Date of Degree

6-2017

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Nathalia Holtzman

Committee Members

Daniel Weinstein

PoKay Ma

Isaac Wirgin

R. Chris Chambers

Moses Chang

Subject Categories

Biology | Environmental Health | Terrestrial and Aquatic Ecology | Toxicology

Keywords

zebrafish, sturgeon, PCBs, TCDD, development, ecotoxicology, cardiaotoxicity

Abstract

There is a long history of damage to natural ecosystems from environmental pollution. Many environmental contaminants are man-made and have been released with abandon over the last 100 years including dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and polychlorinated biphenyls (PCBs). These chemicals act on similar cellular processes and cause skin lesions, cancer, learning disabilities and reproductive problems in many vertebrates. There are many studies exploring various aspects of TCDD and PCB exposure on model and wild organisms. Few studies however, have compared effects of PCB mixtures on ecosystems to effects of individual PCBs in the lab. The first aim of this thesis is to addresses this issue by comparing the impacts of individual PCBs with PCB mixtures (Aroclors) in embryonic zebrafish (Danio rerio). Further, few studies compare contaminant-derived defects in a lab model to a wild species living in contaminated waterways. Aim 2 contrasts cardiac defects seen in the lab model zebrafish to Atlantic sturgeon (Acipenser oxycrinchus oxyrinchus) an endangered fish native to a contaminated ecosystem. Finally, little is known of the cellular and molecular mechanisms governing heart defects caused by contaminant exposure. Aim 3 identifies part of the mechanism contributing to heart defects in zebrafish and Atlantic sturgeon. This thesis aims to fill these gaps and clarify how TCDD and PCBs impact zebrafish and Atlantic sturgeon during early development.

Aim 1: “Toxic effects of PCB congeners and Aroclors on zebrafish growth and development”

- Hypothesis: PCB congeners, PCB 104 and PCB 126, produce more severe defects in early zebrafish development than Aroclors

- Methods: Gross morphological abnormalities early in development were observed. Embryos treated with sublethal concentrations of PCBs and Aroclors were assessed for organ defects and quantified. Surviving embryos were raised to adulthood during which time growth and survival at regular intervals were measured

- Conclusion: While gross morphological impacts were variable by contaminant and concentration, overall, the highest concentrations of exposure resulted in the most severe phenotypes. Organ defects in the cardiovascular and digestive systems were observed in some PCB exposures: PCB 126 elicited the most severe phenotypic responses. PCBs and Aroclors increased mortality, however, interestingly growth of surviving PCB and Aroclor exposed fish did not differ from controls.

Aim 2: “Similarities of the impact of dioxin-like compounds on early heart development in the endangered Atlantic sturgeon and the lab model zebrafish”

- Hypothesis: Atlantic sturgeon treated with TCDD, PCB 126 and an Aroclor mixture will develop cardiac defects similar to those seen in zebrafish.

- Methods: Zebrafish and Atlantic sturgeon embryos were exposed to six concentrations of each toxin, with associated controls for 24 hours during gastrulation and neurulation. To specifically visualize the heart, stage-matched Atlantic sturgeon larvae collected 1 day post hatching and zebrafish embryos collected at 3 days post fertilization were analyzed using immunohistochemistry. Heart morphometrics were measured through image analysis to quantify contaminant effects during cardiac looping and ballooning in both species.

- Conclusion: There are parallels in normal heart development in these different species as well as similarities in response to contaminant exposure. In both TCDD and PCB 126 exposed zebrafish and Atlantic sturgeon there is a decrease of curvature in the heart with increasing dose. Conversely, Aroclor treated fish do have properly curved hearts in both species, but the heart chambers do not balloon as well in hearts exposed to higher concentrations.

Aim 3: “TCDD and PCB 126 derived embryonic cardiac defects result from a novel AhR pathway”

- Hypothesis: TCDD or PCB 126-activated AhR phosphorylates of Src which phosphorylates of the gap junction protein VEcadherin leading to separation of endothelial cells.

- Methods: Hearts were visualized using immunohistochemical staining with primary antibodies for MF20 to mark cardiac muscle, CYP1A as a marker for AhR activation, and an antibody specific to phosphorylated VEcadherin (pVEcad). The pVEcad antibody was used to establish that VEcadherin is in fact phosphorylated in the same tissues impacted by contaminant exposure, which will express CYP1A. Contaminant-exposed embryos were treated with SU6656, an Src inhibitor, to rescue the phenotype and establish that AhR activation and VEcadherin phosphorylation is mediated by Src phosphorylation. These embryos also were stained to verify changes in pVEcad expression.

- Conclusion: Together these data support the hypothesis that Src and VEcadherin contribute to cardiac defects seen in fish exposed to TCDD and PCB 126 through a novel AhR pathway, which is unique from the canonical AhR pathway ending in the expression of cyp1A.

In summary, these Aims successfully explored the impacts of contaminants on two distantly related fish species. This body of data provides needed comparisons within the developmental and ecotoxicological fields comparing individual PCBs with PCB mixtures. Further, the first two aims establish direct connections between two highly evolutionarily divergent fish species, zebrafish and Atlantic sturgeon, which increases our confidence in using zebrafish as a model for cardiac defects in wild species. The final aim establishes a novel mechanism of AhR derived cardiac defects not previously explored.

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