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We previously established that androgen glucuronides are primarily effluxed by MRP2 and MRP3 in liver and intestine. However, no data exist on the mechanism of hepatic uptake of these metabolites. To fill this knowledge gap, the first goal of this study was to explore the role of hepatic uptake transporters and characterize transport kinetics of glucuronides of testosterone (TG), dihydrotestosterone (DHTG), androsterone (AG), and etiocholanolone (EtioG) using cell-lines overexpressing organic anion transporting polypeptide (OATP1B1, OATP1B3, and OATP2B1). Using quantitative proteomics-guided approach, we then estimated the relative contribution (ft) of individual OATPs in hepatic uptake of the androgen glucuronides. The transport screening assays revealed that the glucuronides were primarily influxed by OATP1B1 and OATP1B3. The Km values for OATP1B1-mediated uptake were low for EtioG (6.2 µM), followed by AG, TG, and DHTG (46.2, 56.7, and 71.3 µM, respectively). Whereas, the Km value for OATP1B3-mediated uptake for EtioG, AG, DHTG, and TG were 19.8, 29.3, 69.6, and 110.4 µM, respectively. Both OATP1B1 and OATP1B3 exhibited highest transport rate toward AG as compared to other glucuronides. When adjusted for transporter abundance in human livers, EtioG and DHTG were transported equally by OATP1B1 and OATP1B3, whereas TG and AG were preferentially (>68%) transported by OATP1B3. Collectively, this is the first report that elucidate mechanisms of hepatic uptake of androgen glucuronides. Perturbation in these processes by genetic polymorphisms, disease conditions or drug-interactions can lead to changes in enterohepatic recycling of androgen. The apparent higher selectivity of OATP1B3 towards TG and AG can be leveraged for establishing these metabolites as clinical biomarkers of OATP1B3 activity. SIGNIFICANCE STATEMENT This is the first study to elucidate the mechanism of hepatic uptake of androgen glucuronides and estimate the relative contribution (ft) of individual OATPs using quantitative proteomics. Our results show that both OATP1B1 and OATP1B3 are responsible for the hepatic uptake of major circulating testosterone glucuronides. The apparent higher selectivity of OATP1B3 towards testosterone glucuronide and androsterone glucuronide can be leveraged for establishing these metabolites as clinical biomarkers of OATP1B3 activity.

Membrane transport proteins are involved in the absorption, disposition, efficacy, and/or toxicity of many drugs. Numerous mechanisms (e.g., nuclear receptors, epigenetic gene regulation, microRNAs, alternative splicing, post-translational modifications, and trafficking) regulate transport protein levels, localization, and function. Various factors associated with disease, medications, and dietary constituents, for example, may alter the regulation and activity of transport proteins in the intestine, liver, kidneys, brain, lungs, placenta, and other important sites, such as tumor tissue. This white paper reviews key mechanisms and regulatory factors that alter the function of clinically relevant transport proteins involved in drug disposition. Current considerations with in vitro and in vivo models that are used to investigate transporter regulation are discussed, including strengths, limitations, and the inherent challenges in predicting the impact of changes due to regulation of one transporter on compensatory pathways and overall drug disposition. In addition, translation and scaling of in vitro observations to in vivo outcomes are considered. The importance of incorporating altered transporter regulation in modeling and simulation approaches to predict the clinical impact on drug disposition is also discussed. Regulation of transporters is highly complex and, therefore, identification of knowledge gaps will aid in directing future research to expand our understanding of clinically relevant molecular mechanisms of transporter regulation. This information is critical to the development of tools and approaches to improve therapeutic outcomes by predicting more accurately the impact of regulation-mediated changes in transporter function on drug disposition and response.

To assess efficacy and toxicity of a drug in humans, it is important to measure the tissue concentration of a drug at the target site. For a drug that is transported into or out of the tissue, the tissue unbound steady-state concentration can be dramatically different from its corresponding unbound steady-state plasma concentration. Because routine measurement of drug tissue concentrations is not possible, using rosuvastatin as a model transporter substrate drug, we compared the ability of the proteomics-informed relative expression factor (REF) approach and sandwich-cultured human hepatocytes (SCH) to accurately predict rosuvastatin human hepatobiliary clearances and hepatic concentrations. REF-predicted rosuvastatin biliary clearance (CL

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.

During drug development, in vivo human biliary drug clearances (CL) are usually predicted using human sandwich-cultured hepatocytes (SCH). To do so, SCH are pre-incubated with Ca2+-containing or Ca2+-free buffer to maintain or disrupt canalicular tight junctions (CTJ), respectively. Drug uptake into SCH is then conducted in the presence of Ca2+ (up to 20 min). Under this standard protocol, two key assumptions are made: first, that the CTJ are not reformed during the uptake phase when Ca2+ is repleted, and second, disruption of CTJ by the Ca2+-free buffer does not affect the activity of any of the transporters present in the sinusoidal or canalicular membrane. Here we investigated the validity of these assumptions using rosuvastatin (RSV) and taurocholic acid (TCA) as our model drugs. In human SCH, the disrupted CTJ were “reformed” with just 10-min Ca2+ repletion as reflected in a significant increase in TCA cell accumulation. To avoid CTJ reformation and cell toxicity, the standard SCH protocol was modified by conducting the uptake in the absence of Ca2+ for 10 min. Surprisingly, using this protocol, RSV uptake into SCH, plated hepatocytes, and transporter-expressing cells confirmed that Ca2+ depletion substantially decreased NTCP and not OATP1B1 activity. Collectively, this study provides the first evidence of reformation of CTJ in human SCH with 20-min Ca2+ repletion, whereas Ca2+ depletion, during the uptake phase, leads to a significant reduction in NTCP uptake. Thus, the entire SCH protocol needs to be re-examined and optimized to correctly estimate hepatobiliary CL of drugs including those that are NTCP substrates.

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.

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.

Testosterone (T) is primarily eliminated via glucuronidation upon oral administration. As a result, testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG)...

Testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are the major metabolites of testosterone (T), which are excreted in urine and bile. Glucuronides can be deconjugated to active androgen in gut lumen after biliary excretion, which in turn can affect physiological levels of androgens. The goal of this study was to quantitatively characterize the mechanisms by which TG, AG, EtioG and DHTG are eliminated from liver, intestine, and kidney utilizing relative expression factor (REF) approach. Using vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP, we first identified that TG, AG, EtioG, and DHTG were primarily substrates of MRP2 and MRP3, although lower levels of transport were also observed with MDR1 and BCRP vesicles. The transport kinetic analyses revealed higher intrinsic clearances of TG by MRP2 and MRP3 as compared to that of DHTG, AG, and EtioG. MRP3 exhibited higher affinity for the transport of the studied glucuronides than MRP2. We next quantified the protein abundances of these efflux transporters in vesicles and compared the same with pooled total membrane fractions isolated from human tissues by quantitative LC-MS/MS proteomics. The fractional contribution of individual transporters (f(t)) was estimated by proteomics-based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and corrected for inside-out vesicles (determined by 5’-nucleotidase assay). The glucuronide metabolites of inactive androgens, AG and EtioG were preferentially transported by MRP3, whereas the glucuronides of active androgens, TG and DHTG were mainly transported by MRP2 in liver. Efflux by bile canalicular transport may indicate the potential role of enterohepatic recirculation in regulating the circulating active androgens after deconjugation in the gut. In intestine, MRP3 possibly contributes most to the efflux of these glucuronides. In kidney, all studied glucuronides seemed to be preferentially effluxed by MRP2 and MDR1 (for EtioG). These REF based analysis need to be confirmed with in vivo findings. Overall, characterization of the efflux mechanisms of T glucuronide metabolites is important for predicting the androgen disposition and interindividual variability, including drug-androgen interaction in humans. The mechanistic data can be extrapolated to other androgen relevant organs (e.g. prostate, testis and placenta) by integrating these data with quantitative tissue proteomics data.

This protocol described the procedures of analyzing RNA-Seq data for the expression of long non-coding RNAs (lncRNAs) as well as protein-coding genes (PCGs), as well as identifying the genomic location of lncRNAs and lncRNA-PCG pairs. As an example, we used an existing RNA-Seq dataset generated in our labotratory, which were deposited in Gene Expression Omnibus (GEO) database (accession number: GSE101650) and are available to the public.

This protocol described the procedures of analyzing RNA-Seq data for the expression of long non-coding RNAs (lncRNAs) as well as protein-coding genes (PCGs), as well as identifying the genomic location of lncRNAs and lncRNA-PCG pairs. As an example, we used an existing RNA-Seq dataset generated in our labotratory, which were deposited in Gene Expression Omnibus (GEO) database (accession number: GSE101650) and are available to the public.

The xenobiotic-sensing transcription factors (xeno-sensors) AhR, CAR, and PXR upregulate the expression of many drug-processing genes (DPGs) in liver. Previous studies have unveiled profound changes in the basal expression of DPGs during development; however, knowledge on the ontogeny of the inducibility of DPGs in response to pharmacological activation of xeno-sensors is still limited. The goal of this study was to investigate the age-specific regulation of DPGs by prototypical xeno-sensor ligands: 2,3,7,8-tetrachlorodibenzodioxin (TCDD) for AhR; 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) for CAR; and pregnane-16α-carbonitrile (PCN) for PXR during mouse liver development. The basal mRNAs of most DPGs were low during neonatal age, but gradually increased to adult levels, whereas some DPGs (Cyp1a2, Cyp2b10, Cyp3a11, Gstm2, Gstm3, Papss2, and Oatp1a4) exhibited an adolescent-predominant expression pattern. The inducibility of DPGs was age-specific: 1) during neonatal age, the highest fold increase in the mRNA expression was observed for Cyp1a2, Sult5a1, and Ugt1a9 by TCDD; Cyp3a11 and Mrp2 by TCPOBOP; as well as Gstm2 and Gstm3 by PCN; 2) during adolescent age, the highest fold increase in the mRNA expression was observed for Ugt1a6 and Mrp4 by TCDD, Cyp2b10, Ugt2b34, and Ugt2b35 by TCPOBOP, as well as Gsta1, Gsta4, Sult1e1, Ugt1a1, Mrp3, and Mrp4 by PCN; 3) in adults, the highest fold increase in the mRNA expression was observed for Aldh1a1, Aldh1a7, and Ugt2b36 by TCPOBOP, as well as Papss2 and Oatp1a4 by PCN. In conclusion, the inducibility of hepatic DPGs following the pharmacological activation of xeno-sensors is age specific.

Liver is a major organ for the biotransformation of xenobiotics. Various drugs, dietary factors, and environmental chemicals that require biotransformation for elimination are first taken up into the hepatocytes via uptake transporters, followed by a process that generally converts lipophilic substrates into hydrophilic metabolites, which are readily excreted into urine or bile via efflux transporters. As illustrated in Fig. 1A , drug-metabolizing enzymes can be categorized into two classes: (1) Phase-I drug-metabolizing enzymes catalyze oxidation, reduction, and hydrolysis reactions, which introduce or unmask a small functional group to the substrates and moderately increase the water solubility of the compounds; (2) Phase-II drug-metabolizing enzymes catalyze various conjugation reactions, including glucuronidation, sulfonation, glutathione (GSH) conjugation, acetylation, methylation, amino acid conjugation, and fatty acid conjugation. Except for acetylation, methylation, and fatty acid conjugation, Phase-II conjugation generally leads to a marked increase in the water solubility of the chemicals, which are then recognized as substrates by efflux transporters for their elimination. The present article focuses on Phase-I and -II drug-metabolizing enzymes in the liver.

Epigenetic modifications, defined as heritable changes that do not alter the nucleotide sequence, are key regulators for chromatin architecture and gene expression. Key epigenetic events, such as DNA methylation, histone modifications, and noncoding RNAs, play important roles for normal development and cell differentiation and serve as mechanism of genomic adaption to various environmental stimuli including those that adversely impact human health. Of particular concerns are the environmental exposures to toxicants during early development, which can lead to epigenetic reprogramming and serve as a basis of diseases developed later in life. Studies on the xenobiotic-induced epigenetic events will aid in further understanding of the molecular mechanisms of the pathogenesis of human diseases and more precise risk assessments in human exposure. This article reviews the basic principles of major epigenetic modifications and discusses the epigenetic effects of major classes of human health-related environmental factors, including heavy metals, endocrine disruptors, polycyclic aromatic hydrocarbons, air pollution, and cigarette smoking.

Safety concerns have emerged regarding the potential long-lasting effects due to developmental exposure to xenobiotics. The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are critical xenobiotic-sensing nuclear receptors that are highly expressed in liver. The goal of this study was to test our hypothesis that neonatal exposure to PXR- or CAR-activators not only acutely but also persistently regulates the expression of drug-processing genes (DPGs). A single dose of the PXR-ligand PCN (75 mg/kg), CAR-ligand TCPOBOP (3 mg/kg), or vehicle (corn oil) was administered intraperitoneally to 3-day-old neonatal wild-type mice. Livers were collected 24 h post-dose or from adult mice at 60 days of age, and global gene expression of these mice was determined using Affymetrix Mouse Transcriptome Assay 1.0. In neonatal liver, PCN up-regulated 464 and down-regulated 449 genes, whereas TCPOBOP up-regulated 308 and down-regulated 112 genes. In adult liver, there were 15 persistently up-regulated and 22 persistently down-regulated genes following neonatal exposure to PCN, as well as 130 persistently up-regulated and 18 persistently down-regulated genes following neonatal exposure to TCPOBOP. Neonatal exposure to both PCN and TCPOBOP persistently down-regulated multiple Cyp4a members, which are prototypical-target genes of the lipid-sensor PPARα, and this correlated with decreased PPARα-binding to the Cyp4a gene loci. RT-qPCR, western blotting, and enzyme activity assays in livers of wild-type, PXR-null, and CAR-null mice confirmed that the persistent down-regulation of Cyp4a was PXR and CAR dependent. In conclusion, neonatal exposure to PXR- and CAR-activators both acutely and persistently regulates critical genes involved in xenobiotic and lipid metabolism in liver.