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Tetrabromobisphenol A (TBBPA) is extensively utilized as a brominated flame retardant in numerous chemical products. As an environmental contaminant, the potential human toxicity of TBBPA has been attracting increasing attention. Nonetheless, the exact underlying mechanisms of toxicological effects caused by TBBPA remain uncertain. In this study, we investigated the potential mechanisms of TBBPA toxicity in vitro in the A549 cell line, one of the widely used type II pulmonary epithelial cell models in toxicology research. Cell viability was determined after treatment with varying concentrations of TBBPA. Liquid chromatography-mass spectrometry (LC-MS) metabolomics and metabolic flux approaches were utilized to evaluate metabolite and tricarboxylic acid (TCA) cycle oxidative flux changes. Our findings demonstrated that TBBPA significantly reduced the viability of cells and attenuated mitochondrial respiration in A549 cells. Additionally, LC-MS data showed significant reductions in TCA cycle metabolites including citrate, malate, fumarate, and alpha-ketoglutarate in 50 μM TBBPA-treated A549 cells. Metabolic flux analysis indicated reduced oxidative capacity in mitochondrial metabolism following TBBPA exposure. Moreover, diverse metabolic pathways, particularly alanine, aspartate, and glutamate metabolism and the TCA cycle, were found to be dysregulated. In total, 12 metabolites were significantly changed (p .05) in response to 50 μM TBBPA exposure. Our results provide potential biomarkers of TBBPA toxicity in A549 cells and help elucidate the molecular mechanisms of pulmonary toxicity induced by TBBPA exposure.

Cadmium (Cd) exposure is associated with increased Alzheimer's disease (AD) risks. The human Apolipoprotein E (

It is well-known that the pregnane X receptor (PXR)/Nr1i2 is a critical xenobiotic-sensing nuclear receptor enriched in liver and intestine and is responsible for drug-drug interactions, due to its versatile ligand binding domain (LBD) and target genes involved in xenobiotic biotransformation. PXR can be modulated by various xenobiotics including pharmaceuticals, nutraceuticals, dietary factors, and environmental chemicals. Microbial metabolites such as certain secondary bile acids (BAs) and the tryptophan metabolite indole-3-propionic acid (IPA) are endogenous PXR activators. Gut microbiome is increasingly recognized as an important regulator for host xenobiotic biotransformation and intermediary metabolism. PXR regulates and is regulated by the gut-liver axis. This review summarizes recent research advancements leveraging pharmaco- and toxico-metagenomic approaches that have redefined the previous understanding of PXR. Key topics covered in this review include: (1) genome-wide investigations on novel PXR-target genes, novel PXR-DNA interaction patterns, and novel PXR-targeted intestinal bacteria; (2) key PXR-modulating activators and suppressors of exogenous and endogenous sources; (3) novel bidirectional interactions between PXR and gut microbiome under physiologic, pathophysiological, pharmacological, and toxicological conditions; and (4) modifying factors of PXR-signaling including species and sex differences and time (age, critical windows of exposure, and circadian rhythm). The review also discusses critical knowledge gaps and important future research topics centering around PXR. SIGNIFICANCE STATEMENT: This review summarizes recent research advancements leveraging O'mics approaches that have redefined the previous understanding of the xenobiotic-sensing nuclear receptor pregnane X receptor (PXR). Key topics include: (1) genome-wide investigations on novel PXR-targeted host genes and intestinal bacteria as well as novel PXR-DNA interaction patterns; (2) key PXR modulators including microbial metabolites under physiological, pathophysiological, pharmacological, and toxicological conditions; and (3) modifying factors including species, sex, and time.

Perfluorinated carboxylic acids (PFCAs) are environmental pollutants for which human exposure has been documented. PFCAs at high doses were known regulate xenobiotic transporters partly through PPARα and CAR in rodents. Less is known regarding how various PFCAs at a lower concentration modulate transporters for endogenous substrates such as amino acids in human hepatocytes. Such studies are of particular importance because amino acids are involved in chemical detoxification and their transport system may serve as promising therapeutic targets for structurally similar xenobiotics. The focus of this study was to further elucidate how PFCAs modulate transporters involved in intermediary metabolism and xenobiotic biotransformation. We tested the hepatic transcriptomic response of HepaRG cells exposed to 45 mM PFOA, PFNA, or PFDA in triplicates for 24 h (vehicle: 0.1% DMSO), as well as the prototypical ligands for PPARα (WY-14643, 45 µM) and CAR (CITCO, 2 µM). PFCAs with increasing carbon chain lengths (C8-C10) regulated more liver genes, with amino acid metabolism and transport ranked among the top enriched pathways and PFDA ranked as the most potent PFCA tested. Genes encoding amino acid transporters, which are essential for protein synthesis, were novel inducible targets by all 3 PFCAs, suggesting a potentially protective mechanism to reduce further toxic insults. None of the transporter regulations appeared to be through PPARα or CAR but potential involvement of Nrf2 is noted for all 3 PFCAs. In conclusion, PFCAs with increasing carbon chain lengths up-regulate amino acid transporters and modulate xenobiotic transporters to limit further toxic exposures in HepaRG cells. Significance Statement Little is known regarding how various PFCAs modulate the transporters for endogenous substrates in human liver cells. Using HepaRG cells, this study is among the first to show that PFCAs with increasing carbon chain lengths up-regulate amino acid transporters, which are essential for protein synthesis, and modulate xenobiotic transporters to limit further toxic exposures at concentrations lower than what was used in literature.

Recent evidence suggests that complex diseases can result from early life exposure to environmental toxicants. Polybrominated diphenyl ethers (PBDEs), and polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) and remain a continuing risk to human health despite being banned from production. Developmental BPA exposure mediated-adult onset of liver cancer via epigenetic reprogramming mechanisms has been identified. Here, we investigated whether the gut microbiome and liver can be persistently reprogrammed following neonatal exposure to POPs, and the associations between microbial biomarkers and disease-prone changes in the hepatic transcriptome in adulthood, compared with BPA. C57BL/6 male and female mouse pups were orally administered vehicle, BPA, BDE-99 (a breast milk-enriched PBDE congener), or the Fox River PCB mixture (PCBs), once daily for three consecutive days (postnatal days [PND] 2–4). Tissues were collected at PND5 and PND60. Among the three chemicals investigated, early life exposure to BDE-99 produced the most prominent developmental reprogramming of the gut-liver axis, including hepatic inflammatory and cancer-prone signatures. In adulthood, neonatal BDE-99 exposure resulted in a persistent increase in Akkermansia muciniphila throughout the intestine, accompanied by increased hepatic levels of acetate and succinate, the known products of A. muciniphila. In males, this was positively associated with permissive epigenetic marks H3K4me1 and H3K27, which were enriched in loci near liver cancer-related genes that were dysregulated following neonatal exposure to BDE-99. Our findings provide novel insights that early life exposure to POPs can have a life-long impact on disease risk, which may partly be regulated by the gut microbiome.

Individual variations in xenobiotic metabolism affect the sensitivity to diseases. In this study, the impacts of sex, age, and race/ethnicity on drug-processing genes and nuclear factor erythroid 2–related factor 2 (NRF2) genes in human livers were examined via QuantiGene multiplex suspension array (226 samples) and quantitative polymerase chain reaction (qPCR) (247 samples) to profile the expression of nuclear receptors, cytochrome P450s, conjugation enzymes, transporters, bile acid metabolism, and NRF2-regulated genes. Sex differences were found in expression of about half of the genes, but in general the differences were not large. For example, females had higher transcript levels of catalase, glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase 1 (HO-1), Kelch-like ECH-associated protein 1 (KEAP1), superoxide dismutase 1, and thioredoxin reductase-1 compared with males via qPCR. There were no apparent differences due to age, except children had higher glutamate-cysteine ligase modifier subunit (GCLM) and elderly had higher multidrug resistance protein 3. African Americans had lower expression of farnesoid X receptor (FXR) but higher expression of HO-1, Caucasians had higher expression of organic anion transporter 2, and Hispanics had higher expression of FXR, SULT2A1, small heterodimer partner, and bile salt export pump. An examination of 34 diseased and control human liver samples showed that compared with disease-free livers, fibrotic livers had higher NAD(P)H-quinone oxidoreductase 1 (NQO1), GCLC, GCLM, and NRF2; hepatocellular carcinoma had higher transcript levels of NQO1 and KEAP1; and steatotic livers had lower GCLC, GCLM, and HO-1 expression. In summary, in drug-processing gene and NRF2 genes, sex differences were the major findings, and there were no apparent age differences, and race/ethnicity differences occurred for a few genes. These descriptive findings could add to our understanding of the sex-, age-, and race/ethnicity-dependent differences in drug-processing genes as well as NRF2 genes in normal and diseased human livers. SIGNIFICANCE STATEMENT: In human liver drug-processing and nuclear factor erythroid 2–related factor 2 genes, sex differences were the main finding. There were no apparent differences due to age, except children had higher glutamate-cysteine ligase modifier subunit, and elderly had higher multidrug resistance protein 3. African Americans had lower expression of farnesoid X receptor (FXR) but higher expression of heme oxygenase 1, Caucasians had higher expression of organic anion transporter 2, and Hispanics had higher expression of FXR, small heterodimer partner, SULT2A1, and bile salt export pump.

Maintaining energy homeostasis requires coordinating physiology and behavior both on an acute timescale to adapt to rapid fluctuations in caloric intake and on a chronic timescale to regulate body composition. Hypothalamic agouti-related peptide (AgRP)-expressing neurons are acutely activated by caloric need, and this acute activation promotes increased food intake and decreased energy expenditure. On a longer timescale, AgRP neurons exhibit chronic hyperactivity under conditions of obesity and high dietary fat consumption, likely due to leptin resistance; however, the behavioral and metabolic effects of chronic AgRP neuronal hyperactivity remain unexplored. Here, we use chemogenetics to manipulate Gq signaling in AgRP neurons in mice to explore the hypothesis that chronic activation of AgRP neurons promotes obesity. Inducing chronic Gq signaling in AgRP neurons initially increased food intake and caused dramatic weight gain, in agreement with published data; however, food intake returned to baseline levels within 1 wk, and body weight returned to baseline levels within 60 d. Additionally, we found that, when mice had elevated body weight due to chronic Gq signaling in AgRP neurons, energy expenditure was not altered but adiposity and lipid metabolism were both increased, even under caloric restriction. These findings reveal that the metabolic and behavioral effects of chronic Gq signaling in AgRP neurons are distinct from the previously reported effects of acute Gq signaling and also of leptin insensitivity.

Polychlorinated biphenyls (PCBs) are ubiquitously detected and have been linked to metabolic diseases. Gut microbiome is recognized as a critical regulator of disease susceptibility; however, little is known how PCBs and gut microbiome interact to modulate hepatic xenobiotic and intermediary metabolism. We hypothesized the gut microbiome regulates PCB-mediated changes in the metabolic fingerprints and hepatic transcriptome. Ninety-day-old female conventional and germ-free mice were orally exposed to the Fox River Mixture (synthetic PCB mixture, 6 or 30 mg/kg) or corn oil (vehicle control, 10 ml/kg), once daily for 3 consecutive days. RNA-seq was conducted in liver, and endogenous metabolites were measured in liver and serum by LC-MS. Prototypical target genes of aryl hydrocarbon receptor, pregnane X receptor, and constitutive androstane receptor were more readily upregulated by PCBs in conventional conditions, indicating PCBs, to the hepatic transcriptome, act partly through the gut microbiome. In a gut microbiome-dependent manner, xenobiotic, and steroid metabolism pathways were upregulated, whereas response to misfolded proteins-related pathways was downregulated by PCBs. At the high PCB dose, NADP, and arginine appear to interact with drug-metabolizing enzymes (ie, Cyp1–3 family), which are highly correlated with Ruminiclostridium and Roseburia, providing a novel explanation of gut-liver interaction from PCB-exposure. Utilizing the Library of Integrated Network-based Cellular Signatures L1000 database, therapeutics targeting anti-inflammatory and endoplasmic reticulum stress pathways are predicted to be remedies that can mitigate PCB toxicity. Our findings demonstrate that habitation of the gut microbiota drives PCB-mediated hepatic responses. Our study adds knowledge of physiological response differences from PCB exposure and considerations for further investigations for gut microbiome-dependent therapeutics.

Host cytochrome P450s (P450s) play important roles in the bioactivation and detoxification of numerous therapeutic drugs, environmental toxicants, dietary factors, as well as endogenous compounds. Gut microbiome is increasingly recognized as our “second genome” that contributes to the xenobiotic biotransformation of the host, and the first pass metabolism of many orally exposed chemicals is a joint effort between host drug metabolizing enzymes including P450s and gut microbiome. Gut microbiome contributes to the drug metabolism via two distinct mechanisms: direct mechanism refers to the metabolism of drugs by microbial enzymes, among which reduction and hydrolysis (or deconjugation) are among the most important reactions; whereas indirect mechanism refers to the influence of host receptors and signaling pathways by microbial metabolites. Many types of microbial metabolites, such as secondary bile acids (BAs), short chain fatty acids (SCFAs), and tryptophan metabolites, are known regulators of human diseases through modulating host xenobiotic-sensing receptors. To study the roles of gut microbiome in regulating host drug metabolism including P450s, several models including germ free mice, antibiotics or probiotics treatments, have been widely used. The present review summarized the current information regarding the interactions between microbiome and the host P450s in xenobiotic biotransformation organs such as liver, intestine, and kidney, highlighting the remote sensing mechanisms underlying gut microbiome mediated regulation of host xenobiotic biotransformation. In addition, the roles of bacterial, fungal, and other microbiome kingdom P450s, which is an understudied area of research in pharmacology and toxicology, are discussed.

Polybrominated diphenyl ethers (PBDEs) are persistent environmental toxicants associated with increased risk for metabolic syndrome. Intermediary metabolism is influenced by the intestinal microbiome. To test the hypothesis that PBDEs reduce host-beneficial intermediary metabolites in an intestinal microbiome–dependent manner, 9-week old male conventional (CV) and germ-free (GF) C57BL/6 mice were orally gavaged once daily with vehicle, BDE-47, or BDE-99 (100 μmol/kg) for 4 days. Intestinal microbiome (16S rDNA sequencing), liver transcriptome (RNA-Seq), and intermediary metabolites in serum, liver, as well as small and large intestinal contents (SIC and LIC; LC-MS) were examined. Changes in intermediary metabolite abundances in serum, liver, and SIC, were observed under basal conditions (CV vs. GF mice) and by PBDE exposure. PBDEs altered the largest number of metabolites in the LIC; most were regulated by PBDEs in GF conditions. Importantly, intestinal microbiome was necessary for PBDE-mediated decreases in branched-chain and aromatic amino acid metabolites, including 3-indolepropionic acid, a tryptophan metabolite recently shown to be protective against inflammation and diabetes. Gene-metabolite networks revealed a positive association between the hepatic glycan synthesis gene α-1,6-mannosyltransferase (Alg12) mRNA and mannose, which are important for protein glycosylation. Glycome changes have been observed in patients with metabolic syndrome. In LIC of CV mice, 23 bacterial taxa were regulated by PBDEs. Correlations of certain taxa with distinct serum metabolites further highlight a modulatory role of the microbiome in mediating PBDE effects. In summary, PBDEs impact intermediary metabolism in an intestinal microbiome–dependent manner, suggesting that dysbiosis may contribute to PBDE-mediated toxicities that include metabolic syndrome.

Mammals have developed a variety of antioxidant systems to protect them from the oxygen environment and toxic stimuli. Little is known about the mRNA abundance of antioxidant components during postnatal development of the liver. Therefore, the purpose of this study was to compare the mRNA abundance of antioxidant components during liver development. Livers from male C57BL/6J mice were collected at 12 ages from prenatal to adulthood. The transcriptome was determined by RNA-Seq with transcript abundance estimated by Cufflinks. RNA-Seq provided a complete, more accurate, and unbiased quantification of the transcriptome. Among 33 known antioxidant components examined, three ontogeny patterns of liver antioxidant components were observed: (1) Prenatal-enriched, in which the mRNAs decreased from fetal livers to adulthood, such as metallothionein and heme oxygenase-1; (2) adolescent-rich and relatively stable expression, such as peroxiredoxins; and (3) adult-rich, in which the mRNA increased with age, such as catalase and superoxide dismutase. alternative splicing of several antioxidant genes, such as Keap1, Glrx2, Gpx3, and Txnrd1, were also detected by RNA-Seq. In summary, RNA-Seq revealed the relative abundance of hepatic antioxidant enzymes, which are important in protecting against the deleterious effects of oxidative stress.

To identify host responses induced by commensal microbiota in intestine, transcriptomes of four sections of the intestine were compared between germ-free (GF) mice and conventional (CV) controls using RNA-Seq. Cuffdiff revealed that jejunum had the highest number of differentially expressed genes (over 2000) between CV and GF mice, followed by large intestine (LI), duodenum, and ileum. Gene set association analysis identified section-specific alterations in pathways associated with the absence of commensal microbiota. For example, in GF mice, cytochrome P450 (Cyp)-mediated xenobiotic metabolism was preferably down-regulated in duodenum and ileum, whereas intermediary metabolism pathways such as protein digestion and amino acid metabolism were preferably up-regulated in duodenum, jejunum, and LI. In GF mice, carboxypeptidase A1 (Cpa1), which is important for protein digestion, was the top most up-regulated gene within the entire transcriptome in duodenum (53-fold) and LI (142-fold). Conversely, fatty acid binding protein 6 (Fabp6/Ibabp), which is important for bile acid intestinal reabsorption, was the top most down-regulated gene in jejunum (358-fold), and the drug-metabolizing enzyme Cyp1a1 was the top most down-regulated gene in ileum (40-fold). Section-specific host transcriptomic response to the absence of intestinal microbiota was also observed for other important physiological pathways such as cell junction, the absorption of small molecules, bile acid homeostasis, and immune response. In conclusion, the present study has revealed section-specific host gene transcriptional alterations in GF mice, highlighting the importance of intestinal microbiota in facilitating the physiological and drug responses of the host intestine.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease due to the current epidemics of obesity and diabetes. The pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor known for trans-activating liver genes involved in drug metabolism and transport, and more recently implicated in energy metabolism. The gut microbiota can modulate the host xenobiotic biotransformation and contribute to the development of obesity. While the male sex confers a higher risk for NAFLD than women before menopause, the mechanism remains unknown. We hypothesized that the presence of PXR promotes obesity by modifying the gut-liver axis in a sex-specific manner. Male and female C57BL/6 (wild-type/WT) and PXR-knockout (PXR-KO) mice were fed control or high fat diet (HFD) for 16-weeks. Serum parameters, liver histopathology, transcriptomic profiling, 16S-rDNA sequencing, and bile acid (BA) metabolomics were performed. PXR enhanced HFD-induced weight gain, hepatic steatosis and inflammation especially in males, accompanied by PXR-dependent up-regulation in hepatic genes involved in microbial response, inflammation, oxidative stress, and cancer; PXR-dependent increase in intestinal Firmicutes/Bacteroides ratio (hallmark of obesity) and the pro-inflammatory Lactobacillus, as well as a decrease in the anti-obese Allobaculum and the anti-inflammatory Bifidobacterum, with a PXR-dependent reduction of beneficial BAs in liver. The resistance to NAFLD in females may be explained by PXR-dependent decrease in pro-inflammatory bacteria (Ruminococcus gnavus and Peptococcaceae). In conclusion, PXR exacerbates hepatic steatosis and inflammation accompanied by obesity- and inflammation-prone gut microbiome signature, suggesting that gut microbiome may contribute to PXR-mediated exacerbation of NAFLD.

Pharmacological activation of the xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding their physiological functions on the gut microbiome. In this study, we discovered bivalent hormetic functions of PXR/CAR modulating the richness of the gut microbiome using genetically engineered mice. The absence of PXR or CAR increased microbial richness, and absence of both receptors synergistically increased microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria Helicobacteraceae and Helicobacter. Deficiency in both PXR and CAR increased the relative abundance of Lactobacillus, which has bile salt hydrolase activity, corresponding to decreased primary taurine-conjugated bile acids (BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. The basal effect of PXR/CAR on the gut microbiome was distinct from pharmacological and toxicological activation of these receptors. Common PXR/CAR-targeted bacteria were identified, the majority of which were suppressed by these receptors. hPXR-TG mice had a distinct microbial profile as compared to wild-type mice. This study is the first to unveil the basal functions of PXR and CAR on the gut microbiome.

The gut microbiome is a pivotal player in toxicological responses. We investigated the effects of maternal exposure to 3 human health-relevant toxicants (BDE-47, tetrabromobisphenol [TBBPA], and bisphenol S [BPS]) on the composition and metabolite levels (bile acids [BAs] and short-chain fatty acids [SCFAs]) of the gut microbiome in adult pups. CD-1 mouse dams were orally exposed to vehicle (corn oil, 10 ml/kg), BDE-47 (0.2 mg/kg), TBBPA (0.2 mg/kg), or BPS (0.2 mg/kg) once daily from gestational day 8 to the end of lactation (postnatal day 21). 16S rRNA sequencing and targeted metabolomics were performed in feces of 20-week-old adult male pups (n = 14 − 23/group). Host gene expression and BA levels were quantified in liver. BPS had the most prominent effect on the beta-diversity of the fecal microbiome compared with TBPPA and BDE-47 (QIIME). Seventy-three taxa were persistently altered by at least 1 chemical, and 12 taxa were commonly regulated by all chemicals (most of which were from the Clostridia class and were decreased). The most distinct microbial biomarkers were S24-7 for BDE-47, Rikenellaceae for TBBPA, and Lactobacillus for BPS (LefSe). The community-wide contributions to the shift in microbial pathways were predicted using FishTaco. Consistent with FishTaco predictions, BDE-47 persistently increased fecal and hepatic BAs within the 12α hydroxylation pathway, corresponding to an up-regulation with the hepatic BA-synthetic enzyme Cyp7a1. Fecal BAs were also persistently up-regulated by TBBPA and BPS (liquid chromatography-mass spectrometry). TBBPA increased propionic acid and succinate, whereas BPS decreased acetic acid (gas chromatography-mass spectrometry). There was a general trend in the hepatic down-regulation of proinflammatory cytokines and the oxidative stress sensor target gene (Nqo1), and a decrease in G6Pdx (the deficiency of which leads to dyslipidemia). In conclusion, maternal exposure to these toxicants persistently modified the gut-liver axis, which may produce an immune-suppressive and dyslipidemia-prone signature later in life.

Mammalian polychlorinated biphenyl (PCB) metabolism has not been systematically explored with nontarget high-resolution mass spectrometry (Nt-HRMS). Here we investigated the importance of the gut microbiome in PCB biotransformation by Nt-HRMS analysis of feces from conventional (CV) and germ-free (GF) adult female mice exposed to a single oral dose of an environmental PCB mixture (6 mg/kg or 30 mg/kg in corn oil). Feces were collected for 24 h after PCB administration, PCB metabolites were extracted from pooled samples, and the extracts were analyzed by Nt-HRMS. Twelve classes of PCB metabolites were detected in the feces from CV mice, including PCB sulfates (PCB sulfates), hydroxylated PCB sulfates (OH-PCB sulfates), PCB sulfonates (PCB sulfonates), and hydroxylated methyl sulfone PCBs (OH-MeSO(2)-PCBs) reported previously. We also observed eight additional PCB metabolite classes that were tentatively identified as hydroxylated PCBs (OH-PCBs), dihydroxylated PCBs (diOH-PCBs), monomethoxylated dihydroxylated PCBs (MeO-OH-PCBs), methoxylated PCB sulfates (MeO-PCB sulfates), mono- to tetra-hydroxylated PCB quinones ((OH)(x)-Q, x=1-4), and hydroxylated polychlorinated benzofurans (OH-PCDF). Most metabolite classes were also detected in the feces from GF mice, except for MeO-OH-PCBs, OH-MeSO(2)-PCBs, and OH-PCDFs. Semi-quantitative analyses demonstrate that relative PCB metabolite levels increased with increasing dose and were higher in CV than GF mice, except for PCB sulfates and MeO-PCB sulfates, which were higher in GF mice. These findings demonstrate that the gut microbiome plays a direct or indirect role in the absorption, distribution, metabolism, or excretion of PCB metabolites, which in turn may affect toxic outcomes following PCB exposure.

Background: Pharmacological activation of the host xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding the physiological functions of PXR and CAR on the gut microbiome, which is an important regulator for the host immune surveillance and bile acid (BA) metabolism. We examined the gut microbiome composition and BA metabolites in high vs. low PXR/CAR-expressing mice, and in mice that are deficient in PXR, CAR, or both, at two developmental ages. We also utilized humanized PXR transgenic (hPXR-TG) mice to compare the species-specific effect of PXR on the gut microbiome. Results: We discovered bivalent hormetic functions of PXR and CAR in modulating the richness of the gut microbiome and inflammatory biomarkers: the high PXR/CAR expressers had higher microbial richness, pro-inflammatory bacteria (distinct taxa in Helicobacteraceae and Helicobacter), and fecal pro-inflammatory cytokines, suggesting higher immune surveillance to prevent the colonization of harmful bacteria. Interestingly, the absence of PXR or CAR also increased the microbial richness, and absence of both receptors synergistically increased the microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria (Helicobacteraceae and Helicobacter). Most notably, deficiency in both PXR and CAR markedly increased the relative abundance of Lactobacillus, which is capable of bile salt hydrolase (BSH) activity. This corresponded to a decrease in major primary taurine-conjugated bile acids (BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. hPXR-TG mice had a distinct microbial profile as compared to wild-type mice, including a higher representation of Prevotella. hPXR-TG mice also had higher 12-OH BAs but lower 6-OH BAs, suggesting PXR’s species-specific role in modulating host hepatic BA synthesis. Conclusions: This study is the first to show that the host PXR and CAR profoundly influence the composition of the gut microbiome and its BA metabolites, with a bivalent hormetic relationship between PXR/CAR levels and microbial richness, unveiling the involvement of PXR/CAR-microbiome interactions in host immune surveillance and BA metabolism.

Breast cancer (BC) is a major cause of human morbidity and mortality, especially among women. Despite the important role of metabolism in the molecular pathogenesis of cancer, robust metabolic markers to enable enhanced screening and disease monitoring of BC are still critically needed. In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolic profiling approach is presented for the identification of metabolic marker candidates that could enable highly sensitive and specific detection of all-stage as well as early-stage BC. In this targeted approach, 105 metabolites from >35 metabolic pathways of potential biological relevance were reliably detected in 201 plasma samples taken from two groups of subjects (102 BC patients and 99 healthy controls). The results of our general linear model and partial least squares-discriminant analysis (PLS-DA) informed the construction of a novel 6-metabolite panel of potential biomarkers. A receiver operating characteristic (ROC) curve generated based on an improved PLS-DA model showed relatively high sensitivity (0.80), specificity (0.75), and area under the receiver-operating characteristic curve (AUROC = 0.89). Similar classification performance of the model was observed for detection of early-stage BC (AUROC = 0.87, sensitivity: 0.86, specificity: 0.75). Bioinformatics analyses revealed significant disturbances in arginine/proline metabolism, tryptophan metabolism, and fatty acid biosynthesis. Our univariate and multivariate results indicate the effectiveness of this metabolomics approach for all-stage as well as early-stage BC diagnosis; our bioinformatics results indicate affected pathways related to tumor growth, metastasis, and immune escape mechanisms. Future studies should validate these results using more samples from different locations.

The human PXR (pregnane X receptor), a master regulator of drug metabolism, has essential roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off‐target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows exploiting previously unexplored parts of chemical space. Here, we report functionalized indole derivatives as first‐in‐class non‐cytotoxic PXR agonists as a proof of concept for microbial metabolite mimicry. The lead compound, FKK6 (Felix Kopp Kortagere 6), binds directly to PXR protein in solution, induces PXR‐specific target gene expression in cells, human organoids, and mice. FKK6 significantly represses pro‐inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to underexploited regions of chemical space., This study demonstrates that microbial metabolite mimicry can expand the chemical space in drug discovery. Chemical mimics of microbial indoles interacting with a host nuclear receptor provides a novel and non‐toxic therapeutic approach for treating inflammatory conditions of the intestine.

The microbiome and pregnancy are known to alter drug disposition, yet the interplay of the two physiologic factors on the expression and/or activity of drug metabolizing enzymes and transporters (DMETs) is unknown. This study investigated the effects of microbiome on host hepatic DMETs in mice during pregnancy by comparing four groups of conventional (CV) and germ-free (GF) female mice and pregnancy status, namely, CV nonpregnant, GF non-pregnant, CV pregnant, and GF pregnant mice. Transcriptomic and targeted proteomics of hepatic DMETs were profiled by using multiomics. Plasma bile acid and steroid hormone levels were quantified by liquid chromatography tandem mass spectrometry. CYP3A activities were measured by mouse liver microsome incubations. The trend of pregnancy-induced changes in the expression or activity of hepatic DMETs in CV and GF mice was similar; however, the magnitude of change was noticeably different. For certain DMETs, pregnancy status had paradoxical effects on mRNA and protein expression in both CV and GF mice. For instance, the mRNA levels of Cyp3a11, the murine homolog of human CYP3A4, were decreased by 1.7-fold and 3.3-fold by pregnancy in CV and GF mice, respectively; however, the protein levels of CYP3A11 were increased similarly ∼twofold by pregnancy in both CV and GF mice. Microsome incubations revealed a marked induction of CYP3A activity by pregnancy that was 10-fold greater in CV mice than that in GF mice. This is the first study to show that the microbiome can alter the expression and/or activity of hepatic DMETs in pregnancy. SIGNIFICANCE STATEMENT: We demonstrated for the first time that microbiome and pregnancy can interplay to alter the expression and/or activity of hepatic drug metabolizing enzymes and transporters. Though the trend of pregnancy-induced changes in the expression or activity of hepatic drug metabolizing enzymes and transporters in conventional and germ-free mice was similar, the magnitude of change was noticeably different.

Polybrominated diphenyl ethers (PBDEs) are extensively used as brominated flame retardants in various factory products. As environmental pollutants, the adverse effects of PBDEs on human health have been receiving considerable attention. However, the precise fundamental mechanisms of toxicity induced by PBDEs are still not fully understood. In this study, the mechanism of cytotoxicity induced by 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) was investigated by combining Seahorse XFp analysis and mass spectrometry-based metabolomics and flux approaches in PC12 cells, one of the most widely used neuron-like cell lines for investigating cytotoxic effects. The Seahorse results suggest that BDE-47 significantly attenuated mitochondrial respiration and enhanced glycolysis in PC12 cells. Additionally, metabolomics results revealed the reduction of TCA metabolites such as citrate, succinate, aconitate, malate, fumarate, and glutamate after BDE-47 exposure. Metabolic flux analysis showed that BDE-47 exposure reduced the oxidative metabolic capacity of mitochondria in PC12 cells. Furthermore, various altered metabolites were found in multiple metabolic pathways, especially in glycine-serine-threonine metabolism and glutathione metabolism. A total of 17 metabolic features were determined in order to distinguish potentially disturbed metabolite markers of BDE-47 exposure. Our findings provide possible biomarkers of cytotoxic effects induced by BDE-47 exposure, and elicit a deeper understanding of the intramolecular mechanisms that could be used in further studies to validate the potential neurotoxicity of PBDEs in vivo. Based on our results, therapeutic approaches targeting mitochondrial function and the glycolysis pathway may be a promising direction against PBDE exposure.

Altered expression of long noncoding RNAs (lncRNAs) by environmental chemicals modulates the expression of xenobiotic biotransformation-related genes and may serve as therapeutic targets and novel biomarkers of exposure. The pregnane X receptor (PXR/NR1I2) is a critical xenobiotic-sensing nuclear receptor that regulates the expression of many drug-processing genes, and it has similar target-gene profiles and DNA-binding motifs with another xenobiotic-sensing nuclear receptor, namely, constitutive andronstrane receptor (CAR/Nr1i3). To test our hypothesis that lncRNAs are regulated by PXR in concert with protein-coding genes (PCGs) and to compare the PXR-targeted lncRNAs with CAR-targeted lncRNAs, RNA-Seq was performed from livers of adult male C57BL/6 mice treated with corn oil, the PXR agonist PCN, or the CAR agonist 1, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Among 125,680 known lncRNAs, 3843 were expressed in liver, and 193 were differentially regulated by PXR (among which 40% were also regulated by CAR). Most PXR- or CAR-regulated lncRNAs were mapped to the introns and 3′-untranslated regions (UTRs) of PCGs, as well as intergenic regions. Combining the RNA-Seq data with a published PXR chromatin immunoprecipitation coupled with high-throughput sequencing; cytochrome P450 (P450; ChIP-Seq) data set, we identified 774 expressed lncRNAs with direct PXR-DNA binding sites, and 26.8% of differentially expressed lncRNAs had changes in PXR-DNA binding after PCN exposure. De novo motif analysis identified colocalization of PXR with liver receptor homolog (LRH-1), which regulates bile acid synthesis after PCN exposure. There was limited overlap of PXR binding with an epigenetic mark for transcriptional activation (histone-H3K4-di-methylation, H3K4me2) but no overlap with epigenetic marks for transcriptional silencing [H3 lysine 27 tri-methylation (H3K27me3) and DNA methylation]. Among differentially expressed lncRNAs, 264 were in proximity of PCGs, and the lncRNA-PCG pairs displayed a high coregulatory pattern by PXR and CAR activation. This study was among the first to demonstrate that lncRNAs are regulated by PXR and CAR activation and that they may be important regulators of PCGs involved in xenobiotic metabolism.

The gut microbiome regulates important host metabolic pathways including xenobiotic metabolism and intermediary metabolism, such as the conversion of primary bile acids (BAs) into secondary BAs. The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are well-known regulators for xenobiotic biotransformation in liver. However, little is known regarding the potential effects of PXR and CAR on the composition and function of the gut microbiome. To test our hypothesis that activation of PXR and CAR regulates gut microbiota and secondary BA synthesis, 9-week-old male conventional and germ-free mice were orally gavaged with corn oil, PXR agonist PCN (75 mg/kg), or CAR agonist TCPOBOP (3 mg/kg) once daily for 4 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. In liver and small intestinal content of germ-free mice, there was a TCPOBOP-mediated increase in total, primary, and conjugated BAs corresponding with increased Cyp7a1 mRNA. Bifidobacterium, Dorea, Peptociccaceae, Anaeroplasma, and Ruminococcus positively correlated with T-UDCA in LIC, but negatively correlated with T-CDCA in serum. In conclusion, PXR and CAR activation downregulates BA-metabolizing bacteria in the intestine and modulates BA homeostasis in a gut microbiota-dependent manner.

This article is a report on a symposium entitled “Physiological Regulation of Drug Metabolism and Transport” sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2017 meeting in Chicago, IL. The contributions of physiologic and pathophysiological regulation of drug-metabolizing enzymes and transporters to interindividual variability in drug metabolism are increasingly recognized but in many cases are not well understood. The presentations herein discuss the phenomenology, consequences, and mechanism of such regulation. CYP2D6 transgenic mice were used to provide insights into the mechanism of regulation of this enzyme in pregnancy, via hepatocyte nuclear factor 4α, small heterodimer partner, and retinoids. Regulation of intestinal and hepatic drug-processing enzymes by the intestinal microbiota via tryptophan and its metabolites was investigated. The potential impact of parasitic infections on human drug metabolism and clearance was assessed in mice infected with Schistosoma mansoni or Plasmodium chabaudi chabaudi AS, both of which produced widespread and profound effects on murine hepatic drug-metabolizing enzymes. Finally, the induction of Abcc drug efflux transporters by fasting was investigated. This was demonstrated to occur via a cAMP, protein kinase A/nuclear factor-E2-related factor 2/Sirtuin 1 pathway via antioxidant response elements on the Abcc genes.

The gut microbiome is a novel frontier in xenobiotic metabolism. Polybrominated diphenyl ethers (PBDEs), especially BDE-47 (2, 2′, 4, 4′-tetrabromodiphenyl ether) and BDE-99 (2, 2′, 4, 4′,5-pentabromodiphenyl ether), are among the most abundant and persistent environmental contaminants that produce a variety of toxicities. Little is known about how the gut microbiome affects the hepatic metabolism of PBDEs and the PBDE-mediated regulation of drug-processing genes (DPGs) in vivo. The goal of this study was to determine the role of gut microbiome in modulating the hepatic biotransformation of PBDEs. Nine-week-old male C57BL/6J conventional (CV) or germ-free (GF) mice were treated with vehicle, BDE-47 or BDE-99 (100 μmol/kg) for 4 days. Following BDE-47 treatment, GF mice had higher levels of 5-OH-BDE-47 but lower levels of four other metabolites in liver than CV mice; whereas following BDE-99 treatment GF mice had lower levels of four minor metabolites in liver than CV mice. RNA sequencing demonstrated that the hepatic expression of DPGs was regulated by both PBDEs and enterotypes. Under basal conditions, the lack of gut microbiome upregulated the Cyp2c subfamily but downregulated the Cyp3a subfamily. Following PBDE exposure, certain DPGs were differentially regulated by PBDEs in a gut microbiome–dependent manner. Interestingly, the lack of gut microbiome augmented PBDE-mediated upregulation of many DPGs, such as Cyp1a2 and Cyp3a11 in mouse liver, which was further confirmed by targeted metabolomics. The lack of gut microbiome also augmented the Cyp3a enzyme activity in liver. In conclusion, our study has unveiled a novel interaction between gut microbiome and the hepatic biotransformation of PBDEs.

Recent technological advancements including metagenomics sequencing and metabolomics have allowed the discovery of critical functions of gut microbiota in obesity, malnutrition, neurological disorders, asthma, and xenobiotic metabolism. Classification of the human gut microbiome into distinct “enterotypes” has been proposed to serve as a new paradigm for understanding the interplay between microbial variation and human disease phenotypes, as many organs are affected by gut microbiota modifications during the pathogenesis of diseases. Gut microbiota remotely interacts with liver and other metabolic organs of the host through various microbial metabolites that are absorbed into the systemic circulation. The present review summarizes recent literature regarding the importance of gut microbiota in modulating the physiological and pathological responses of various host organs and describes the functions of the known microbial metabolites that are involved in this remote sensing process, with a primary focus on the gut microbiota-liver axis. Under physiological conditions, gut microbiota modulates the hepatic transcriptome, proteome, and metabolome, most notably downregulating cytochrome P450 3a-mediated xenobiotic metabolism. Gut microbiome also modulates the rhythmicity in liver gene expression, likely through microbial metabolites, such as butyrate and propionate that serve as epigenetic modifiers. Additionally, the production of host hormones such as primary bile acids and glucagon-like peptide 1 is altered by gut microbiota to modify intermediary metabolism of the host. Dysregulation of gut microbiota is implicated in various liver diseases such as alcoholic liver disease, non-alcoholic steatohepatitis, liver cirrhosis, cholangitis, and liver cancer. Gut microbiota modifiers such as probiotics and prebiotics are increasingly recognized as novel therapeutic modalities for liver and other types of human diseases.

Polybrominated diphenyl ethers (PBDEs) were formally used as flame-retardants and are chemically stable, lipophlic persistent organic pollutants which are known to bioaccumulate in humans. Although its toxicities are well characterized, little is known about the changes in transcriptional regulation caused by PBDE exposure. Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of transcriptional and translational processes. It is hypothesized that lncRNAs can regulate nearby protein-coding genes (PCGs) and changes in the transcription of lncRNAs may act incisto perturb gene expression of its neighboring PCGs. The goals of this study were to 1) characterize PCGs and lncRNAs that are differentially regulated from exposure to PBDEs; 2) identify PCG-lncRNA pairs through genome annotation and predictive binding tools; and 3) determine enriched canonical pathways caused by differentially expressed lncRNA-PCGs pairs. HepaRG cells, which are human-derived hepatic cells that accurately represent gene expression profiles of human liver tissue, were exposed to BDE-47 and BDE-99 at a dose of 25 μM for 24 hours. Differentially expressed lncRNA-PCG pairs were identified through DESeq2 and HOMER; significant canonical pathways were determined through Ingenuity Pathway Analysis (IPA). LncTar was used to predict the binding of 19 lncRNA-PCG pairs with known roles in drug-processing pathways. Genome annotation revealed that the majority of the differentially expressed lncRNAs map to PCG introns. PBDEs regulated overlapping pathways with PXR and CAR such as protein ubiqutination pathway and PPARα-RXRα activation but also regulate distinctive pathways involved in intermediary metabolism. BDE-47 uniquely regulated signaling by Rho Family GTPases and PBDE-99 uniquely regulates JAK/Stat signaling, bile acid biosynthesis, sirtuin signaling pathway, and autophagy. In conclusion, lncRNAs play essential roles in modifying important pathways involved in intermediary metabolism such as carbohydrate and lipid metabolism.

Arsenic is a known human carcinogen. Early-life exposure to inorganic arsenic induces tumors in humans and in C3H mice. We hypothesized that arsenic exposure in utero may induce epigenetic changes at the level of DNA methylation and miRNA alterations that could lead to greater postnatal susceptibility to cancer. To test this hypothesis, pregnant C3H mice were given sodium arsenite at doses known to cause liver cancer (42.5 and 85 ppm in the drinking water) from gestation day 8–19, and the livers from male fetal mice were collected for analysis. The antibody against 5-methylcytosine was used to perform chromatin-immunoprecipitation coupled with sequencing (ChIP-Seq) to determine genome-wide methylation alterations. In utero arsenic exposure produced global DNA hypomethylation and an array of gene-specific DNA methylation changes, including hypomethylation of Cyclin D1 and hypermethylation of Tp53. Illumina Correlation Engine analysis revealed 260 methylation alterations that would affect 143 microRNAs. MicroRNA array further revealed 140 aberrantly expressed miRNAs out of the 718 miRNAs. The increased expression of miR-205, miR-203, miR-215, miR-34a, and decreased expression of miR-217 were confirmed by qPCR. Comparison of the methylation changes to those of microarray analyses indicates little if any correspondence between gene methylation and gene expression. The increased expression of Xist, Prrc2, Krit1, Nish, and decreased expression of Prss2, Spp1, Col1a2, and Lox were confirmed by qPCR. In summary, in utero arsenic exposure induced global alterations in DNA methylation and aberrant miRNA expression that might contribute to adult adverse outcomes including liver cancer.

1. Phase-II enzymes are important in metabolizing many xenobiotics including prescription drugs and chemical carcinogens. Whereas it is known that diet can alter the expression of phase-II conjugation enzymes, the previous studies are limited in using only two or three diets and examining only a few enzymes. 2. Adult male C57BL6 mice were fed one of nine diets for 3 weeks. Of the 87 genes encoding major hepatic phase-II enzymes, approximately one-half (43) were altered by at least one diet. Diet restriction altered the hepatic expression of the most genes encoding phase-II enzymes (27), followed by lab chow (15), atherogenic diet (13), high-fat diet (10), western diet (7), high-fructose diet (5), and essential fatty acid-deficient diet (3), whereas the low n-3 fatty acid diet had no effect on the hepatic expression of these phase-II enzymes. 3. This comprehensive study provides detailed information on which conjugation enzymes are changed by these diets, and these data can be used to further investigate the mechanism for these changes in messenger RNAs, and whether these changes result in alterations in enzyme activity and drug action.

Previous reports on tissue distribution of xenobiotic-processing genes (XPGs) have limitations, because many non–cytochrome P450 phase I enzymes have not been investigated, and one cannot compare the real mRNA abundance of multiple XPGs using conventional quantification methods. Therefore, this study aimed to quantify and compare the mRNA abundance of all major XPGs in the liver and intestine using RNA sequencing. The mRNA profiles of 304 XPGs, including phase I, phase II enzymes, phase II cosubstrate synthetic enzymes, xenobiotic transporters, as well as xenobiotic-related transcription factors, were systematically examined in the liver and various sections of the intestine in adult male C57BL/6J mice. By two-way hierarchical clustering, over 80% of the XPGs had tissue-divergent expression, which partitioned into liver-predominant, small intestine–predominant, and large intestine–predominant patterns. Among the genes, 54% were expressed highest in the liver, 21% in the duodenum, 4% in the jejunum, 6% in the ileum, and 15% in the large intestine. The highest-expressed XPG in the liver was Mgst1; in the duodenum, Cyp3a11; in the jejunum and ileum, Ces2e; and in the large intestine, Cyp2c55. Interestingly, XPGs in the same family usually exhibited highly different tissue distribution patterns, and many XPGs were almost exclusively expressed in one tissue and minimally expressed in others. In conclusion, the present study is among the first and the most comprehensive investigations of the real mRNA abundance and tissue-divergent expression of all major XPGs in mouse liver and intestine, which aids in understanding the tissue-specific biotransformation and toxicity of drugs and other xenobiotics.

Histone methylation is important in modulating the accessibility of transcription factors to target genes and the subsequent changes in transcription. The site-specific methylation and demethylation of histone residues are catalyzed by methyltransferases and demethylases, respectively. In general, transcriptional activation marks increase the permissibility of gene transcription, whereas transcriptional silencing marks promote heterochromatin formation. These processes are tightly regulated by methyltransferases and demethylases. Human diseases and toxicological responses from exposure to environmental chemicals are associated with aberrant histone methylation patterns and increased risks of adverse effects. In this chapter, we will introduce various histone methylation marks and their functions on gene transcription, writers and erasers that regulate specific sites of histone methylation patterns, cofactors and other regulators, and human diseases and environmental chemicals associated with dysregulation of histone methylation.