Molecular Mechanisms of Polycyclic Aromatic Hydrocarbon-induced Teratogenesis in

184 Pages·2011·1.65 MB·English

by  Hugh Crumley

Molecular Mechanisms of Polycyclic Aromatic Hydrocarbon-induced Teratogenesis in
Zebrafish (Danio rerio)
by
Lindsey Anne Van Tiem
Department of Environment
Duke University

Date: ______ _______
Approved:

___________________________
Richard T. Di Giulio, Supervisor

___________________________
David E. Hinton

___________________________
Margaret L. Kirby

___________________________
Joel N. Meyer

___________________________
Heather M. Stapleton

Dissertation submitted in partial fulfillment of
the requirements for the degree of Doctor of Philosophy in the Department of
Environment in the Graduate School
of Duke University

2011

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ABSTRACT
Molecular Mechanisms of Polycyclic Aromatic Hydrocarbon-induced Teratogenesis in
Zebrafish (Danio rerio)
by
Lindsey Anne Van Tiem
Department of the Environment
Duke University

Date: ______ ______
Approved:

___________________________
Richard T. Di Giulio, Supervisor

___________________________
David E. Hinton

___________________________
Margaret L. Kirby

___________________________
Joel N. Meyer

___________________________
Heather M. Stapleton

An abstract of a dissertation submitted in partial
fulfillment of the requirements for the degree
of Doctor of Philosophy in the Department of
Environment in the Graduate School
of Duke University

2011
i
v
Copyright by
Lindsey Anne Van Tiem
2011
Abstract
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental
contaminants formed from the incomplete combustion of fossil fuels and are found in
the environment as complex mixtures. PAHs are developmentally toxic to fish, causing
yolk sac edema, hemorrhaging, craniofacial malformations and cardiac defects including
impaired heart looping, elongated heart, decreased blood flow, and pericardial effusion.
Previous research has shown that many of the toxic effects of PAHs are mediated
through the aryl hydrocarbon receptor (AHR), which upregulates phase I and II
metabolic genes, but the underlying mechanisms of PAH-induced toxicity are not yet
known. The primary goal of this dissertation was to better understand the molecular
mechanisms by which PAH mixtures cause developmental toxicity in fish. To this end,
the zebrafish (Danio rerio) was used as a developmental model. Simple mixtures
consisting of a PAH that is an AHR agonist (benzo[a]pyrene or benzo[k]fluoranthene)
and a PAH that is a cytochrome P450 1 (CYP1) inhibitor (fluoranthene) were used in
these experiments along with the dioxin-like compound 3,3',4,4',5-pentachlorobiphenyl
(PCB-126). Morpholino gene knockdown was used to examine the role of specific genes
in response to PAHs, gene expression changes in response to PAH exposures were
examined via QPCR, quantification of pericardial effusion was used as a metric for
cardiac toxicity, and CYP1 activity was measured as an indication of AHR pathway

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induction. First, PAH mixtures consisting of an AHR agonist (BkF) and a CYP1 inhibitor
(FL) induced cardiac toxicity that was preceded by upregulation of CYP1 and redox-
responsive gene expression, and these effects were dependent upon the AHR2. Second,
knockdown of glutathione s-transferase pi class 2 (GSTp2), part of phase II metabolism,
exacerbated PAH-induced toxicity but did not affect PCB-126-induced toxicity. Third,
knockdown of another isoform of the AHR, AHR1, exacerbated PAH- and PCB-126-
induced toxicity and increased CYP1 activity but did not affect CYP expression in
response to these agonists. Simultaneous knockdown of AHR1A and AHR2 did not
exacerbate nor ameliorate PAH-induced toxicity but did prevent PCB-126-induced
toxicity. Fourth, to examine AHR2-dependent and AHR2-independent gene induction
in zebrafish hearts in response to PAHs, microarrays were used. Gene expression
changes caused by PAHs were largely AHR2-dependent and consisted of genes
involved in cell adhesion, oxidation-reduction, and TGF- signaling processes as well as
genes involved in heart structure and function. These findings help to elucidate how
PAHs elicit deformities during development and highlight differences between PAHs
and other AHR agonists. Additionally, these experiments have identified other genes in
addition to AHR2 that are involved in mediating or responding to the toxicity of PAHs.

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Dedication
To my remarkable parents. This work and everything else in my life would not
be possible without your love, encouragement, and faith in me.

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Contents
Abstract .................................................................................................................................... iv
List of Tables........................................................................................................................... xii
List of Figures ........................................................................................................................xiii
Acknowledgements ............................................................................................................... xv
1. Introduction ................................................................................................................. 1
1.1 Polycyclic aromatic hydrocarbons ............................................................................ 1
1.2 The aryl hydrocarbon receptor pathway.................................................................. 4
1.2.1 Basic biology........................................................................................................ 4
1.2.2. Role of the AHR in mediating toxicity ............................................................. 8
1.2.3 The AHR pathway and oxidative stress.......................................................... 10
1.3 Zebrafish ................................................................................................................... 11
1.3.1 Basic biology...................................................................................................... 11
1.3.2. Use as a developmental model ....................................................................... 12
1.4 Dissertation Objective and Outline......................................................................... 14
2. AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-
associated gene induction in zebrafish (Danio rerio)............................................................ 19
2.1 Introduction .............................................................................................................. 19
2.2 Materials and Methods ............................................................................................ 22
2.2.1 Fish care ............................................................................................................. 22
2.2.2 Chemicals and exposure................................................................................... 23
2.2.3 Morpholino injection ........................................................................................ 24

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2.2.4 Deformity assessment ....................................................................................... 25
2.2.5 In vivo EROD assay .......................................................................................... 25
2.2.6 RNA extraction and reverse transcription ...................................................... 26
2.2.7 QPCR ................................................................................................................. 26
2.2.8 Statistical analysis ............................................................................................. 28
2.3 Results ....................................................................................................................... 28
2.3.1 Deformities and EROD activity ....................................................................... 28
2.3.2 Gene expression ................................................................................................ 29
2.3.3 Effect of AHR2 knockdown on deformities and EROD activity ................... 30
2.3.4 Effect of AHR2 knockdown on gene expression ............................................ 31
2.4 Discussion ................................................................................................................. 32
3. Glutathione s-transferase pi class 2 (GSTp2) protects against the cardiac deformities
caused by exposure to PAHs but not PCB-126 in zebrafish embryos ................................ 49
3.1 Introduction .............................................................................................................. 49
3.2 Material and methods .............................................................................................. 52
3.2.1 Fish care ............................................................................................................. 52
3.2.2 Chemicals .......................................................................................................... 52
3.2.3 Morpholino injection ........................................................................................ 53
3.2.4 Dosing ................................................................................................................ 54
3.2.4.1 Dosing for deformities ................................................................................... 54
3.2.4.2 Dosing for reverse transcription PCR and QPCR ........................................ 54
3.2.5 Confirmation of GSTp2 splice-mo efficacy by reverse transcription PCR
analysis ....................................................................................................................... 55

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3.2.6 QPCR ................................................................................................................. 56
3.2.7 Deformity assessment ....................................................................................... 57
3.2.8 Statistical analysis ............................................................................................. 58
3.3 Results ....................................................................................................................... 58
3.3.1 GSTp2 mRNA expression in response to PAH and CPB-126 exposure ....... 58
3.3.2 Confirmation of morpholino efficacy by reverse transcription PCR analysis
..................................................................................................................................... 59
3.3.3 Deformity Assessment...................................................................................... 59
3.3.4 Influence of GSTp2 knockdown on deformities caused by BkF + FL co-
exposure ..................................................................................................................... 60
3.3.5 Influence of GSTp2 knockdown on deformities caused by BaP + FL co-
exposure ..................................................................................................................... 61
3.3.6 Influence of GSTp2 knockdown on deformities caused by PCB-126 ............ 62
3.4 Discussion ................................................................................................................. 63
4. The role of AHR1 isoforms in PAH- and PCB-126-induced toxicity ............................. 72
4.1 Introduction .............................................................................................................. 72
4.2 Materials and Methods ............................................................................................ 75
4.2.1 Fish Husbandry ................................................................................................. 75
4.2.2 Chemicals and Dosing ...................................................................................... 75
4.2.3 Morpholino Injection ........................................................................................ 77
4.2.4 Deformity Assessment...................................................................................... 78
4.2.5 In vivo EROD Assay ......................................................................................... 79

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4.2.6 Confirmation of AHR1A and AHR1B splice-mo efficacy by reverse
transcription PCR analysis ........................................................................................ 79
4.2.7 QPCR ................................................................................................................. 81
4.2.8 Statistical Analysis ............................................................................................ 82
4.3 Results ....................................................................................................................... 83
4.3.1 Confirmation of AHR1A and AHR1B splice-mo efficacy by reverse
transcription PCR analysis ........................................................................................ 83
4.3.2 Preliminary experiments investigating the effect of AHR1A and AHR1B
knockdown on PAH and PCB-126 toxicity.............................................................. 83
4.3.3 Effect of AHR1A knockdown on PAH and PCB-126 toxicity ....................... 85
4.3.4 Effect of AHR1A knockdown on PAH- and PCB-126-induced CYP1 gene
expression .................................................................................................................. 86
4.3.5 Effect of AHR1A-mo, AHR2-mo, and AHR1A/2-mo on PAH and PCB-126
toxicity ........................................................................................................................ 87
4.3.6 Effect of AHR2-mo, AHR1A-mo, and AHR1A/2-mo on PAH- and PCB-126-
induced CYP1 activity ............................................................................................... 90
4.4 Discussion ................................................................................................................. 92
5. Heart-specific microarray identification of AHR-dependent and AHR-independent
genes involved in the synergistic developmental toxicity of PAHs ................................. 109
5.1 Introduction ............................................................................................................ 109
5.2 Materials and Methods .......................................................................................... 112
5.2.1 Fish care ........................................................................................................... 112
5.2.2 Morpholino injection ...................................................................................... 113
5.2.3 Chemicals and dosing..................................................................................... 113
5.2.4 Deformity Assessment.................................................................................... 115

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