Understanding and developing biomolecular tools for studying epigenetic toxicity of per- and polyfluoroalkyl substances

Wen, Yi

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https://hdl.handle.net/2142/116085 Copy

Description

Title

Understanding and developing biomolecular tools for studying epigenetic toxicity of per- and polyfluoroalkyl substances

Author(s)

Wen, Yi

Issue Date

2022-07-15

Director of Research (if dissertation) or Advisor (if thesis)

Irudayaraj, Joseph MK

Doctoral Committee Chair(s)

Irudayaraj, Joseph MK

Committee Member(s)

• Flaws, Jodi A
• Nie, Shuming
• Underhill, Gregory H

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Epigenetics
• Toxicology
• PFAS

Abstract

Per- and polyfluoroalkyl substances (PFAS) are a group of synthetic chemicals used in manufacturing industries and consumer products around the world since the 1940s for their exceptional hydrophobic and oleophobic properties as surfactants. Their high stability owing to strong carbon-fluorine bonds has caused widespread accumulation in soil and water sources around the globe. Long chain PFAS especially perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are the most commonly used and found to be highly accumulative in the environment. PFOA and PFOS have long half-lives ranging from 2.4 to 5.4 years in humans. Considering their high bioaccumulation potential and ubiquitous exposure, all humans on earth are vulnerable to the toxicity caused by PFAS. As primary detoxification organs, the liver and kidney act to protect the body from xenobiotic toxicants. However, PFAS are especially accumulative in the liver and kidney, hindering their vital functions. PFAS were also found to inflict toxicity through disrupting hormonal balance, and therefore are classified as endocrine disrupting chemicals (EDCs). Epigenetic mechanisms were found to play important roles in EDC-induced toxicity. Despite extensive toxicological studies on PFAS, much research is needed to unravel the mechanisms through which they exert their toxicity, particularly epigenetic mechanisms. Herein, we have identified significant cytotoxicity in human hepatocyte HepG2 cells exposed to PFOA and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoic acid (GenX). PFOA affected the expression of cell cycle and lipid metabolism genes and induced fatty acid synthesis and accumulation. Global DNA methylation of HepG2 cells was found to be inversely proportional to PFOA. Ten-eleven translocation methylcytosine dioxygenases (TETs) were significantly affected by PFOA. PFOS dose-dependent decrease in viability was noted in embryonic kidney cells HEK293 upon acute exposure. At non-lethal dosage, genes related to embryonic development, carcinogenesis, glycolysis, lipids, and xenobiotics metabolism were found to be altered in expression in PFOS treated cells compared to control. Although no significant change in global DNA methylation in PFOS-treated cells compared to control was found in HEK293. Gene-specific DNA methylation reprogramming was identified through bisulfite sequencing and pyrosequencing CpG assays. DNA methyltransferases (DNMTs) were significantly affected by PFOS compared to control. In animal studies, PFOA was found to induce hepatocellular steatosis in mouse liver. Decreased global DNA methylation and altered expression of Dnmt3a and Tet1 in response to PFOA were noted. Gene expression in mTOR pathway were altered and Pten expression was decreased with PFOA treatment compared to control. Changes in liver tissue-specific RNA binding proteins and alternative splicing were noted upon PFOA exposure compared to control. On the other hand, kidney injury markers including Acta2 and Bcl2l2 were found to be affected in PFOS exposed mouse kidney compared to control. The expression of numerous transcription factors including nuclear receptors such as Ppara, Pparg and Ppard was disrupted by PFOS exposure compared to control. Bioinformatics analysis on the transcriptome identified significantly upregulated pathways in response to PFOS such as kidney fibrosis, adipogenesis and beta oxidation. Similar to the PFOA exposed liver, global DNA methylation decreased in the PFOS exposed kidney compared to control. Finally, to study the epigenetic mechanisms and potentially revert the epigenetic effect of environmental toxicants on specific gene targets, a CRISPR-based biomolecular toolbox composed of loci-specific sgRNA and dCas9 fused to a catalytic domain of epigenetic regulators such as DNMT3A and TET1 was designed and constructed through molecular cloning. Targeted epigenome editing was performed on embryonic kidney cells and future perspectives on utilizing epigenome editing in toxicology studies were discussed.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

© 2022 Yi Wen

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