Document Type

Dissertation

Date of Degree

Fall 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Human Toxicology

First Advisor

Gabriele Ludewig

Abstract

Polychlorinated biphenyls (PCBs) are the persistent environmental pollutants with the continuous concerns over adverse human health effects. As semi-volatile compounds, PCBs were found in indoor and outdoor air. The observation of high levels of airborne PCBs in old school buildings raised the concerns of inhalation exposure and toxicity of PCBs. Lower chlorinated PCBs (LC-PCBs), major components of airborne PCBs, are subject to biotranformation. In vitro and in vivo studies revealed that reactive metabolites of LC-PCBs formed covalent adducts on DNA and proteins. The hypothesis of the project is that the reactive metabolites of LC-PCBs are able to form adducts on proteins or even protein crosslinks, and the formation of protein adducts and crosslinks causes the dysfunction of the target proteins. In addition, the objectives of the project are also to identify protein targets by PCB metabolites, which may be related to the mechanism of toxicity of LC-PCBs. The alkaline permethylation (AP) was established and optimized to identify and measure the protein adducts from LC-PCB metabolites. The AP method evidenced PCB metabolites formed protein adducts through the sulfhydryl groups and also one molecule of PCB quinoid metabolites was able to bind to more than one protein. Application of cytochrome c as the model protein revealed PCB quinoid metabolites also formed adducts on lysine and glutamic acid. The adduct formation and crosslinks caused the dysfunction of cytochrome c. In addition, the quinone protein adducts still kept the ability for redox reactions, which may lead to unexpected toxicity. The SILAC method was applied to identify the target proteins in the samples of in vitro proteome incubation. The instability of PCB quinone protein adducts was found by further reaction of quinone protein adducts. This may be the reason why cysteine-PCB quinone adducts were not frequently identified by proteomics method. The further understanding of protein adducts by reactive PCB metabolites helps to identify the target proteins, and ultimately reveal the role of protein adducts impacting on human health.

Public Abstract

Polychlorinated biphenyls (PCBs) are environmental pollutants that resist breakdown in the environment, and are generally persistent. PCBs are known to cause adverse health effects in humans including an increased likelihood of infections, diabetes, and cancer, just to name a few. Traditionally, we think humans are exposed to PCBs through our food. Recently, PCBs were found in indoor air in buildings and outdoor air in major big cities of North America. The observation of PCBs in old school buildings especially raised concerns of adverse health effects of PCBs in air to children. Previous research reported that some PCB metabolites, which are formed in our bodies from inhaled or eaten PCBs, can attach to DNA or proteins through chemical reactions. This attachment may lead to the reported health damage to humans. The objective of this project was to establish methods to measure the attachment of these PCB metabolites into proteins, and also to find out how the reaction between these metabolites and proteins may lead to adverse health effects. The application of a derivatization and an analytical chemistry method ‘AP method’ allowed me to determine how much of a metabolite was attached to proteins. The proteomics technology revealed types of attachments of PCB metabolites to proteins. My research proved that PCBs react with proteins through specific reactions, and also showed the malfunction of a protein, cytochrome c, which is important in energy production in cells. I also observed that these metabolites can tether several proteins together and change their ability to function normally. In addition, a sophisticated variation of the proteomics method (SILAC) allowed me to identify preferred target proteins of these PCB metabolites in a protein mixture. In future experiments, the identification of target proteins of these PCB metabolites by proteomics technology and the properties of the interactions between PCBs and proteins will help us to better explain and predict the various adverse effects caused by PCBs from air and other contaminants. The findings and observations in this research will ultimately help us to better protect our environment and human health.

Keywords

publicabstract, Polychlorinated Biphynels, Protein Adducts, Proteomics, Quinone, Reactive Metabolites, SILAC

Pages

xvi, 139 pages

Bibliography

Includes bibliographical references (pages 125-139).

Copyright

Copyright 2015 Miao Li

Included in

Toxicology Commons

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