UDP-glucuronosyltransferase Expression Research Articles

Isoform-level expression of the constitutive androstane receptor (CAR or NR1I3) transcription factor better predicts the mRNA expression of the cytochrome P450s in human liver samples.

Many factors cause inter-person variability in the activity and expression of liver cytochrome P450 (CYP) drug-metabolizing enzymes, leading to variable drug exposure and treatment outcomes. Several liver-enriched transcription factors (TFs) are associated with CYP expression, with estrogen receptor alpha (ESR1) and constitutive androstane receptor (CAR or NR1I3) being the two top factors. ESR1 and NR1I3 undergo extensive alternative splicing that results in numerous splice isoforms, but how these splice isoforms associate with CYP expression is unknown. Here, we quantified 18 NR1I3 splice isoforms and the three most abundant ESR1 isoforms in 260 liver samples derived from African Americans (AA, n=125) and European Americans (EA, n=135). Our results showed variable splice isoform populations in the liver for both NR1I3 and ESR1. Multiple linear regression analyses revealed that, compared to gene-level NR1I3, isoform-level NR1I3 expression better predicted the mRNA expression of most CYPs and three UDP-glucuronosyltransferases (UGTs), while ESR1 isoforms improved predictive models for the UGTs and CYP2D6, but not for most CYPs. Also, different NR1I3 isoforms were associated with different CYPs, and the associations varied depending on sample ancestry. Surprisingly, non-canonical NR1I3 isoforms having retained introns (introns 2 or 6) were abundantly expressed and associated with the expression of most CYPs and UGTs, whereas the reference isoform (NR1I3-205) only associated with CYP2D6. Moreover, NR1I3 isoform diversity increased during the differentiation of induced pluripotent stem cells to hepatocytes, paralleling increasing CYP expression. These results suggest that isoform-level TF expression may help to explain variation in CYP or UGT expression between individuals. Significance Statement We quantified 18 NR1I3 splice isoforms and three ESR1 splice isoforms in 260 liver samples derived from AA and EA donors and found variable NR1I3 and ESR1 splice isoform expression in the liver. Multiple linear regression analysis showed that, compared to gene-level expression, isoform-level expression of NR1I3 and ESR1 better predicted the mRNA expression of some CYPs and UGTs, highlighting the importance of isoform-level analyses to enhance our understanding of gene transcriptional regulatory networks controlling the expression of drug-metabolizing enzymes.

Cytochrome P450 and UDP-Glucuronosyltransferase Expressions, Activities, and Induction Abilities in 3D-Cultured Human Renal Proximal Tubule Epithelial Cells.

The role of the kidney as an excretory organ for exogenous and endogenous compounds is well recognized, but there is a wealth of data demonstrating that the kidney has significant metabolizing capacity for a variety of exogenous and endogenous compounds that in some cases surpass the liver. The induction of drug-metabolizing enzymes by some chemicals can cause drug-drug interactions and intraindividual variability in drug clearance. In this study, we evaluated the expression and induction of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) isoforms in 3D-cultured primary human renal proximal tubule epithelial cells (RPTEC) to elucidate their utility as models of renal drug metabolism. CYP2B6, CYP2E1, CYP3A4, CYP3A5, and all detected UGTs (UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) mRNA levels in 3D-RPTEC were significantly higher than those in 2D-RPTEC and HK-2 cells and were close to the levels in the human kidney cortex. CYP1B1 and CYP2J2 mRNA levels in 3D-RPTEC were comparable to those in 2D-RPTEC, HK-2 cells, and the human kidney cortex. Midazolam 1'-hydroxylation, trifluoperazine N-glucuronidation, serotonin O-glucuronidation, propofol O-glucuronidation, and morphine 3-glucuronidation in the 3D-RPTEC were significantly higher than the 2D-RPTEC and comparable to those in the HepaRG cells, although bupropion, ebastine, and calcitriol hydroxylations were not different between the 2D- and 3D-RPTEC. Treatment with ligands of the aryl hydrocarbon receptor and farnesoid X receptor induced CYP1A1 and UGT2B4 expression, respectively, in 3D-RPTEC compared with 2D-RPTEC. We provided information on the expression, activity, and induction abilities of P450s and UGTs in 3D-RPTEC as an in vitro human renal metabolism model. SIGNIFICANCE STATEMENT: This study demonstrated that the expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in 3D-cultured primary human renal proximal tubule epithelial cells (3D-RPTEC) was higher than those in 2D-cultured primary human renal proximal tubule epithelial cells and HK-2 cells. The results were comparable to that in the human kidney cortex. 3D-RPTEC are useful for evaluating the induction of kidney P450s, UDP-glucuronosyltransferases, and human renal drug metabolism in cellulo.

A Comprehensive Evaluation of the Effects of RNA-Editing Proteins ADAR and ADARB1 on the Expression of the Drug-Metabolizing Enzymes in HepaRG Cells

Two RNA-editing proteins, the adenosine deaminase acting on RNA, ADAR, and ADARB1, broadly regulate gene expression in editing-dependent and editing-independent manners. Previous studies showed that the expression of the drug-metabolizing cytochrome P450s (P450s) and UDP glucuronosyltransferases (UGTs) changes upon knockdown (KD) of ADAR or ADARB1 in different hepatic cell lines. To systematically survey the effects of these two ADARs on the expression of P450s and UGTs, we used small interfering RNA in HepaRG cells and tested the association between the expression of the P450s and ADARs in a liver sample cohort (n = 246). KD of ADAR increased the expression of the CYP3As and CYP2C9 and reduced the expression of the others, whereas KD of ADARB1 reduced the expression of nearly all genes tested. ADAR KD also suppressed the induction of most P450s, whereas ADARB1 KD had mixed effects depending on the inducer/gene combination. P450 expression was positively associated with both ADARs in liver samples, consistent with the KD results. However, after adjusting for the expression of transcription factors (TFs) known to regulate P450 expression, the associations disappeared, indicating that the effects of ADAR or ADARB1 primarily occur through TFs. Moreover, we found that the expression of normally spliced CYP3A5 transcripts is increased in both KDs, indicating a direct effect of the ADARs on promoting the usage of the cryptic splice site generated by CYP3A5*3. Taken together, our results revealed the nonoverlapping regulatory effects of ADAR and ADARB1 and supported their broad roles in controlling the expression of drug-metabolizing enzymes in the liver. SIGNIFICANCE STATEMENT: Here, this study systematically surveyed the roles of ADAR and ADARB1 in both basal and induced expression of drug-metabolizing enzymes and assessed their coexpression in liver samples. This study's results support that ADAR and ADARB1 regulate the expression of the drug-metabolizing enzymes in the liver, suggesting that factors affecting ADAR expression also have the potential to impact drug metabolism.

Dysregulated hepatic UDP-glucuronosyltransferases and flavonoids glucuronidation in experimental colitis.

Glucuronidation catalyzed by UDP-glucuronosyltransferases (UGTs) is one of the most important phase II mechanisms, facilitating drug clearance via conjugation of glucuronic acid with polar groups of xenobiotics. Accumulating evidence suggests that IBDs impact drug disposition, but whether and how IBDs regulate UGTs and drug glucuronidation remains undefined. In this study, we aim to investigate the expression of UGTs and drug glucuronidation in experimental colitis. Given that glucuronidation occurs primarily in the liver, we analyzed the mRNA changes in hepatic UGTs with a DSS-induced mouse colitis model. Twelve UGTs were downregulated in the liver of colitis mice including UGT1A1 and UGT1A9 (two representative UGTs). Colitis in mice downregulated UGT1A1 and UGT1A9 in the liver but not in small intestine, colon, and kidney. We also established that the downregulation of UGTs was attributed to the disease itself rather than the DSS compound. Moreover, colitis-reduced UGT1A1 and UGT1A9 lead to dampened baicalein and puerarin glucuronidation. PXR was the only UGT regulator significantly downregulated in colitis mice, suggesting dysregulation of PXR is associated with the downregulation of UGT1A1 and UGT1A9, thereby potentially resulting in dysfunction of baicalein and puerarin glucuronidation. Collectively, we establish that UGTs and glucuronidation are dysregulated in colitis, and this effect may cause variation in drug responsiveness in IBDs.

Characterization of Hepatic UDP-Glucuronosyltransferase Enzyme Abundance-Activity Correlations and Population Variability Using a Proteomics Approach and Comparison with Cytochrome P450 Enzymes.

The expression of ten major drug-metabolizing UDP-glucuronosyltransferase (UGT) enzymes in a panel of 130 human hepatic microsomal samples was measured using a liquid chromatography-tandem mass spectrometry-based approach. Simultaneously, ten cytochromes P450 and P450 reductase were also measured, and activity-expression relationships were assessed for comparison. The resulting data sets demonstrated that, with the exception of UGT2B17, 10th to 90th percentiles of UGT expression spanned 3- to 8-fold ranges. These ranges were small relative to ranges of reported mean UGT enzyme expression across different laboratories. We tested correlation of UGT expression with enzymatic activities using selective probe substrates. A high degree of abundance-activity correlation (Spearman's rank correlation coefficient > 0.6) was observed for UGT1As (1A1, 3, 4, 6) and cytochromes P450. In contrast, protein abundance and activity did not correlate strongly for UGT1A9 and UGT2B enzymes (2B4, 7, 10, 15, and 17). Protein abundance was strongly correlated for UGTs 2B7, 2B10, and 2B15. We suggest a number of factors may contribute to these differences including incomplete selectivity of probe substrates, correlated expression of these UGT2B isoforms, and the impact of splice and polymorphic variants on the peptides used in proteomics analysis, and exemplify this in the case of UGT2B10. Extensive correlation analyses identified important criteria for validating the fidelity of proteomics and enzymatic activity approaches for assessing UGT variability, population differences, and ontogenetic changes. SIGNIFICANCE STATEMENT: Protein expression data allow detailed assessment of interindividual variability and enzyme ontogeny. This study has observed that expression and enzyme activity are well correlated for hepatic UGT1A enzymes and cytochromes P450. However, for the UGT2B family, caution is advised when assuming correlation of expression and activity as is often done in physiologically based pharmacokinetic modeling. This can be due to incomplete probe substrate specificities, but may also be related to presence of inactive UGT protein materials and the effect of splicing variations.

Glucuronidation of Efavirenz and its Cytochrome P450‐dependent Metabolites in Brain Microsomes and Neurons

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) anti-HIV medication that is glucuronidated by UDP-glucuronosyl transferases (UGTs). Brain penetrant anti-HIV drugs, such as EFV, are essential to combatting HIV in the brain. Persons with HIV often suffer from neurocognitive deficits, known as HIV-associated neurocognitive disorder (HAND). In addition to HAND, EFV itself has been shown to lead to severe neurological adverse events that can result the discontinuation of EFV-based therapies. Despite the importance of this drug and the observed adverse side effects, its metabolism in the brain is virtually unknown. Furthermore, UGT expression and activity in the brain remains poorly understood. We tested whether EFV and/or its cytochrome P450-dependent metabolites –8-hydroxyefavirenz (8-OHEFV) and 8,14-dihydroxyefavirenz (8,14-diOHEFV) undergo glucuronidation in the brain. To investigate this, brain microsomes and primary mouse neurons in culture were incubated with EFV, 8-hydroxyefavirenz (8-OHEFV), or 8,14-dihydroxy efavirenz (8,14-diOHEFV). Glucuronidated metabolites were detected using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC MS/MS). Incubating pooled brain microsomes from mice, cynomolgus macaques, and humans with 10µM EFV or one of its cytochrome P450-dependent metabolites for 30 min, we observed the formation of glucuronidated metabolites of 8-OHEFV and 8,14-diOHEFV for all three species. Interestingly, the cynomolgus macaque microsomes were the only microsomes to yield the EFV N-glucuronide metabolite, indicating interspecies differences in UGT activity toward EFV. In primary C57BL6/J neurons treated with a drug concentration of 50µM for 3-4 hours, we observed glucuronidation of 8-OHEFV, but not EFV. This was commensurate with our findings using mouse brain microsomes. These data lend insight into EFV disposition in brain across humans, mice, and cynomolgus macaques, and can be leveraged to inform understanding of EFV-mediated neurotoxicity.

The N6-methyladenosine modification posttranscriptionally regulates hepatic UGT2B7 expression

UDP-glucuronosyltransferases (UGTs) are enzymes catalyzing the glucuronidation of various endogenous and exogenous compounds. In this study, we examined the possibility that N6-methyladenosine (m6A) modification affects hepatic UGT expression. Treatment of HepaRG cells with 3-deazaadenosine, an inhibitor of RNA methylation, significantly increased UGT1A1, UGT1A3, UGT1A4, UGT1A9, UGT2B7, UGT2B10, and UGT2B15 mRNA levels (1.3- to 2.6-fold). Among them, we focused on UGT2B7 because it most highly contributes to glucuronidation of clinically used drugs. Methylated RNA immunoprecipitation assays revealed that UGT2B7 mRNA in HepaRG cells and human livers is subjected to m6A modification mainly at the 5′ untranslated region (UTR) and secondarily at the 3′UTR. UGT2B7 mRNA and protein levels in Huh-7 cells were significantly increased by double knockdown of methyltransferase-like 3 (METTL3) and METTL14, whereas those were decreased by knockdown of fat mass and obesity-associated protein (FTO) or alkB homolog 5, RNA demethylase (ALKBH5), suggesting that m6A modification downregulates UGT2B7 expression. By experiments using actinomycin D, an inhibitor of transcription, it was demonstrated that ALKBH5-mediated demethylation would attenuate UGT2B7 mRNA degradation, whereas METTL3/METTL14 or FTO-mediated m6A modification would alter the transactivity of UGT2B7. Luciferase assays revealed that the promoter region at −118 to −106 has a key role in the decrease in transactivity of UGT2B7 by FTO knockdown. We found that hepatocyte nuclear factor 4α (HNF4α) expression was significantly decreased by knockdown of FTO, indicating that this would be the underlying mechanism of the decreased transactivity of UGT2B7 by knockdown of FTO. Interestingly, treatment with entacapone, which is used for the treatment of Parkinson’s disease and is an inhibitor of FTO, decreased HNF4α and UGT2B7 expression. In conclusion, this study clarified that RNA methylation posttranscriptionally controls hepatic UGT2B7 expression.

The function of uterine UDP-glucuronosyltransferase 1A8 (UGT1A8) and UDP-glucuronosyltransferase 2B7 (UGT2B7) is involved in endometrial cancer based on estrogen metabolism regulation.

The progression of endometrial cancer (EC) is closely related to estrogen levels. UDP-glucuronosyltransferases (UGTs) are an essential class of phase II metabolizing enzymes that play a pivotal role in detoxifying steroid hormone. In this study, we aimed to uncover the role of UGTs in estrogen metabolism and the pathogenesis of EC. A total of 100 unrelated EC patients (mean age 52.15 ± 10.04y) and 100 healthy subjects (mean age 50.26 ± 8.80y) were recruited for analysis of the UGT gene polymorphism and estrogen level. In six cases of EC, EC-adjacent tissues and cancer tissues were collected for detection of UGT expression. Our results showed that the estrogen homeostasis profile was disturbed in EC patients, with carcinogenic catechol estrogens (4-OHE1, 2-OHE1, 2-OHE2) significantly accumulated in the serum of these patients. Also, levels of estrogen-glucuronides were decreased significantly, and the expression of UGT1A8 and UGT2B7 in uterine tissues was downregulated in EC patients. Consistent with this, we observed that the distribution of genotypes and allele frequencies in UGT1A8 rs1042597 and UGT2B7 rs7439366 was significantly different between EC patients and healthy volunteers. These results indicated that UGT1A8 and UGT2B7 may contribute to the estrogen signaling pathway and the pathogenesis of EC.

Dysregulation of UDP-glucuronosyltransferases in CCl4 induced liver injury rats

UDP-glucuronosyltransferases (UGTs) are a family of phase II drug metabolizing enzymes that catalyze glucuronidation of numerous endogenous and exogenous substrates. Carbon tetrachloride (CCl4) is widely used to develop liver injuries mimicking human liver diseases. However, effects of CCl4 on the expression and activities of UGTs and the mechanism have not been fully elucidated. The present study aims to elucidate the dysregulation patterns of major UGTs induced by CCl4. Biochemical and histopathological results showed that CCl4 exerted hepatotoxicity in rats. The mRNA levels of UGTs were all significantly reduced in acute liver injury rats. However, mRNA levels of UGT1A1, 1A6, 2B1 and 2B2 were up-regulated while the UGT2B3, 2B6 and 2B12 levels were reduced in chronic CCl4-induced liver fibrosis rats. The protein expression of UGT1A1, 1A6 and 2B were decreased in acute liver injury rats. UGT1A1 and 1A6 proteins were increased, whereas UGT2B protein was reduced in liver fibrosis rats. In addition, CCl4 inhibited the enzyme activities of UGTs in rats. Moreover, the dysregulation of UGTs was accompanied by the decreased mRNA expression of Nrf2, CAR, FXR, PXR, PPAR-α and their corresponding target genes, except for Nrf2, HO-1, AhR and CYP1A1 in liver fibrosis rats. These findings suggest that dysregulation of UGTs under CCl4 exposure is isoform-specific, which could have a complex impact on drug efficacy and endogenous metabolism. Different exposure durations of CCl4 (single vs multiple doses) could have differential effects on rat hepatic UGTs expression.

SOX1 promotes differentiation of nasopharyngeal carcinoma cells by activating retinoid metabolic pathway

Undifferentiation is a key feature of nasopharyngeal carcinoma (NPC), which presents as a unique opportunity for intervention by differentiation therapy. In this study, we found that SOX1 inhibited proliferation, promoted differentiation, and induced senescence of NPC cells, which depended on its transcriptional function. RNA-Seq-profiling analysis showed that multiple undifferentiated markers of keratin family, including KRT5, KRT13, and KRT19, were reduced in SOX1 overexpressed NPC cells. Interestingly, gene ontology (GO) analysis revealed genes in SOX1 overexpressed cells were enriched in extracellular functions. The data of LC/MS untargeted metabolomics showed that the content of retinoids in SOX1 overexpressed cells and culture medium was both higher than that in the control group. Subsequently, we screened mRNA level of genes in retinoic acid (RA) signaling or metabolic pathway and found that the expression of UDP-glucuronosyltransferases was significantly decreased. Furtherly, UGT2B7 could rescue the differentiation induced by SOX1 overexpression. Inhibition of UGTs by demethylzeylasteral (T-96) could mimic SOX1 to promote the differentiation of NPC cells. Thus, we described a mechanism by which SOX1 regulated the differentiation of NPC cells by activating retinoid metabolic pathway, providing a potential target for differentiation therapy of NPC.

Genistein protects against acetaminophen-induced liver toxicity through augmentation of SIRT1 with induction of Nrf2 signalling

Previous studies suggest that genistein protects liver from acetaminophen (APAP)-induced injury, however, the detailed mechanism of the process is still incompletely. Therefore, present study was to investigate the potential mechanism of the genistein mediated protection against APAP-induced hepatotoxicity. As shown, supplementation with 150 mg/kg genistein greatly alleviated the increase in serum alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, hepatic malondialdehyde (MDA) contents, and reversed the decrease in hepatic GSH levels in response to overdose APAP. At the same time, hepatic SIRT1 protein and activity were markedly upregulated in mouse receiving genistein. However, the amelioration was almost abolished by the knockdown of hepatic SIRT1 expression using lentivirus carrying specific shRNA targeting SIRT1. These results were further validated by histopathology examination. Moreover, depletion of hepatic SIRT1 prevented the accumulation of Nrf2 in nucleus and the upregulation of the antioxidant gene expression in the presence of genistein and/or APAP. Concomitantly, the induced mRNA expression of UDP-glucuronosyltransferases (UGTs) by genistein was largely dependent on the SIRT1 expression and activity. Together, our results support the notion that the strong elevation of SIRT1 expression and activity may represent a potential mechanism of protection against APAP-induced liver injury by genistein.

Arsenic modifies serotonin metabolism through glucuronidation in pancreatic β-cells

In arsenic-endemic regions of the world, arsenic exposure correlates with diabetes mellitus. Multiple animal models of inorganic arsenic (iAs, as As3+) exposure have revealed that iAs-induced glucose intolerance manifests as a result of pancreatic β-cell dysfunction. To define the mechanisms responsible for this β-cell defect, the MIN6-K8 mouse β-cell line was exposed to environmentally relevant doses of iAs. Exposure to 0.1-1 µM iAs for 3 days significantly decreased glucose-induced insulin secretion (GIIS). Serotonin and its precursor, 5-hydroxytryptophan (5-HTP), were both decreased. Supplementation with 5-HTP, which loads the system with bioavailable 5-HTP and serotonin, rescued GIIS, suggesting that recovery of this pathway was sufficient to restore function. Exposure to iAs was accompanied by an increase in mRNA expression of UDP-glucuronosyltransferase 1 family, polypeptide a6a (Ugt1a6a), a phase-II detoxification enzyme that facilitates the disposal of cyclic amines, including serotonin, via glucuronidation. Elevated Ugt1a6a and UGT1A6 expression levels were observed in mouse and human islets, respectively, following 3 days of iAs exposure. Consistent with this finding, the enzymatic rate of serotonin glucuronidation was increased in iAs-exposed cells. Knockdown by siRNA of Ugt1a6a during iAs exposure restored GIIS in MIN6-K8 cells. This effect was prevented by blockade of serotonin biosynthesis, suggesting that the observed iAs-induced increase in Ugt1a6a affects GIIS by targeting serotonin or serotonin-related metabolites. Although it is not yet clear exactly which element(s) of the serotonin pathway is/are most responsible for iAs-induced GIIS dysfunction, this study provides evidence that UGT1A6A, acting on the serotonin pathway, regulates GIIS under both normal and pathological conditions.

Regulation of Mammalian UDP-Glucuronosyltransferases.

UDP-glucuronosyltransferases (UGTs) are a class of important phase II drug metabolizing enzymes (DMEs), playing essential roles in the homeostasis of endobiotics as well as the dispositional behavior of exogenous compounds. The expression and enzyme activity of UGTs are regulated by multiple dimensions of mechanisms and can be influenced by diverse factors. Thus, the intensive research of its regulatory network is pivotal for better understanding about the physiological, pathological, and therapeutic significance of UGTs. Despite the lag to the research in cytochrome P450s, extensive efforts have been made to advance the understanding of the regulatory network of UGTs in recent years. This review presents a comprehensive summary and intensive discussion about the recent advancement on the regulatory network of UGTs. UGTs can be regulated at the epigenetic level via DNA methylation and histone modification. Various nuclear receptors can influence the mRNA levels of UGTs in a ligand dependent manner. Some general transcriptional factors such as AP-1, NF-κB, and p53, and some tissue specific transcriptional factors including HFN1α and HNF4α can also regulate UGTs at the transcriptional level. Multiple miRNAs have been found to be involved in the regulation of UGTs at post-transcriptional level. UGT proteins can be directly regulated via various post-translational modifications, protein-protein interactions, and protein-chemicals interactions, leading to the alternation of enzyme activities. In addition to the well-defined genetic polymorphism that induce individual variation of UGTs, this review reinforces the importance of other mechanisms that are critical for the regulation of UGTs.

The Ontogeny of UDP-glucuronosyltransferase Enzymes, Recommendations for Future Profiling Studies and Application Through Physiologically Based Pharmacokinetic Modelling.

Limited understanding of drug pharmacokinetics in children is one of the major challenges in paediatric drug development. This is most critical in neonates and infants owing to rapid changes in physiological functions, especially in the activity of drug-metabolising enzymes. Paediatric physiologically based pharmacokinetic models that integrate ontogeny functions for cytochrome P450 enzymes have aided our understanding of drug exposure in children, including those under the age of 2years. Paediatric physiologically based pharmacokinetic models have consequently been recognised by the European Medicines Agency and the US Food and Drug Administration as innovative tools in paediatric drug development and regulatory decision making. However, little is currently known about age-related changes in UDP-glucuronosyltransferase-mediated metabolism, which represents the most important conjugation reaction for xenobiotics. Therefore, the objective of the review was to conduct a thorough literature survey to summarise our current understanding of age-related changes in UDP-glucuronosyltransferases as well as associated clinical and experimental sources of variance. Our findings indicate that there are distinct differences in UDP-glucuronosyltransferase expression and activity between isoforms for different age groups. In addition, there is substantial variability between individuals and laboratories reported for human liver microsomes, which results in part from a lack of standardised experimental conditions. Therefore, we provide a number of best practice recommendations for experimental conditions, which ultimately may help improve the quality of data used for quantitative clinical pharmacology approaches, and thus for safe and effective pharmacotherapy in children.

Cooperative Regulation of Intestinal UDP-Glucuronosyltransferases 1A8, -1A9, and 1A10 by CDX2 and HNF4α Is Mediated by a Novel Composite Regulatory Element

The gastrointestinal tract expresses several UDP-glucuronosyltransferases (UGTs) that act as a first line of defense against dietary toxins and contribute to the metabolism of orally administered drugs. The expression of UGT1A8, UGT1A9, and UGT1A10 in gastrointestinal tissues is known to be at least partly directed by the caudal homeodomain transcription factor, CDX2. We sought to further define the factors involved in regulation of the UGT1A8-1A10 genes and identified a novel composite element located within the proximal promoters of these three genes that binds to both CDX2 and the hepatocyte nuclear factor (HNF) 4α, and mediates synergistic activation by these factors. We also show that HNF4α and CDX2 are required for the expression of these UGT genes in colon cancer cell lines, and show robust correlation of UGT expression with CDX2 and HNF4α levels in normal human colon. Finally, we show that these factors are involved in the differential expression pattern of UGT1A8 and UGT1A10, which are intestinal specific, and that of UGT1A9, which is expressed in both intestine and liver. These studies lead to a model for the developmental patterning of UGT1A8, UGT1A9, and UGT1A10 in hepatic and/or extrahepatic tissues involving discrete regulatory modules that may function (independently and cooperatively) in a context-dependent manner.

Involvement of UDP-Glucuronosyltransferases and Sulfotransferases in the Excretion and Tissue Distribution of Resveratrol in Mice.

Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. It is unknown whether the expression of metabolizing enzymes correlates with resveratrol levels in organs and tissues. Therefore, we investigated the metabolism and tissue distribution of resveratrol in mice and assessed its association with the expression of UDP-glucuronosyltransferase (Ugt) and sulfotransferase (Sult) genes. Plasma, urine, feces, and various organs were analyzed using high-performance liquid chromatography at up to 8 h after intragastric resveratrol administration. The metabolism of resveratrol was pronounced, leading to the formation of resveratrol glucuronides and sulfates. Concentrations of resveratrol and its metabolites were high in the gastrointestinal organs, urine, and feces, but low in the liver and kidneys. In lung, heart, thymus, and brain tissues, parent resveratrol levels exceeded the sulfate and glucuronide concentrations. The formation of resveratrol conjugates correlated with the expression of certain Ugt and Sult genes. Reverse transcription quantitative PCR (RT-qPCR) analysis revealed high mRNA expression of Ugt1a1 and Ugt1a6a in the liver, duodenum, jejunum, ileum, and colon, leading to high concentrations of resveratrol-3-O-glucuronide in these organs. Strong correlations of resveratrol-3-O-sulfate and resveratrol-3-O-4′-O-disulfate formation with Sult1a1 mRNA expression were also observed, particularly in the liver and colon. In summary, our data revealed organ-specific expression of Sults and Ugts in mice that strongly affects resveratrol concentrations; this may also be predictive in humans following oral uptake of dietary resveratrol.

Disturbance of Mammary UDP-Glucuronosyltransferase Represses Estrogen Metabolism and Exacerbates Experimental Breast Cancer

The progression of breast cancer is closely related to the levels of estrogens within the body. UDP-glucuronosyltransferase (UGT) is an important class of phase II metabolizing enzymes, playing a pivotal role in detoxifying steroid hormone. In the present study, we aim at uncovering the potential dysregulation pattern of UGT and its role in estrogen metabolism and in the pathogenesis of breast cancer. Female Sprague-Dawley rats were treated with 100 mg/kg dimethylbenz(a)anthracene (DMBA) to induce breast cancer. Our results showed that the expression and activity of UGT in mammary tissues were downregulated significantly in DMBA rats. Consistent with this, levels of estradiol, 4-hydroxylated estradiol, and 2-hydroxylated estradiol were increased in both mammary tissues and serum, supporting a notable accumulation of toxic estrogen species in the target tissue of breast cancer. In addition, we also observed the decreased cell migration, cell proliferation, and DNA damage in UGT-transfected MCF-7 cells, suggesting a protective role of UGT against estrogen-induced mammary carcinogenesis. Taken together, these results indicated that accumulation of estrogens induced by UGT deficiency is a critical factor to induce the development of breast cancer. UGT contributes to estrogen elimination, and its glucuronidation capacity influences the estrogen signaling pathway and the pathogenesis of breast cancer.

Constitutive androstane receptor activation promotes bilirubin clearance in a murine model of alcoholic liver disease.

Increased plasma levels of bilirubin have been reported in rat models and patients with alcoholic liver disease (ALD). The constitutive androstane receptor (CAR) is a known xenobiotic receptor, which induces the detoxification and transport of bilirubin. In the present study, the bilirubin transport regulatory mechanisms, and the role of CAR activation in hepatic and extrahepatic bilirubin clearance were investigated in a murine model of ALD. The mice were fed a Lieber-DeCarli ethanol diet or an isocaloric control diet for 4 weeks, followed by the administration of CAR agonists, 1,4-bis-[2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP) and phenobarbital (PB), and their vehicles to examine the effect of the pharmacological activation of CAR on serum levels of bilirubin and on the bilirubin clearance pathway in ALD by serological survey, western blotting and reverse transcription-quantitative polymerase chain reaction. The results showed that chronic ethanol ingestion impaired the nuclear translocation of CAR, which was accompanied by elevated serum levels of bilirubin, suppression of the expression of hepatic and renal organic anion transporting polypeptide (OATP) 1A1 and hepatic multidrug resistance-associated protein 2 (MRP2), and induction of the expression of UDP-glucuronosyltransferase (UGT) 1A1. The activation of CAR by TCPOBOP and PB resulted in downregulation of the serum levels of bilirubin followed by selective upregulation of the expression levels of OATP1A1, OATP1A4, UGT1A1 and MRP2 in ALD. These results revealed the bilirubin transport regulatory mechanisms and highlighted the importance of CAR in modulating the bilirubin clearance pathway in the ALD mouse model.

Hepatic and renal metabolism of genistein: An individual-based model to predict glucuronidation behavior of genistein in different organs

The present study aims to develop an individual-based model to predict the glucuronidation characteristics of genistein in 45 human livers. The model was validated in 18 human kidneys. Ten UDP-glucuronosyltransferases (UGTs) were expressed in liver abundantly. Among them, UGT1A1, UGT1A3, UGT1A6, UGT1A9 and UGT2B7 contributed 95.6% to genistein glucuronidation in human liver, with significant correlation between gene expression of the five UGTs and the glucuronidation of genistein. The genistein glucuronidation differences between individuals were varied enormously; meanwhile the expression variations can explain 24.7% total metabolic variation in livers. In kidney, UGT1A6, UGT1A9 and UGT2B7 were amply expressed. Their gene expressions and glucuronidation rates of genistein had high correlations. Expression variations of UGT1A9/2B7 can explain up to 40.9% of the total variation of genistein glucuronidation in kidney. The model was therefore established based on UGT expression between individuals taking into account an important assessment which is the ratio of metabolic rate of each recombinant UGT isoform. Excellent linear correlations between predicted and observed glucuronidation rate revealed that the model could predict metabolic activity of genistein using quite a small size of tissue. In conclusion, the individual-based model can be employed for predicting individual glucuronidation behavior of genistein in different organs.

Integrated analysis of multiomic data reveals the role of the antioxidant network in the quality of sea buckthorn berry.

Berries of sea buckthorn, known as the "king of vitamin C," are abundant in antioxidants, have attractive colors, and are an excellent material with which to study the relationships between berry color, antioxidants, and berry quality. No study has yet determined the molecular basis of the relationship between sea buckhorn berries and their color and antioxidant levels. By using RNA-seq, LC-MS/MS, and LC/GC-MS technology and selecting red (darkest colored) and yellow (lightest colored) sea buckthorn berries at different development stages, this study showed that the red and yellow berry resulted from a higher ratio of lycopene to β-carotene and of β-carotene to lycopene content, respectively. The uronic acid pathway-a known animal pathway-in ascorbic acid synthesis was found in sea buckthorn berries, and the higher expression of UDP-glucuronosyltransferase in red berries was consistent with the higher content of ascorbic acid. In summary, multiomic data showed that the color of sea buckthorn berries is mainly determined by β-carotene and lycopene; red sea buckthorn berries were richer than yellow berries in antioxidants, such as carotenoids, flavonoids, and ascorbic acid; and the animal pathway might be operating in sea buckthorn.-He, C., Zhang, G., Zhang, J., Zeng, Y., Liu, J. Integrated analysis of multiomic data reveals the role of the antioxidant network in the quality of sea buckthorn berry.