Regulation of Hepatic Drug Metabolism by the Interaction of Host- and Gut Microbiome-Derived Bile Acids and Hepatic LncRNAs

Thesis (Ph.D.)--University of Washington, 2020Liver is a critical organ for nutrient homeostasis and xenobiotic biotransformation. The gut microbiome regulates important host metabolic pathways in liver including xenobiotic metabolism and intermediary metabolism, such as the conversion of primary bile acids (BAs) into secondary BAs and is an important factor of healthy development. The nuclear receptors pregnane X receptor (PXR/NR1I2) and constitutive androstane receptor (CAR/Nr1i3) are well-known regulators for xenobiotic metabolism and disposition in liver. However, little is known regarding the age specificity of secondary BA metabolism or other microbial metabolites and the potential effects of PXR and CAR on the composition and function of the gut microbiome. Additionally, altered expression of long noncoding RNAs (lncRNAs) by environmental chemicals modulates the expression of xenobiotic metabolizing genes and transporters; yet little is known regarding how lncRNAs are regulated by PXR, affecting xenobiotic metabolism. Therefore, the goal of my dissertation was to use a multi-omics approach, including transcriptomics (RNA-seq), targeted metabolomics (UPLC-MS/MS for BAs and GC-MS/MS for short-chain fatty acids), targeted proteomics (LC-MS), and 16S rRNA gene sequencing as well as bioinformatics, to strategically investigate the modulation of hepatic xenobiotic metabolizing genes through gut microbiome-mediated, age-dependent, and transcriptional mechanisms. To achieve this goal, multi-omics analysis of various tissues were performed in male and female conventional (CV) and germ-free (GF) mice at various ages: postnatal days 1, 5, 10, 15, 25, 60, and 120. With increasing age, the gut microbiome in mice become more diverse and had increased functional capacity to produce secondary BAs, including the family S24-7. Twenty-seven bacteria were positively associated with secondary unconjugated, deoxy, and keto BAs with nine of these BAs only produced in CV mice. Short-chain fatty acids (SCFAs) were found in livers of CV mice only with six SCFAs increasing with age. Global analysis of hepatic RNA expression showed an age-specific enrichment of KEGG pathways, including xenobiotic metabolism for adult CV and GF mice. Differentially expressed drug-metabolizing enzymes and transporters exhibited age and sex specific expression patters with more genes differentially expressed with increasing age. Overall, the age-specific gut microbiome impacts host xenobiotic metabolism likely through circulation of metabolites (e.g. BAs and SCFAs). Inversely, to test how the liver (specifically PXR and CAR) regulates gut microbiota and secondary BA synthesis, nine-week-old male CV and GF mice were orally gavaged with corn oil, PXR agonist pregnenolone-16α-carbonitrile (PCN; 75 mg/kg), or CAR agonist 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, 3,3′,5,5′-tetrachloro-1,4 bis(pyridyloxy)benzene (TCPOBOP; 3 mg/kg) once daily for four days. PCN and TCPOBOP decreased two taxa in the Bifidobacterium genus, which corresponded with decreased gene abundance of the BA-deconjugating enzyme bile salt hydrolase (bsh). In liver and small intestinal content of GF mice, there was a TCPOBOP-mediated increase in total, primary, and conjugated BAs corresponding with increased Cyp7a1 mRNA. Bacteria from the genera Bifidobacterium, Dorea, Peptociccaceae, Anaeroplasma, and Ruminococcus positively correlated with T-UDCA in LIC, but negatively correlated with T-CDCA in serum. Therefore, PXR and CAR activation down-regulates BA-metabolizing bacteria in the intestine and modulates BA homeostasis in a gut microbiota-dependent manner. To understand how lncRNAs may impact hepatic xenobiotic metabolism, RNA-Seq was performed from livers of adult male C57BL/6 mice treated with corn oil, the PXR agonist PCN, or the CAR agonist TCPOBOP. PXR activation differentially regulated 193 lncRNAs with 40% also regulated by CAR. Among differentially expressed lncRNAs, the lncRNA-PCG pairs displayed a high co-regulatory pattern by PXR and CAR activation. Combining the RNA-Seq data with a published PXR ChIP-Seq dataset (Cui et al., 2010b), 774 expressed lncRNAs with direct PXR-DNA binding sites, and 26.8% of differentially expressed lncRNAs had changes in PXR-DNA binding following PCN exposure. Therefore, some lncRNAs are regulated by PXR and CAR activation and that they may be important regulators of xenobiotic metabolism. Taken together, there is an age-dependent interaction in the gut-liver axis that exhibits a circulatory patter such that BA homeostasis is both governed by both the gut microbiome and the host liver, with modifications to either biocompartment impacting the other biocompartment. Targeting the gut-liver axis and lncRNAs may lead to the design of novel therapies alleviated toxicities through control of hepatic xenobiotic metabolism