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Author ORCID Identifier

https://orcid.org/0000-0002-2572-3227

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded

2020

Month Degree Awarded

May

First Advisor

Alicia R. Timme-Laragy

Subject Categories

Developmental Biology | Endocrine System Diseases | Maternal and Child Health | Toxicology

Abstract

The glutathione (GSH) system evolved to combat oxygen toxicity in nearly all living organisms, and serves as the predominant cellular redox buffer in vertebrates. This dissertation explores the roles of GSH during embryogenesis using the zebrafish (Danio rerio). The first agnostic in vivo characterization of GSH utilization in the embryo revealed strict spatiotemporal GSH profiles in developing organ systems. Interruption of these GSH profiles by exogenous xenobiotics led to organ specific adverse developmental outcomes; pancreatic β-cells were found to be especially susceptible and developed atypical morphologies following redox active xenobiotic exposures. Pancreatic β-cells lacked expression of Nrf2a (Nuclear factor erythroid (2) like 2; Nrf2a – zebrafish Nrf2 co-ortholog), a transcription factor that regulates antioxidant genes. Using a novel application of BioGEE (biotinylated glutathione ethyl ester) to visualize in situ protein glutathionylation at the whole organism level, experiments demonstrated the lack of Nrf2 expression was accompanied by oxidized GSH pools. Moreover, altered Nrf2a signaling and differential protein glutathionylation patterns were found to underlie aberrant pancreatic β-cell morphologies. Using the techniques developed here, the mode of action of PFOS, a ubiquitous environmental toxicant known to disrupt β-cell morphogenesis, was interrogated. PFOS disrupted GSH profiles in the developing pancreas and yolk, and, induced Nrf2a expression in the endocrine pancreas, where it is normally absent. Notably, bolstering cellular GSH pools rescued these changes, indicating PFOS toxicity in the pancreas is redox mediated. Experiments done in cell culture to better resolve the mechanism were inconclusive, and merit further investigation. This dissertation demonstrates that organ systems develop in specific redox microenvironments, and consequently, have separate resilience to toxicant mediated redox perturbations that change with developmental stage. Further, embryonic pancreatic β-cells have oxidized GSH redox conditions, and many xenobiotics disrupt redox signaling in these cells potentially altering their reserve antioxidant capacity and rendering them vulnerable to later life exposures. Given the multitude of exogenous xenobiotics people are exposed to everyday, this work has broad implications for identifying exposed populations and their underlying susceptibility to metabolic disorders. Although these exposures are impossible to avoid, by identifying vulnerable populations, lifestyle interventions can be implemented, minimizing the risk of disease outcomes.

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

Available for download on Saturday, May 08, 2021

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