Investigating the Toxicity of
Oxygenated Polycyclic Aromatic
Hydrocarbons (OPAHs) in
Developing Zebrafish
Researched by
Annika Swanson
Mentor
Dr. Robert Tanguay

PAHs
• Polycyclic aromatic hydrocarbons
– 2 or more rings
– Named by functional groups
Naphthalene http://www.sigmaaldrich.com

PAHs
• Environmental contaminants
– Carcinogenic
– Mutagenic
– Teratogenic
• PAH production
– Automobile exhaust
– Industry emissions
– Wood burning
– Tobacco smoke http://www.platinum.matthey.com

OPAHs
• Oxygenated polycyclic aromatic hydrocarbons
• PAHs with oxygen additions
– Functional groups
– Carbonyl, hydroxyl, carboxyl http://chemed.chem.purdue.edu/genchem/topicreview/bp/2organic/carbonyl.html

OPAH Formation
• Incomplete combustion processes
• Transformation of PAHs
– Chemical oxidation
– Photo-oxidation
– Biological/microbial transformation

Bacteria Degradation of Naphthalene http://www.mdpi.com/1660-4601/6/1/278/htm

Research Objectives
• Analyze the toxicity of OPAHs in developing zebrafish
– AHR activation
– CYP1A induction
– Molecular structures
• Expand the limited scientific knowledge about
OPAH toxicity

• Zebrafish
• Development and toxicity

The AHR Pathway
AHR Ligand
• Three
Zebrafish
AHR receptors
– AHR2 (liver, vasculature)
– AHR1B
(vasculature)
– AHR1A (liver)
AhR
AhR
ARNT
Cytoplasm
AhR
ARNT
Nucleus
AhR
ARNT
DRE
Transcription of genes
CYP1A
Translation
• CYP1A protein expression
•
Xenobiotic metabolism

Experiment Setup
• Dechorionate embryos
• Expose to the chemical in solution
• Make 24 hour and 5 day evaluations
• Fix for IHC analysis

Stages of Development
3 min
19 hr
1.25 hr
48 hr
4 hr
24 hr
5 day
6 hr

9,10-Phenanthrenequinone
Five Day Observations
1% DMSO (Control)
2.0μM PQO in 1% DMSO

Immunohistochemistry
• IHC
– Tag CYP1A protein http://www.badrilla.com/ihc.html

IHC
10 µM Leflunomide 25 µM Benz(a)anthracene
Positive Controls (Vehicle: 1% DMSO)
Negative Control (1% DMSO)

9,10-Phenanthrenequinone (PQO)
• C
14
H
18
O
2
• Toxic chemical in diesel exhaust emissions (DEP)
• Quinone Toxicity
1.6µM http://www.sigmaaldrich.com

• C
14
H
8
O
2
• Quinone
9,10-Anthraquinone
Toxicity
20µM http://en.wikipedia.org/wiki/File:Anthraquinone_acsv.svg

Xanthone
• C
13
H
8
O
2
• Very little information known
20µM
Toxicity http://en.wikipedia.org/wiki/File:Xanthone.svg

1,9-Benz-10-anthrone (BAO)
• C
17
H
10
O
• Very little information known
Toxicity
20µM http://www.sigmaaldrich.com

Benz[a]anthracene-7,12-dione (BADO)
• C
18
H
10
O
2
• Quinone
Toxicity
5µM http://www.sigmaaldrich.com

AHR2 Knockout
• 5µM Benz[a]anthracene-7,12-dione (BADO)
Wild-type Fish AHR2 Knockout Fish http://learn.genetics.utah.edu/content/labs/pcr/

2,3-Dihydroxynaphthalene
• C
10
H
8
O
2
• 2-ring, more volatile
• Strong unique CYP1A expression
Toxicity
20µM http://www.sigmaaldrich.com

Experiment
• Polymerase Chain Reaction
• (PCR)
• Gene expression changes
• qPCR http://learn.genetics.utah.edu/content/labs/pcr/

Experiment
• Quantitative PCR
• Gene expression differences between products
CYP1A (5dpf)
140
120
100
80
60
40
20
0
0.1%_DMSO OPAH7_5uM OPAH10_20uM OPAH17_1uM OPAH20_20uM OPAH21-5uM BKF_10uM
Sample

Structural Dependence of OPAH
Toxicity
Toxicity
5µM
Benz[a]anthracene-7,12-dione (BADO)
Toxicity
1,9-Benz-10-anthrone (BAO)
10µM

9,10-Phenanthrenequinone (PQO)
2µM
9,10-Anthraquinone
20µM
Xanthone 20µM
Toxicity
Toxicity
Toxicity

Conclusion
• Oxygenated polycyclic aromatic hydrocarbons
– Toxic compounds
– Structural relationships
– Present in environment
• Future
– 2-ring OPAHs
– Injections into chorion

Acknowledgements
• Howard Hughes Medical Institute
• URISC
• Oregon State University Honors College
• CRIPPS
• Dr. Robert Tanguay and Lab
• Britton Goodale and Andrea Knecht
• Dr. Kevin Ahern