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
3
2
4
5
1
O
6
14
7
13
12
8
11
10
9
Cytochrome P450
and epoxide
hydrolase
D BP
Cytochrome P450
HO
OH
DBP-dihydrodiol
CH2OH
O
HO
N
N
N
N
NH
HO
HO
OH
Adenosine (blue) adduct formed by reaction of
Dibenzo[a,I]pyrene (red) with DNA
HO
OH
DBP-diolepoxide
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
Bath exposure
embryo
BP or DBP
24hr after exposure
Postlabeling
24 hr post fertilization
DNA adduct analysis
Determining DNA Adduct
Formation
 Treat fish with carcinogen
 Isolate DNA
 Digest and label DNA with
 Sep-Pak purification
 HPLC Analysis
33P
33P
Postlabeling of DNA
Adducts
Sep-pak
Results: DBP-DNA Adduct
Profiles
DBP
DBPDE
4
2b
1
2a
BP-DNA Adducts
BP
BPDE
BPDE-dG adduct
DNA Adduct Formation in
Zebrafish
165.70
12.65
4.10
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