Characterizing the impact of phthalates on the female gastrointestinal tract and reproductive system during adulthood

Chiu, Karen

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https://hdl.handle.net/2142/115381 Copy

Description

Title

Characterizing the impact of phthalates on the female gastrointestinal tract and reproductive system during adulthood

Author(s)

Chiu, Karen

Issue Date

2022-04-12

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

Flaws, Jodi A

Doctoral Committee Chair(s)

Swanson, Kelly S

Committee Member(s)

• Mei, Wenyan
• Nowak, Romana A

Department of Study

Nutritional Sciences

Discipline

Nutritional Sciences

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Di-isononyl phthalate (DiNP)
• di(2-ethylhexyl) phthalate (DEHP)
• colon
• cecum
• intestines
• gut
• reproduction

Abstract

The gastrointestinal (GI) tract is a continuous passageway composed of the mouth, esophagus, stomach, small intestine, large intestine (colon), and anus. The primary focus of this dissertation is the colon, which is segmented into several parts—cecum, appendix, ascending colon, transverse colon, descending colon, and rectum. The function of the colon is to absorb or remove water and minerals, eliminate waste from the body, store feces, synthesize vitamin K, and ferment indigestible food matter. Synthesis of vitamin K and fermentation of ingestible food matter like beta-glucan, pectin, inulin, and other select fibers are achieved by anaerobic gut microbes known as the gut microbiota. The gut microbiota are defined as all the microorganisms (e.g., bacteria, viruses, archaea, eukaryotes, and fungi) present in the gut, whereas the gut microbiome includes the microorganisms and its genetic material. The gut microbiome has been termed a separate “organ” due to its distinct immune and metabolic activity. There are 10x more microbial cells in the human gut than the whole human body, weighing about 4 pounds, and totaling approximately 100 trillion microbes. These microbial cells have the power to influence the host’s immune system and metabolic state. Although the microbial cells have the ability to influence the host’s immune and metabolic states, the gut microbiome is influenced by many factors including diet, age, sex, geographical location, mode of infant delivery method, medication and drug use, and physical activity. In addition, the gut microbiome is impacted by exposure to environmental chemicals, including phthalates. Phthalates are commonly used as plasticizers or stabilizers that increase the resiliency and flexibility of plastic materials. Phthalates account for approximate two-thirds of the global plasticizer market. Di-isononyl phthalate (DiNP) and di-(2-ethylhexyl) phthalate (DEHP) are some of the most widely used phthalates. The application of DiNP is most commonly used for cable wires, roofing, flooring, wall coverings, coated fabrics, and polyvinyl chloride (PVC), and the application of DEHP is in PVC and medical devices (e.g., medical tubing and catheters). Because DiNP and DEHP are noncovalently bonded to materials, they can leach out of the materials and into the environment. From the environment, phthalates end up in the human body by ingestion (most common route of exposure), inhalation, intravenous absorption, and dermal contact. Since the most common route of exposure to phthalates is by ingestion of phthalates, we wanted to investigate the impacts of DiNP and DEHP on the GI tract—specifically the colon. Surprisingly, information was limited on the effects of high-molecular weight phthalates, including DiNP and DEHP, on the female GI tract and whether and how changes in the gut impact pregnancy outcomes. Therefore, the purpose of my doctoral dissertation work was to investigate the effects of high-molecular weight phthalates on GI health during adulthood and to determine whether changes in the gut microbiome affect pregnancy outcomes in female mice. Specifically, I investigated the effects of subacute exposure to DiNP or DEHP during adulthood on inflammation, gut integrity, intestinal cell populations, cell health, sex steroid hormones, and fertility. The first aim of my dissertation tested the hypothesis that subacute exposure to DiNP dysregulates cellular, endocrine, and immunological aspects in the colon of adult female mice. After treatment with corn oil vehicle control or DiNP, colon tissue samples were subjected to morphological, biochemical, and hormone assays. Assessing morphological, biochemical, and hormonal changes in the colon can give insight to possible avenues through which DiNP may disrupt the gastrointestinal system. I discovered that DiNP exposure significantly increased histological damage in the colon compared to control. The histological data agreed with gene expression data that showed DiNP exposure significantly decreased a cell cycle regulator (Ccnb1) and increased apoptotic factors (Aifm1 and Bcl2l10) in the colon compared to control. Exposure to DiNP also significantly decreased sICAM-1 levels and increased Tnf expression compared to control. Further, analysis of colon-extracted lipids revealed that subacute exposure to DiNP significantly decreased estradiol levels compared to control. Collectively, these data indicate that subacute exposure to DiNP alters colon morphology, endocrine function, and the immune microenvironment in adult female mice. Next, I tested the hypothesis that DiNP exposure alters immune responses and impacts specialized epithelial cells in the colon of adult female mice. After treatment with vehicle control or DiNP, colon tissues and sera were collected for histological, genomic, and proteomic analysis of various immune factors and specialized epithelial cells. Subacute exposure to DiNP significantly increased protein levels of Ki67 and MUC2, expression of a lysozyme marker (Lyz1), and estradiol levels in sera compared to control. Gene expression of mucins (Muc1, Muc2, Muc3a, and Muc4), Toll-like receptors (Tlr4 and Tlr5), and specialized epithelial cells (ChgA, Lgr5, Cd24a, and Vil1) were not significantly different between treatment groups and control. Cytokine levels of IL-1RA and CXCL12 were also not significantly different between DiNP treatment groups and control. These data reveal that DiNP exposure increases circulating estradiol levels and gene expression in specialized epithelial cells with immune response capabilities (e.g., goblet cells) in the mouse colon, which may initiate immune responses to prevent further damage in the colon. The third aim of my dissertation tested the hypothesis that subacute exposure to environmentally relevant doses of DiNP alters the gut microbiota and that the colon harbors anaerobic bacteria capable of using DiNP as the sole carbon source. Female mice were dosed with control or environmentally relevant doses of DiNP (20 or 200 µg/kg) for 10-14 days. After the treatment period, colon contents were collected for full-length 16S rRNA gene sequencing and Sanger sequencing. The data revealed that DiNP exposure significantly altered the colon microbiota and that several anaerobic bacteria can use DiNP as the sole carbon source, suggesting that the gut microbiome may be partly responsible for mediating phthalate-induced changes in the colon. The final objective of my dissertation was to investigate the effects of DEHP exposure during pregnancy on the cecal microbiome and its impact on pregnancy and birth outcomes. I tested the hypothesis that subacute exposure to an environmentally-relevant dose of DEHP during pregnancy alters the cecal microbiome in pregnant mice, leading to changes in birth outcomes. To test this hypothesis, pregnant dams were orally dosed with corn oil vehicle control or 20 µg/kg DEHP for 10 days and euthanized 21 days after their last dose. Cecal contents were collected for 16S Illumina and shotgun metagenomic sequencing. Fertility studies were also conducted to examine whether DEHP exposure impacted birth outcomes. Subacute exposure to an environmentally relevant dose of DEHP in pregnant dams significantly increased alpha diversity and significantly altered beta diversity. Furthermore, DEHP exposure during pregnancy significantly increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Deferribacteres compared with controls. The affected taxonomic families included Deferribacteraceae, Lachnospiraceae, and Mucisprillum. In addition to changes in the gut microbiota, DEHP exposure significantly altered 14 functional pathways compared with the control. Although there were significant changes in the cecal microbiome and functional profiles of DEHP-exposed mice compared to control, DEHP exposure did not significantly impact the fertility and birth outcomes compared with the control. These data indicate that DEHP exposure during pregnancy shifts the cecal microbiome, but the shifts do not impact fertility and birth outcomes. Collectively, these data from my dissertation indicate that subacute exposure to phthalates have a variety of consequences on the female gastrointestinal tract ranging from the endocrine, immunological, and cellular perspective.

Graduation Semester

2022-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Karen Chiu

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