not Highly Toxic

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

15. 2,5-Hexanedione Not Highly Toxic
Results
2,5-Hexanedione Not Highly Toxic

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

17. 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.

18. 1,2-Diacetylbenzene Concentration-Dependent
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.

19. 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.

20. 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%

21. Control and dosing solutions prepared and changed daily
Methods
Control and dosing solutions prepared and changed daily

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

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

24. Representative Images Of Adults
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.

25. 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

26. 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

27. 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

28. Questions?

29. Proposed Mechanism for 1,2-DAB