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