DNA Adduct Formation in Zebrafish

Early Development and DNA Damage

Eric Brooks Mentor: Dr. William Baird

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.

Relevance  Early vertebrate development and DNA damage  DNA Repair

Malformations caused by Exposure to Carcinogen Untreated embryo.

Carcinogen treated embryo: Abnormal curvature of the tail.

Why Zebrafish?

 Key similarities to humans.

 Easily observable developmental stages.

 Less expensive than standard rodent model systems.

Photo Courtesy of the University of Oregon

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)

DNA Adduct Formation 1 3 1 2 1 4 2 1 1 1 3 1 0 4 5 9 8 6 7

D B P O

Cytochrome P450 and epoxide

H

hydrolase

O DBP-dihydrodiol

Cytochrome P450

H O DBP-diolepoxide

CH 2 OH O HO N N N N NH HO HO OH

Adenosine (blue) adduct formed by reaction of Dibenzo[a,I]pyrene (red) with DNA

B-DNA Adducted B-DNA Chen J,

et. al.

"

MMDB: Entrez's 3D-structure database

",

Nucleic Acids Res. 2003 Jan; 31(1)

: 474-7.

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

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

33 P Postlabeling of DNA Adducts Sep-pak

Results: DBP-DNA Adduct Profiles

DBP DBPDE

1 2a 2b 4

BP-DNA Adducts

BP BPDE

BPDE-dG adduct

DNA Adduct Formation in Zebrafish 165.70

4.10

12.65

26.39

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

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