1. DNA Adduct Formation in Zebrafish Early Development and DNA Damage Eric Brooks Mentor: Dr. William Baird

2. Goals  To examine the effects of environmental carcinogens on early developmental stages in zebrafish  To examine the effect of gene knockdown of nucleotide excision repair (NER) enzymes on levels of DNA adduct formation.

3. Relevance  Early vertebrate development and DNA damage  DNA Repair

4. Malformations caused by Exposure to Carcinogen Carcinogen treated embryo: Abnormal curvature of the tail. Untreated embryo.

5. Why Zebrafish?  Key similarities to humans.  Easily observable developmental stages.  Less expensive than standard rodent model systems. Photo Courtesy of the University of Oregon

6. Polycyclic Aromatic Hydrocarbons  Found in environmental pollutants such as coal tar.  Are the products of incomplete combustion of organic compounds.  Human exposure linked to cancer incidence.  Benzopyrene (BP) and Dibenzopyrene (DBP)

7. DNA Adduct Formation NH N N N N O HO OH HO Adenosine (blue) adduct formed by reaction of Dibenzo[a,I]pyrene (red) with DNA CH 2 OH

8. Chen J, et. al. "MMDB: Entrez's 3D-structure database", Nucleic Acids Res. 2003 Jan; 31(1): 474-7. B-DNAAdducted B-DNA

9. Experimental Scheme 24 hr post fertilization Bath exposure embryo BP or DBP Dose 0, 10 ppm 24hr after exposure Postlabeling DNA adduct analysis

10. Determining DNA Adduct Formation  Treat fish with carcinogen  Isolate DNA  Digest and label DNA with 33 P  Sep-Pak purification  HPLC Analysis

11. 33 P Postlabeling of DNA Adducts Sep-pak

12. Results: DBP-DNA Adduct Profiles DBPDBPDE 1 2a 2b 4

13. BP-DNA Adducts BP BPDE BPDE-dG adduct

14. DNA Adduct Formation in Zebrafish 4.10 26.39 12.65 165.70

15. In Progress  Knockdown of the NER enzymes  Comparison of adduct formation in knockdown fish to non-knockdown fish

16. Acknowledgements Dr. William M. Baird Dr. Brinda Mahadevan Dr. Robert Tanguay Mark Reimers Jennifer Atkin Dr. Kevin Ahern Howard Hughes Medical Institute