METABOLOMICS REVEALS THE IMPACT OF XENOBIOTICS ON THE HOST-METABOLITES-MICROBIOME INTERACTION
Open Access
- Author:
- Cai, Jingwei
- Graduate Program:
- Molecular Toxicology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 25, 2018
- Committee Members:
- Andrew D. Patterson, Dissertation Advisor/Co-Advisor
Andrew D. Patterson, Committee Chair/Co-Chair
Gary H. Perdew, Committee Member
Curtis J. Omiecinski, Committee Member
Matam Vijay-Kumar, Outside Member - Keywords:
- microbiome
metabolomics
short chain fatty acid
bile acid
xenobiotics
LC-MS
GC-MS
NMR
TEMPOL
in vitro - Abstract:
- The diversity and composition of the bacterial community inhabiting the human gastrointestinal tract contributes to the evolutionary fitness of the host through its role in extracting energy from diet and producing signaling molecules (e.g., short chain fatty acid [SCFA] and bile acid) to regulate metabolic and immunological function. Further, the gut microbiome composition and function can be perturbed by environmental stressors (xenobiotics, toxicants, drugs), change in diet (nutrition) or lifestyle (smoking, exercise, stress), and thus greatly influence the host metabolic phenotype and disease risk. A better understanding of how the xenobiotic-microbiome-host interaction contributes to disease risk may identify new therapeutic targets for metabolic and inflammatory disorders like obesity and diabetes. High-throughput metabolomics approaches including liquid chromatography coupled with mass spectrometry (LC-MS), gas chromatography coupled with mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy inform metabolic changes by creating a “metabolite dictionary” to decipher the metabolite chatter between the host and the gut microbiota. Moreover, robust genomics approaches, including 16S rRNA gene sequencing, metagenomics and metatranscriptomics, provide an additional perspective to view and understand the microbiome community structure and function. By combining those approaches, the correlation between microbial community structure, metabolic profiles and phenotypes of microbiome and host can be established to develop a deeper understanding of microbiota-host interaction. Therefore, the central hypothesis of the dissertation is metabolomics in addition with other informative techniques enables the comprehensive and complementary understanding of the mechanistic interplay between the host and microbiome. Given the biological and clinical significance of microbiota and microbial-derived metabolites like SCFAs and bile acids, reliable and efficient metabolomics platforms and methods to provide robust detection and quantitation results with improved analytical confidence is highly demanded. Four different methods for SCFA extraction and quantitation were evaluated and compared using two independent platforms GC-MS and 1H NMR spectroscopy. MS-based methods, especially after derivatization, have incomparable sensitivity and precision thus they are highly recommended for trace/ultratrace detection. GC-MS acidified water method, because of the easier sample preparation and short run time is most suitable for studies with large sample numbers. Alternatively, NMR-based methods, while exhibiting high repeatability and relatively low sensitivity, are suitable for cecal and fecal samples with both global and target analysis purpose. The application of three mutually independent methods, GC-MS, NMR, and bomb calorimetry in the germ free (GF) mice study showed consistent results, demonstrating the feasibility of the techniques used in metabolomics studies and the critical role that gut microbiome play in host energy balance and metabolic status. To investigate the metabolic functional roles of gut microbiome and how to target the microbiome for potential pharmaceutical application, a typical xenobiotic and antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) with anti-obesity and microbiome-modulation effect was investigated in conventionally-raised (CONV-R) and GF mouse models. The metabolic changes were evaluated with metabolomics tools combined with biochemistry and molecular biological techniques. The results demonstrated tempol exerts its metabolic regulatory role on host through changing gut microbiota metabolism. Tempol decreases gut energy availability by inhibiting bacterial SCFAs production in a dose-dependent manner, and the restricted gut SCFAs availability impacts overall host metabolism by promoting energy expenditure. This study provides insight into a possible mechanism for the anti-obesity effect of tempol mediated by gut-microbiota, which sheds light on the pharmaceutical and therapeutic potential of tempol for obesity treatment and prevention. The gut microbiome affects the bioavailability and toxicity of xenobiotics and can be modulated physiologically, compositionally and metabolically by xenobiotics. To further investigate the causal relationship between xenobiotic exposure and changes in gut microbiota metabolism, a novel approach combining in vitro bacterial incubation, single-cell flow cytometry, and global metabolomics tools including Orbitrap LC-MS and 1H NMR were developed to elucidate the direct impact of xenobiotics on the microbiome physiology and metabolism. This multi-platform approach identified the unique physiological and metabolic biomarkers for microbial membrane damage and metabolism disruption. The result also revealed that the disrupted metabolic activity of the gut microbiota is strongly correlated with the bacterial membrane damage by direct xenobiotic exposure. Importantly, in vitro and in vivo results were highly consistent thus indicating the in vitro methods can be a convenient, economic approach to better understand and/or predict in vivo physiological and metabolic responses to xenobiotics for future screening and risk assessment application. Together, the research presented in the dissertation demonstrates valuable metabolomics tools combined with other techniques are elegant approaches to study xenobiotics-microbiome-host interactions, therefore opening up avenues for better risk assessment and toxicity study during drug discovery to minimize undesirable side effects.