UNDERSTANDING THE EFFECTS OF ENVIORNMENTAL POLLUTANTS ON THE GUT MICROBIOME
Open Access
- Author:
- Nichols, Robert
- Graduate Program:
- Molecular Toxicology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 21, 2019
- Committee Members:
- Andrew David Patterson, Dissertation Advisor/Co-Advisor
Andrew David Patterson, Committee Chair/Co-Chair
Gary H Perdew, Committee Member
Curtis John Omiecinski, Committee Member
Istvan Albert, Outside Member
Curtis John Omiecinski, Program Head/Chair - Keywords:
- Microbiome
16S sequencing
Metatranscriptoics
AHR
TCDF
Flow Cytometry - Abstract:
- The human microbiome is made up of distinct regions (e.g., intestinal, skin, oral or vaginal) that differ in composition and functionality. The human microbiome may be modulated with prebiotics, probiotics, and postbiotics to potentially aid in the treatment of diseases like irritable bowel syndrome, bacterial vaginosis, atopic dermatitis, gingivitis, obesity, or cancer. There is also potential for many members of the human microbiome to directly modulate host gene expression and modulate host detoxifying enzyme activity like cytochrome P450s (CYPs), dehydrogenases, and carboxylesterases. Therefore, the microbiome is important to consider during drug discovery, risk assessment, and dosing regimens for various diseases given that the human microbiome has been shown to impact host detoxification processes. In order to study the human microbiome, bioinformatic techniques, like 16S ribosomal RNA (rRNA) gene sequencing, sequencing the functional capability of a microenvironment with metagenomics, and studying the functional changes by sequencing the actively transcribed messenger RNA through metatranscriptomics, must be included and combined with traditional toxicological techniques like metabolomics. Currently, the intestinal microbiome is an important focus of the human microbiome due to the role the intestinal microbiome plays in drug and xenobiotic metabolism, in addition to the number of xenobiotics that humans are exposed to on a daily basis through food and water intake. An important class of xenobiotics are persistent organic pollutants (POPs) which are environmental concerns and continued study of their mechanism of action remains a high priority. POPs include among others 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and polychlorinated biphenyls (PCBs) and are byproducts of pesticides and herbicides production streams and other human-made chemicals. Some POPs, including TCDF, TCDD, and PCB-126, are agonists for the aryl hydrocarbon receptor (AHR). Activation of the AHR is reported to modulate the intestinal microbiome, host immunity, and the host metabolome. Modulation of the intestinal microbiome from exposure to TCDD, TCDF and PCB-126 was measured with a combination of 16S rRNA sequencing, metatranscriptomics, metabolomics and assay-based approaches. TCDF was further explored with two different studies where male C57BL6/J mice were exposed to vehicle, or 5 µg/kg body weight (BW) TCDF, or 24 µg/kg BW of TCDF every day for 5 days. The functional and structural changes caused by TCDF exposure to the intestinal microbiome and host metabolome was measured with 16S rRNA gene amplicon sequencing, metabolomics, and bacterial metatranscriptomics. Statistically significant changes in bacterial enzyme gene expression revealed increases in lipopolysaccharide (LPS) biosynthesis after exposure to 24 µg/kg BW of TCDF. Increases in LPS biosynthesis were confirmed with metabolomics and LPS assays using serum obtained from TCDF-treated mice. Significant increases in gene expression within aspartate and glutamate metabolism were noted after exposure to 24 µg/kg BW of TCDF. Together these results suggest that after exposure to 24 µg/kg BW of TCDF the intestinal microbiome increases the production of LPS and glutamate to promote localized gut inflammation, potentially using glutamate as a stress response. Due to the compositional and functional changes in the intestinal microbiome imparted by TCDF, PCB-126, and TCDD, flow cytometry was introduced to help characterize the damage on the intestinal microbiome. Isolated cecal contents were exposed to four antibiotics with different modes of action (ampicillin, rifampicin, tetracycline, and ciprofloxacin) at three different doses (4.5 mg, 0.45mg, and 0.045 mg of ampicillin, ciprofloxacin and tetracycline, and 0.14 mg, 0.014 mg, and 0.0014 mg of rifampicin), within an anaerobic chamber and incubated for 4 hours. Through flow cytometry (to investigate the antibiotic damage profile to the bacterial cell), 16S rRNA gene sequencing (to investigate the change in bacterial composition) and metabolomics (to investigate the modulated metabolic profile), distinct damage profiles were created for each antibiotic. This dissertation examines the value of using multiple analytical techniques to create a systems approach to fully understand the effects of environmental chemicals on the intestinal microbiome. Additionally, it examines how this strategy can be employed to predict and identify the effects of other unknown compounds on the intestinal microbiome.