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Objective: We aimed to investigate the ambiguous findings of earlier research regarding the reduction of quetiapine plasma levels when combined with lamotrigine, most likely via UDP-glucuronosyltransferase induction by lamotrigine. Methods: One thousand one hundred and fifty samples, divided into four groups of patients receiving either quetiapine immediate- (IR) or extended-release (XR) without or in combination with lamotrigine were compared regarding absolute and dose-adjusted plasma concentrations. Furthermore, samples of intra-individual controls were analyzed. Results: Patients receiving quetiapine IR in combination with lamotrigine showed 31% lower plasma (p = 0.002) and 23% lower dose-adjusted plasma concentrations (p = 0.004) compared to those receiving IR monotherapy. The proportion of patients with quetiapine plasma concentrations below the lower limit of the therapeutic reference range was 50% and 30% in the combination group and in patients receiving monotherapy, respectively (p = 0.03). However, no significant differences regarding plasma concentration (p = 0.13) and dose-adjusted plasma concentration (p = 0.42) were observed in patients with combination vs. monotherapy with the XR formulation of quetiapine. In the intra-individual controls, no trends could be identified, possibly due to insufficient number of samples (p > 0.05). Conclusions: The combination of quetiapine IR with lamotrigine is associated with significantly lower drug concentrations of quetiapine, potentially impacting quetiapine effectiveness. For quetiapine ER, a significant interaction is less likely. [ABSTRACT FROM AUTHOR]

Purpose: Predicting the quantitative fraction of glucuronidation (fgluc) by individual UDP-glucuronosyltransferase enzymes (UGTs) is challenging due to the lack of selective inhibitors and inconsistent activity of recombinant UGT systems (rUGTs). Our study compares the relative expression versus activity factors (REF versus RAF) to predict fgluc based on rUGT data to human liver and intestinal microsomes (HLM and HIM). Methods: REF scalars were derived from a previous in-house proteomics study for eleven UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17), whereas RAF was calculated by measuring activities in rUGTs to microsomes of selective UGT probe substrates. Protein-normalized activity factor (pnAF) values were generated after correcting activity of individual UGTs to their corresponding protein abundance. The utility of REF and RAF in predicting fgluc was assessed for three UGT substrates—diclofenac, vorinostat, and raltegravir. Results: The REF values ranged from 0.02 to 1.75, RAF based on activity obtained in rUGTs to HLM/HIM were from 0.1 to 274. pnAF values were ~ 5 to 80-fold, except for UGT2B4 and UGT2B15, where pnAF was ~ 180 and > 1000, respectively. The results revealed confounding effect of differential specific activities (per pmol) of rUGTs in fgluc prediction. Conclusion: The data suggest that the activity of UGT enzymes was significantly lower when compared to their activity in microsomes at the same absolute protein amount (pmol). Collectively, results of this study demonstrate poor and variable specific activity of different rUGTs (per pmol protein), as determined by pnAF values, which should be considered in fgluc scaling. [ABSTRACT FROM AUTHOR]

Liver-related side effects are a known complication of treatment with elexacaftor/tezacaftor/ivacaftor (ETI) for cystic fibrosis (CF). Gilbert's syndrome is caused by a genetic mutation that reduces activity of the enzyme UDP glucuronosyltransferase 1 polypeptide A1 (UGT1A1), causing elevated levels of unconjugated bilirubin in the blood and duodenal bile. The presence of Gilbert's syndrome and CF might represent additive risk factors for liver-related adverse events during ETI treatment. This case series describes six people with CF (pwCF) in whom previously unknown Gilbert's syndrome was unmasked after initiation of treatment with ETI. Although all patients had some level of hepatic dysfunction and/or elevated levels of bilirubin after initiation of ETI, the clinical course varied. Only one patient had to stop ETI therapy altogether, while the others were able to continue treatment (some at a reduced dosage and others at the full recommended daily dosage). All patients, even those using a lower dosage, experienced clinical benefit during ETI therapy. Gilbert's syndrome is not a contraindication for ETI therapy but may be mistaken for a risk factor for liver-related adverse events in pwCF. This is something that physicians need to be aware of in pwCF who show liver adverse events during ETI therapy. [ABSTRACT FROM AUTHOR]

Icaritin is a prenylflavonoid derivative of the genus Epimedium (Berberidaceae) and has a variety of pharmacological actions. Icaritin is approved by the National Medical Products Administration as an anticancer drug that exhibits efficacy and safety advantages in patients with hepatocellular carcinoma cells. This study aimed to evaluate the inhibitory effects of icaritin on UDP‐glucuronosyltransferase (UGT) isoforms. 4‐Methylumbelliferone (4‐MU) was employed as a probe drug for all the tested UGT isoforms using in vitro human liver microsomes (HLM). The inhibition potentials of UGT1A1 and 1A9 in HLM were further tested by employing 17β‐estradiol (E2) and propofol (PRO) as probe substrates, respectively. The results showed that icaritin inhibits UGT1A1, 1A3, 1A4, 1A7, 1A8, 1A10, 2B7, and 2B15. Furthermore, icaritin exhibited a mixed inhibition of UGT1A1, 1A3, and 1A9, and the inhibition kinetic parameters (Ki) were calculated to be 3.538, 2.117, and 0.306 (μM), respectively. The inhibition of human liver microsomal UGT1A1 and 1A9 both followed mixed mechanism, with Ki values of 2.694 and 1.431 (μM). This study provides supporting information for understanding the drug–drug interaction (DDI) potential of the flavonoid icaritin and other UGT‐metabolized drugs in clinical settings. In addition, the findings provide safety evidence for DDI when liver cancer patients receive a combination therapy including icaritin. [ABSTRACT FROM AUTHOR]

Triclosan (TCS) is an antimicrobial toxicant found in a myriad of consumer products and has been detected in human tissues, including breastmilk. We have evaluated the impact of lactational TCS on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) neonatal mice. In hUGT1 mice, expression of the hepatic UGT1A1 gene is developmentally delayed resulting in elevated total serum bilirubin (TSB) levels. We found that newborn hUGT1 mice breastfed or orally treated with TCS presented lower TSB levels along with induction of hepatic UGT1A1. Lactational and oral treatment by gavage with TCS leads to the activation of hepatic nuclear receptors constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor alpha (PPARα), and stress sensor, activating transcription factor 4 (ATF4). When CAR-deficient hUGT1 mice (hUGT1/Car−/−) were treated with TCS, TSB levels were reduced with a robust induction of hepatic UGT1A1, leaving us to conclude that CAR is not tied to UGT1A1 induction. Alternatively, when PPARα-deficient hUGT1 mice (hUGT1/Pparα−/−) were treated with TCS, hepatic UGT1A1 was not induced. Additionally, we had previously demonstrated that TCS is a potent inducer of ATF4, a transcriptional factor linked to the integrated stress response. When ATF4 was deleted in liver of hUGT1 mice (hUGT1/Atf4ΔHep) and these mice treated with TCS, we observed superinduction of hepatic UGT1A1. Oxidative stress genes in livers of hUGT1/Atf4ΔHep treated with TCS were increased, suggesting that ATF4 protects liver from excessive oxidative stress. The increase oxidative stress may be associated with superinduction of UGT1A1. The expression of ATF4 in neonatal hUGT1 hepatic tissue may play a role in the developmental repression of UGT1A1. [ABSTRACT FROM AUTHOR]

Inorganic arsenic (iAs) is an environmental toxicant that can lead to severe health consequences, which can be exacerbated if exposure occurs early in development. Here, we evaluated the impact of oral iAs treatment on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) mice. We found that oral administration of iAs to neonatal hUGT1 mice that display severe neonatal hyperbilirubinemia leads to induction of intestinal UGT1A1 and a reduction in total serum bilirubin values. Oral iAs administration accelerates neonatal intestinal maturation, an event that is directly associated with UGT1A1 induction. As a reactive oxygen species producer, oral iAs treatment activated the Keap-Nrf2 pathway in the intestinal tract and liver. When Nrf2-deficient hUGT1 mice (hUGT1/Nrf2-/-) were treated with iAs, it was shown that activated Nrf2 contributed significantly toward intestinal maturation and UGT1A1 induction. However, hepatic UGT1A1 was not induced upon iAs exposure. We previously demonstrated that the nuclear receptor PXR represses liver UGT1A1 in neonatal hUGT1 mice. When PXR was deleted in hUGT1 mice (hUGT1/Pxr-/-), derepression of UGT1A1 was evident in both liver and intestinal tissue in neonates. Furthermore, when neonatal hUGT1/Pxr-/- mice were treated with iAs, UGT1A1 was superinduced in both tissues, confirming PXR release derepressed key regulatory elements on the gene that could be activated by iAs exposure. With iAs capable of generating reactive oxygen species in both liver and intestinal tissue, we conclude that PXR deficiency in neonatal hUGT1/Pxr-/- mice allows greater access of activated transcriptional modifiers such as Nrf2 leading to superinduction of UGT1A1.

Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) is expressed ubiquitously in cancer cells and can metabolize exogenous substances. Studies show higher UGT1A1 levels in pancreatic cancer cells than normal cells. Therefore, we need a method to monitor the activity level of UGT1A1 in pancreatic cancer cells and in vivo. Here, we report a fluorescent probe, BCy-panc, for UGT1A1 imaging in cells and in vivo. Compared with other molecular probes, this probe is readily prepared, with high selectivity and sensitivity for the detection of UGT1A1. Our results show that BCy-panc rapidly detects UGT1A1 in pancreatic cancer. In addition, there is an urgent need for evidence to clarify the relationship between UGT1A1 and pancreatic cancer development. The present investigation found that the increase of UGT1A1 by chrysin was effective in inducing apoptosis in pancreatic cancer cells. These results indicate that the synergistic effect of chrysin and cisplatin at the cellular level is superior to that of cisplatin alone. The UGT1A1 level may be a biomarker for early diagnosis of cancer. Meanwhile, UGT1A1 plays a crucial role in pancreatic cancer, and the combination of chrysin and cisplatin may provide effective ideas for pancreatic cancer treatment. [ABSTRACT FROM AUTHOR]

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), bisphenol A (BPA) analogs, are endocrine-disrupting chemicals predominantly metabolized into glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in humans and rats. In the present study, TBBPA and TCBPA glucuronidation by the liver microsomes of humans and laboratory animals (monkeys, dogs, minipigs, rats, mice, and hamsters) and recombinant human hepatic UGTs (10 isoforms) were examined. TBBPA glucuronidation by the liver microsomes followed the Michaelis–Menten model kinetics in humans, rats, and hamsters and the biphasic model in monkeys, dogs, minipigs, and mice. The CLint values based on the Eadie–Hofstee plots were mice (147) > monkeys (122) > minipigs (108) > humans (100) and rats (98) > dogs (81) > hamsters (47). TCBPA glucuronidation kinetics by the liver microsomes followed the biphasic model in all species except for minipigs, which followed the Michaelis–Menten model. The CLint values were monkeys (172) > rats (151) > mice (134) > minipigs (104), dogs (102), and humans (100) > hamsters (88). Among recombinant human UGTs examined, UGT1A1 and UGT1A9 showed higher TBBPA and TCBPA glucuronidation abilities. The kinetics of TBBPA and TCBPA glucuronidation followed the substrate inhibition model in UGT1A1 and the Michaelis–Menten model in UGT1A9. The CLint values were UGT1A1 (100) > UGT1A9 (42) for TBBPA glucuronidation and UGT1A1 (100) > UGT1A9 (53) for TCBPA glucuronidation, and the activities at high substrate concentration ranges were higher in UGT1A9 than in UGT1A1 for both TBBPA and TCBPA. These results suggest that the glucuronidation abilities toward TBBPA and TCBPA in the liver differ extensively across species, and that UGT1A1 and UGT1A9 expressed in the liver mainly contribute to the metabolism and detoxification of TBBPA and TCBPA in humans. [ABSTRACT FROM AUTHOR]

Simple Summary: UGT enzymes metabolize and detoxify numerous small molecules that are important in cancer, including carcinogens, endogenous growth regulators, and anticancer drugs. Alternatively spliced UGT transcripts can encode truncated proteins that inhibit canonical UGTs, thus reducing detoxification activity. We assessed the expression of specific variant transcripts, designated as UGT1A_v2 and _v3, in six different cancers using RNA-seq datasets with large cohorts of paired normal and tumor tissues. Our results show high interindividual variation in v2 and v3 transcript abundance, as well as tissue- and tumor-specific expression patterns. These findings suggest that the variants have tissue-specific impacts on glucuronidation and may have a more significant role in tumors than in normal tissues. The high interindividual variability is likely relevant to differing personalized drug metabolisms through the UGT conjugation pathway. Finally, our discovery of novel UGT1A variant transcripts further highlights the diversity of the UGT1A transcriptome and proteome. The UGT1A locus generates over 60 different alternatively spliced transcripts and 30 circular RNAs. To date, v2 and v3 transcripts are the only variant UGT1A transcripts that have been functionally characterized. Both v2 and v3 transcripts encode the same inactive variant UGT1A proteins (i2s) that can negatively regulate glucuronidation activity and influence cancer cell metabolism. However, the abundance and interindividual variability in the expression of v2 and v3 transcripts in human tissues and their potential deregulation in cancers have not been comprehensively assessed. To address this knowledge gap, we quantified the expression levels of v1, v2, and v3 transcripts using RNA-seq datasets with large cohorts of normal tissues and paired normal and tumor tissues from patients with six different cancer types (liver, kidney, colon, stomach, esophagus, and bladder cancer). We found that v2 and v3 abundance varied significantly between different tissue types, and that interindividual variation was also high within the same tissue type. Moreover, the ratio of v2 to v3 variants varied between tissues, implying their differential regulation. Our results showed higher v2 abundance in gastrointestinal tissues than liver and kidney tissues, suggesting a more significant negative regulation of glucuronidation by i2 proteins in gastrointestinal tissues than in liver and kidney tissues. We further showed differential deregulation of wildtype (v1) and variant transcripts (v2, v3) in cancers that generally increased the v2/v1 and/or v3/v1 expression ratios in tumors compared to normal tissues, indicating a more significant role of the variants in tumors. Finally, we report ten novel UGT1A transcripts with novel 3′ terminal exons, most of which encode variant proteins with a similar structure to UGT1A_i2 proteins. These findings further emphasize the diversity of the UGT1A transcriptome and proteome. [ABSTRACT FROM AUTHOR]

The microRNA miR399 plays an important role in phosphorus signal transduction pathways in plants. Previously, miR399 was shown to be closely associated with berry ripening in grape (Vitis vinifera). The objective of the present study was to elucidate the evolutionary characteristics of the miR399 gene family in grape and to verify the cleavage effect on the target genes. Grape miR399s were identified by miRNA sequencing and retrieval from the miRBase database. The mature sequences and precursor sequences were subjected to phylogenetic analysis to reconstruct evolutionary trees, as well as secondary structure analysis of the precursor sequence, and prediction of target genes. The cisacting elements in the miR399 promoter were predicted and the cleavage effect of grape miR399b on its target genes was verified. The grape miR399 family comprised nine precursor sequences and nine mature sequences. The precursor sequences formed a typical and stable stemeloop structure. The minimum folding free energy ranged from -55.70 kcal • mol-1 to -37.40 kcal • mol-1. Multiple sequence alignment revealed that the miR399 family was highly conserved. The grape miR399 family was phylogenetically closely related to peach, apple, and citrus miR399s. Grape miR399s were predicted to target inorganic phosphate transporter 1-3, phospholipase D delta-like, and beta-glucuronosyltransferase. The cleavage effect of grape miR399b on the target genes was verified by means of a dual-luciferase assay and 50 RLM-RACE. Histochemical GUS staining showed that the promoter activity of miR399b was promoted by GA3 treatment. [ABSTRACT FROM AUTHOR]

Phytocannabinoid‐rich hemp extracts containing cannabidiol (CBD) and cannabidiolic acid (CBDA) are increasingly being used to treat various disorders in dogs. The objectives of this study were to obtain preliminary information regarding the in vitro metabolism of these compounds and their capacity to inhibit canine cytochrome P450 (CYP)‐mediated drug metabolism and canine P‐glycoprotein‐mediated transport. Pure CBD and CBDA, and hemp extracts enriched for CBD and for CBDA were evaluated. Substrate depletion assays using pooled dog liver microsomes showed CYP cofactor‐dependent depletion of CBD (but not CBDA) and UDP‐glucuronosytransferase cofactor‐dependent depletion of CBDA (but not CBD) indicating major roles for CYP and UDP‐glucuronosytransferase in the metabolism of these phytocannabinoids, respectively. Further studies using recombinant canine CYPs demonstrated substantial CBD depletion by the major hepatic P450 enzymes CYP1A2 and CYP2C21. These results were confirmed by showing increased CBD depletion by liver microsomes from dogs treated with a known CYP1A2 inducer (β‐naphthoflavone) and with a known CYP2C21 inducer (phenobarbital). Cannabinoid‐drug inhibition experiments showed inhibition (IC50 = 4.6–8.1 μM) of tramadol metabolism via CYP2B11‐mediated N‐demethylation (CBD and CBDA) and CYP2D15‐mediated O‐demethylation (CBDA only) by dog liver microsomes. CBD and CBDA did not inhibit CYP3A12‐mediated midazolam 1′‐hydroxylation (IC50 > 10 μM). CBD and CBDA were not substrates or competitive inhibitors of canine P‐glycoprotein. Results for cannabinoid‐enriched hemp extracts were identical to those for pure cannabinoids. These in vitro studies indicate the potential for cannabinoid–drug interactions involving certain CYPs (but not P‐glycoprotein). Confirmatory in vivo studies are warranted. [ABSTRACT FROM AUTHOR]

Background and Objective: Early investigations into drug–drug interactions (DDIs) involving cytochrome P450 2C8 (CYP2C8) have highlighted the complexity of interactions between CYP2C8 substrate drugs, including montelukast, desloratadine, pioglitazone, repaglinide, and cerivastatin (the latter two being OATP1B1 substrates), and standardized CYP2C8 inhibitors such as clopidogrel (Clop) and gemfibrozil (Gem). These interactions have proven challenging to predict based solely on simple CYP inhibition. A hypothesis has emerged suggesting that these substrate drugs first distribute to UDP-glucuronosyltransferase (UGT) before undergoing oxidation by CYP2C8, resulting in bidirectional elimination. The process of drug distribution to UGT is believed to significantly impact these DDIs. This study aims to explore the intricate interplay between UGT and CYP2C8 in the context of DDIs involving CYP2C8 substrates affected by Clop and Gem. Methods: Plasma-level data for the unchanged drug and its metabolite, drawn from the respective literature, formed the basis of our analysis. We evaluated the enzymatic inhibitory activities of DDIs and utilized simulations to estimate plasma levels of the unchanged victim drug and its metabolite in each DDI. This was accomplished by employing a functional relationship that considered the fractional contributions of CYP2C8 and UGT to clearance, perpetrator-specific inhibitory activities against CYP2C8, and drug distribution to UGT. Results: Our findings emphasize the pivotal role of UGT-mediated distribution in the context of CYP2C8 substrate metabolism, particularly in the complex DDIs induced by Clop and Gem. In these DDIs, Gem exerts inhibitory effects on both UGT and CYP2C8, whereas Clop (specifically its metabolite, Clop-COOH) solely targets CYP2C8. Importantly, the inhibition of CYP2C8 by both Clop and Gem is achieved through a non-competitive mechanism, driven by the actions of their acyl-glucuronides. Clop and Gem exhibit inhibition activities accounting for 85% (pAi,CYP2C8 = 7) and 93% (pAi,CYP2C8 = 15), respectively. In contrast, Gem's inhibition of UGT is relatively modest (50%, pAi,UGT(d) = 2), and it operates through a non-specific, competitive process in drug distribution to UGT. Within this context, our UGT-CYP2C8 interplay model offers an accurate means of predicting the alterations resulting from DDIs, encompassing changes in plasma levels of the unchanged drug and its metabolites, as well as shifts in metabolite formation rates. Our analysis highlights the critical importance of considering the fractional contributions of CYP2C8 and UGT to the victim drug's clearance (fm,CYP2C8; fm,UGT) in DDI prediction. Furthermore, our examination of DDIs involving OATP1B1 substrate drugs underscores that accounting for the hepatic uptake transporters' role in the liver is superfluous in DDI prediction. Conclusion: These findings substantially enhance our comprehension of CYP2C8-mediated oxidation and DDIs, holding crucial implications for drug development and the planning of clinical trials involving these inhibitors. [ABSTRACT FROM AUTHOR]

UDP‐glucuronosyltransferases (UGTs) are phase II drug metabolizing enzymes that play important roles in the detoxification of endogenous and exogenous substrates. The 22 human UGTs belong to four families (UGT1, UGT2, UGT3, and UGT8) and differ in their expression, substrate specificity, UDP‐sugar preference, and physiological functions. Differential expression/activity of the UGTs contributes to interperson variability in drug responses and toxicity, hormone homeostasis, and disease/cancer risks. However, in normal tissues, the tissue‐specific expression profiles and transcriptional regulation of the UGTs are still not fully understood. In this study, we comprehensively analyzed the transcriptome of 22 UGTs in 54 human tissues/regions using RNAseq data from GTEx. We then validated the findings in the liver and small intestine samples using real‐time PCR. Our results showed large interindividual variability across tissues in the expression of each UGT and the overall composition of UGT pools, consisting of different UGTs and their splice isoforms. Our results also revealed coexpression of the UGTs, Cytochrome P450s, and many transcription factors in the liver, suggesting potential coregulation or functional coordination. Our results provide the groundwork for future studies to detail further the regulation of the expression and activity of the UGTs. [ABSTRACT FROM AUTHOR]

Liposomal irinotecan is a liposomal formulation of irinotecan, which prolongs circulation of irinotecan and its active metabolite SN-38. A population pharmacokinetic (PK) model was developed based on data from seven studies (N = 440). Adequacy of the model was assessed using multiple methods, including visual predictive check. Associations between PK exposure and the incidence of diarrhea (grade ≥3) and neutropenia adverse events (AEs) (grade ≥3) at first event in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) were investigated using logistic regression based on data from two studies (the phase III NAPOLI-1 [N = 260] and phase I/II NCT02551991 [N = 56] trials). The PKs of total irinotecan was described by a two-compartment model with first-order elimination, with SN-38 formed directly by a first-order constant from the central compartment of irinotecan or after using a transit compartment. Clearance was 17.9 L/week (0.107 L/h) and 19,800 L/week (118 L/h) for total irinotecan and SN-38, respectively. The UGT1A1*28 7/7 homozygous genotype had no significant impact on SN-38 clearance. Model evaluation was satisfactory for both irinotecan and SN-38. The incidence of diarrhea (grade ≥3) at first event was significantly higher with increasing average concentrations of total irinotecan and SN-38; there was no significant association between an increased risk of neutropenia AEs (grade ≥3) at first event and average SN-38 concentrations. In summary, the PKs of total irinotecan and SN-38 after administration of liposomal irinotecan were well-described by the model. The UGT1A1*28 status had no significant impact on the PKs of liposomal irinotecan.

The predominant catechin in green tea, epigallocatechin gallate (EGCG), may be hepatotoxic in high doses. Our objective was to investigate the influence of catechol-O-methyltransferase (COMT) and uridine 5'-diphospho-glucuronosyltransferase 1A4 (UGT1A4) genotypes on changes in liver injury biomarkers in response to long-term, high-dose green tea extract (GTE) supplementation among postmenopausal women. A secondary analysis was conducted using data from the Minnesota Green Tea Trial (N = 1,075), in which participants were randomized to consume high-dose GTE (843 mg/day EGCG) or placebo capsules for 12 months. Analysis of covariance adjusting for potential confounders was performed to examine changes in aspartate aminotransferase (AST), alanine aminotransferase (ALT), AST: ALT ratio, and alkaline phosphatase from baseline to months 3, 6, 9, and 12 across COMT and UGT1A4 genotypes. Mean age and BMI within the GTE group (n = 400) were 59.8 yrs and 25.1 kg/m2, respectively, and 98% of subjects were white. From baseline to month 3, mean AST: ALT ratio change was +1.0% in the COMT (rs4680) A/G genotype versus −4.8% in the A/A genotype (p = 0.03). From baseline to months 6 and 9, respectively, mean ALT change was +78.1% and +82.1% in the UGT1A4 (rs6755571) A/C genotype versus +28.0% and +30.1% in the C/C genotype (p < 0.001 and p = 0.004, respectively). The UGT1A4 (rs6755571) A/C genotype may be an important risk factor for clinically-relevant serum transaminase elevations with 6-9 months of high-dose GTE supplementation among postmenopausal women. Understanding the genetic underpinnings of GTE-related hepatotoxicity may allow for a genetically-informed paradigm for therapeutic use of GTE. [ABSTRACT FROM AUTHOR]

Green tea is a popular beverage worldwide. The abundant green tea catechin (−)‐epigallocatechin gallate (EGCG) is a potent in vitro inhibitor of intestinal UDP‐glucuronosyltransferase (UGT) activity (Ki ~2 μM). Co‐consuming green tea with intestinal UGT drug substrates, including raloxifene, could increase systemic drug exposure. The effects of a well‐characterized green tea on the pharmacokinetics of raloxifene, raloxifene 4′‐glucuronide, and raloxifene 6‐glucuronide were evaluated in 16 healthy adults via a three‐arm crossover, fixed‐sequence study. Raloxifene (60 mg) was administered orally with water (baseline), with green tea for 1 day (acute), and on the fifth day after daily green tea administration for 4 days (chronic). Unexpectedly, green tea decreased the geometric mean green tea/baseline raloxifene AUC0–96h ratio to ~0.60 after both acute and chronic administration, which is below the predefined no‐effect range (0.75–1.33). Lack of change in terminal half‐life and glucuronide‐to‐raloxifene ratios indicated the predominant mechanism was not inhibition of intestinal UGT. One potential mechanism includes inhibition of intestinal transport. Using established transfected cell systems, a green tea extract normalized to EGCG inhibited 10 of 16 transporters tested (IC50, 0.37–12 μM). Another potential mechanism, interruption by green tea of gut microbe‐mediated raloxifene reabsorption, prompted a follow‐up exploratory clinical study to evaluate the potential for a green tea–gut microbiota–drug interaction. No clear mechanisms were identified. Overall, results highlight that improvements in current models and methods used to predict UGT‐mediated drug interactions are needed. Informing patients about the risk of co‐consuming green tea with raloxifene may be considered. [ABSTRACT FROM AUTHOR]

ObjectivesTo investigate if the nicotine metabolite ratio (NMR, the ratio of nicotine metabolites 3'-hydroxycotinine/cotinine) is a reliable phenotypic biomarker for nicotine clearance across races, and as a function of differences in the rate of nicotine, cotinine and 3'-hydroxycotinine glucuronidation and UGT genotypes.MethodsParticipants [Caucasians (Whites), African Americans (Blacks) and Asian-Americans (Asians)] received an oral solution of deuterium-labeled nicotine and its metabolite cotinine. Plasma and saliva concentrations of nicotine and cotinine were used to determine oral clearances. Rates of glucuronidation were assessed from urine glucuronide/parent ratios, and UGT2B10 and UGT2B17 genotypes from DNA.ResultsAmong the 227 participants, 96 (42%) were White, 67 (30%) Asian and 64 (28%) Black. Compared to the other two races, Whites had higher nicotine and cotinine total oral clearance, Blacks had lower nicotine and cotinine glucuronidation rates and Asians had lower 3'-hydroxycotinine glucuronidation rates. A strong positive correlation (correlations coefficients 0.77-0.84; P

BackgroundVariation in CYP2A6 activity influences tobacco smoking behaviors and smoking-related health outcomes. Plasma Nicotine Metabolite Ratio (NMR) is a robust phenotypic biomarker of CYP2A6 activity and nicotine clearance. In urine, the NMR has been calculated as a ratio of free trans-3'-hydroxycotinine to free cotinine (NMRF/F), total trans-3'-hydroxycotinine to free cotinine (NMRT/F), or total trans-3'-hydroxycotinine to total cotinine (NMRT/T). We evaluated these three urinary NMR versions relative to plasma NMR and nicotine clearance and elucidated mechanisms of discrepancies among them.MethodsBaseline plasma and urine biomarker data were available from two smoking cessation clinical trials and one nicotine pharmacokinetic study (total N = 768). NMRs were compared using Pearson correlations, linear regressions and ANOVA analyses. UGT2B10 and UGT2B17 were genotyped.ResultsUrinary NMRT/F was the most highly related to plasma NMR (R2 = 0.70, P urinary NMRT/F > NMRF/F > NMRT/T (R2 = 0.41 > 0.37 > 0.35 > 0.25 respectively).ConclusionUrinary NMRT/F followed by NMRF/F are the best urinary alternatives to plasma NMR or nicotine clearance. NMRT/T has the least utility as it is influenced substantially by variation in cotinine glucuronidation.ImpactThis work highlighted the variation in urinary NMRs, and identified mechanisms for disparities among them, which facilitates their use in predicting smoking-related outcomes.

The initial isolation of adrenal steroids from large quantities of animal adrenals resulted in an amorphous fraction resistant to crystallization and identification and had potent effects on electrolyte transport. Aldosterone was eventually isolated and identified in the fraction and was soon shown to cause hypertension when in excess. The autonomous and excessive production of aldosterone, primary aldosteronism, is the most common cause of secondary hypertension. Aldosterone is metabolized in the liver and kidney, and its metabolites are conjugated with glucuronic acid for excretion. The most common liver metabolite is 3α,5β-tetrahydroaldosterone-3-glucuronide, while that of the kidney is aldosterone-18-oxo-glucuronide. In terms of their value, especially the aldosterone-18-oxo-glucuronide, is commonly used for the diagnosis of primary aldosteronism because they provide an integrated value of the total daily production of aldosterone. Conversion of aldosterone to 18-oxo-glucuronide is impeded by drugs, like some common non-steroidal anti-inflammatory drugs that compete for UDP-glucuronosyltransferase-2B7, the most important glucuronosyltransferase for aldosterone metabolism. Tetrahydroaldosterone is the most abundant metabolite and the most reliable for the diagnosis of primary aldosteronism, but it is not commonly measured. [ABSTRACT FROM AUTHOR]

Background and Objective: The impact of liver cirrhosis on the activity of UDP-glucuronosyltransferases (UGTs) is currently not well characterized. We investigated the glucuronidation capacity and glucuronide accumulation in patients with liver cirrhosis. Methods: We administered the Basel phenotyping cocktail (caffeine, efavirenz, flurbiprofen, omeprazole, metoprolol, midazolam) to patients with liver cirrhosis (n = 16 Child A, n = 15 Child B, n = 5 Child C) and n = 12 control subjects and obtained pharmacokinetic profiles of substrates and primary metabolites and their glucuronides. Results: Caffeine and its metabolite paraxanthine were only slightly glucuronidated. The metabolic ratio (AUCglucuronide/AUCparent, MR) was not affected for caffeine but decreased by 60% for paraxanthine glucuronide formation in Child C patients. Efavirenz was not glucuronidated whereas 8-hydroxyefavirenz was efficiently glucuronidated. The MR of 8-hydroxyefavirenz-glucuronide formation increased three-fold in Child C patients and was negatively correlated with the glomerular filtration rate. Flurbiprofen and omeprazole were not glucuronidated. 4-Hydroxyflurbiprofen and 5-hydroxyomeprazole were both glucuronidated but the corresponding MRs for glucuronide formation were not affected by liver cirrhosis. Metoprolol, but not α-hydroxymetoprolol, was glucuronidated, and the MR for metoprolol-glucuronide formation dropped by 60% in Child C patients. Both midazolam and its metabolite 1′-hydroxymidazolam underwent glucuronidation, and the corresponding MRs for glucuronide formation dropped by approximately 80% in Child C patients. No relevant glucuronide accumulation occurred in patients with liver cirrhosis. Conclusions: Detailed analysis revealed that liver cirrhosis may affect the activity of UGTs of the UGT1A and UGT2B subfamilies according to liver function. Clinically significant glucuronide accumulation did not occur in the population investigated. Clinical Trial Registration: NCT03337945. [ABSTRACT FROM AUTHOR]

Aims: Letermovir, a cytomegalovirus (CMV) DNA terminase complex inhibitor, is a substrate of ABCB1 (P‐glycoprotein; P‐gp), organic anion transporting polypeptide (OATP)1B1/3, UDP‐glucuronosyltransferase (UGT)1A1, UGT1A3 and possibly ABCG2 (breast cancer resistance protein; BCRP). A study was conducted to evaluate the effects of itraconazole, a prototypic ABCB1/ABCG2 inhibitor, on letermovir pharmacokinetics (PK) and the effects of letermovir on itraconazole PK. Methods: In an open‐label, fixed‐sequence study in 14 healthy participants, 200 mg oral itraconazole was administered once daily for 4 days. Following a 10‐day washout, 480 mg oral letermovir was administered once daily for 14 days (Days 1–14) and then coadministered with 200 mg itraconazole once daily for 4 days (Days 15–18). Intensive PK sampling was performed for letermovir and itraconazole. PK and safety were evaluated. Results: Letermovir geometric mean ratio (GMR; 90% confidence interval [CI]) for area under the concentration–time curve from time 0 to 24 h (AUC0–24) was 1.33 (1.17, 1.51) and for maximum concentration (Cmax) was 1.21 (1.05, 1.39) following administration with/without itraconazole. Itraconazole GMR (90% CI) for AUC0–24 was 0.76 (0.71, 0.81) and for Cmax was 0.84 (0.76, 0.92) following administration with/without letermovir. Coadministration of letermovir with itraconazole was generally well tolerated. Conclusions: The increase in letermovir exposure with coadministration of itraconazole is likely predominantly due to inhibition of intestinal ABCB1 and potentially ABCG2 transport. The mechanism for the decrease in itraconazole exposure is unknown. The modest changes in letermovir and itraconazole PK are not considered clinically meaningful. [ABSTRACT FROM AUTHOR]

Liver X receptors (LXRs), LXRα and LXRβ, are nuclear receptors that regulate the metabolism of cholesterol and bile acids and are activated by oxysterols. Humanized UGT1 (hUGT1) mice express the 9-human UGT1A genes associated with the UGT1 locus in a Ugt1-null background. The expression of UGT1A1 is developmentally delayed in the liver and intestines, resulting in the accumulation of serum bilirubin during the neonatal period. Induction of UGT1A1 in newborn hUGT1 mice leads to rapid reduction in total serum bilirubin (TSB) levels, a phenotype measurement that allows for an accurate prediction on UGT1A1 expression. When neonatal hUGT1 mice were treated by oral gavage with the LXR agonist T0901317, TSB levels were dramatically reduced. To determine the LXR contribution to the induction of UGT1A1 and the lowering of TSB levels, experiments were conducted in neonatal hUGT1/Lxrα -/- , hUGT1/Lxrβ -/- , and hUGT1/Lxrαβ -/- mice treated with T0901317. Induction of liver UGT1A1 was dependent upon LXRα, with the induction pattern paralleling induction of LXRα-specific stearoyl CoA desaturase 1. However, the actions of T0901317 were also shown to display a lack of specificity for LXR, with the induction of liver UGT1A1 in hUGT1/Lxrαβ -/- mice, a result associated with activation of both pregnane X receptor and constitutive androstane receptor. However, the LXR agonist GW3965 was highly selective toward LXRα, showing no impact on lowering TSB values or inducing UGT1A1 in hUGT1/Lxrα -/- mice. An LXR-specific enhancer site on the UGT1A1 gene was identified, along with convincing evidence that LXRα is crucial in maintaining constitutive expression of UGT1A1 in adult hUGT1 mice. SIGNIFICANCE STATEMENT: It has been established that activation of LXRα, and not LXRβ, is responsible for the induction of liver UGT1A1 and metabolism of serum bilirubin in neonatal hUGT1 mice. Although induction of the human UGT1A1 gene is initiated at a newly characterized LXR enhancer site, allelic deletion of the Lxrα gene drastically reduces the constitutive expression of liver UGT1A1 in adult hUGT1 mice. Combined, these findings indicate that LXRα is critical for the developmental expression of UGT1A1.

Environmental toxicants such as heavy metals from contaminated water or soil and isothiocyanates (ITC) from dietary sources act as pro-oxidants by directly generating reactive oxygen species (ROS) or through depleting cellular antioxidants such as glutathione. Toxicants can alter drug metabolism, and it was reported that CYP2B10 and UGT1A1 are induced by phenethyl isothiocyanate (PEITC) through the constitutive androstane receptor (CAR). The possibility that nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of the antioxidant response, could coactivate CAR was investigated in neonatal hUGT1/Nrf2 -/- mice. Neonatal mice were treated with PEITC or cadmium (Cd2+) by oral gavage for 2 days. Both PEITC and Cd2+ induced UGT1A1 RNA and protein in intestinal tissues in both hUGT1/Nrf2 +/- and hUGT1/Nrf2 -/- neonates, indicating NRF2-independent regulation of UGT1A1. Increases in CYP2B10 RNA in intestinal tissues were observed following PEITC or Cd2+ exposure. Activation of intestinal CAR by Cd2+ exposure was directly assessed by nuclear fractionation and Western blot analyses at 0.5, 1, 2, and 4 hours after treatment in hUGT1 neonates and after 48 hours in hUGT1/Nrf2 +/- and hUGT1/Nrf2 -/- neonates. CAR localized to the nucleus independently of NRF2 48 hours after exposure. Substantial CAR localization to the nucleus occurred at the 2- and 4-hour time points, coinciding with a decrease in phosphorylation of cytoplasmic extracellular signal-regulated kinases 1 and 2 and a nuclear increase in P38/p-P38 content. This suggests that a novel oxidative stress-MAPK-CAR axis exists with phenotypic consequences. SIGNIFICANCE STATEMENT: Pro-oxidant toxicants can alter drug metabolism through activation of CAR, independent of the NRF2-KEAP1 signaling pathway. Changes in proteins associated with drug metabolism and linked to increases in intestinal maturation are mediated through an oxidative stress-MAPK-CAR axis.

Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemo‐enzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α‐glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthase‐mediated coupling reactions as key steps. The xylo‐oligosaccharide donors were protected at the non‐reducing end with a 4‐O‐tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3‐O‐methylether substituent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyl‐transferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI‐MS/MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long‐chain acceptors for assaying biosynthetic glycosyltransferases. [ABSTRACT FROM AUTHOR]

OBJECTIVES: Carbamazepine (CBZ), an anti-seizure drug, is widely prescribed for the management of focal seizures. At a given therapeutic dose, CBZ exhibits marked interindividual variation in the plasma CBZ levels. The aim wasto study the influence of EPHX1 c.337 T>C and UGT2B7*2 genetic polymorphisms on plasma carbamazepine (CBZ) levels in persons with epilepsy (PWE) from South India. METHODS: 115 PWE belong to South India origin who are on carbamazepine monotherapy were recruited. Genotyping of the two variants weredone using RT-PCR method. PWE who had seizure freedom for one year and their last dose which was not changed for one year duration were included and their plasma levels of CBZ and its active metabolite CBZ 10,11 epoxide were analysed by reverse phase HPLC. RESULTS: In EPHX1 c. 337 (T>C) polymorphism, the PWE carrying CC had lower plasma CBZ levels when compared to CT genotype (2.45 µg/ml vs 3.15 µg/ml. In UGT2B7*2, PWE carrying homozygous mutant TT had higher levels when compared with CT (3.09 µg/ml vs 2.74 µg/ml) genotype but found no statistical significance. Mutant genotype of EPHX1 (CC) had higher metabolic ratio compared to TT genotype (1.33 vs 1.17) but not found to be statistically significant. Mutant genotype of UGT2B7*2 (TT) was found to be having lower metabolic ratio when compared with CC genotype (1.18 vs 1.35; p value =0.08). CONCLUSION: PWE carrying EPHX1 c.337 T>C (rs1051740) and UGT2B7*2 (rs7439366) genetic polymorphisms did not affect the plasma CBZ levels and metabolic ratio of PWE of South Indian origin. However, this finding should be confirmed in a larger sample size which may help in optimization and personalized CBZ therapy in South Indians. [ABSTRACT FROM AUTHOR]

The adverse effects (diarrhea and neutropenia) of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) are associated with genetic variants of uridine diphosphate glucuronosyltransferase 1A subfamilies (UGT1As). UGT1As are enzymes that metabolize the active form of irinotecan, 7-ethyl-10 hydroxycamptothecin (SN-38), by glucuronidation in the liver. They are widely known as predictive factors of severe adverse effects, such as neutropenia and diarrhea. Some studies have suggested that variants of UGT1As affect SN-38 glucuronidation activities, thus exerting severe adverse effects. We aimed to identify UGT1A isoforms that show SN-38 glucuronidation activity and determine the relationship between UGT1A variants and SN-38 glucuronidation in vitro. We found that UGT1A1 and UGT1A6–UGT1A10 displayed SN-38 glucuronidation activity. Among these, UGT1A1 was the most active. Furthermore, the variants of these isoforms showed decreased SN-38 glucuronidation activity. In our study, we compared the different variants of UGT1As, such as UGT1A1.6, UGT1A1.7, UGT1A1.27, UGT1A1.35, UGT1A7.3, UGT1A8.4, UGT1A10M59I, and UGT1A10T202I, to determine the differences in the reduction of glucuronidation. Our study elucidates the relationship between UGT1A variants and the level of glucuronidation associated with each variant. Therefore, testing can be done before the initiation of irinotecan treatment to predict potential toxicities and adverse effects. [ABSTRACT FROM AUTHOR]

Inorganic arsenic (iAs) is an environmental toxicant that can lead to severe health consequences, which can be exacerbated if exposure occurs early in development. Here, we evaluated the impact of oral iAs treatment on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) mice. We found that oral administration of iAs to neonatal hUGT1 mice that display severe neonatal hyperbilirubinemia leads to induction of intestinal UGT1A1 and a reduction in total serum bilirubin values. Oral iAs administration accelerates neonatal intestinal maturation, an event that is directly associated with UGT1A1 induction. As a reactive oxygen species producer, oral iAs treatment activated the Keap-Nrf2 pathway in the intestinal tract and liver. When Nrf2-deficient hUGT1 mice (hUGT1/Nrf2-/-) were treated with iAs, it was shown that activated Nrf2 contributed significantly toward intestinal maturation and UGT1A1 induction. However, hepatic UGT1A1 was not induced upon iAs exposure. We previously demonstrated that the nuclear receptor PXR represses liver UGT1A1 in neonatal hUGT1 mice. When PXR was deleted in hUGT1 mice (hUGT1/Pxr-/-), derepression of UGT1A1 was evident in both liver and intestinal tissue in neonates. Furthermore, when neonatal hUGT1/Pxr-/- mice were treated with iAs, UGT1A1 was superinduced in both tissues, confirming PXR release derepressed key regulatory elements on the gene that could be activated by iAs exposure. With iAs capable of generating reactive oxygen species in both liver and intestinal tissue, we conclude that PXR deficiency in neonatal hUGT1/Pxr-/- mice allows greater access of activated transcriptional modifiers such as Nrf2 leading to superinduction of UGT1A1. [ABSTRACT FROM AUTHOR]

An association between genetic variants in the vitamin D receptor (VDR) gene and epithelial ovarian cancer (EOC) was previously reported in women of African ancestry (AA). We sought to examine associations between genetic variants in VDR and additional genes from vitamin D biosynthesis and pathway targets (EGFR, UGT1A, UGT2A1/2, UGT2B, CYP3A4/5, CYP2R1, CYP27B1, CYP24A1, CYP11A1, and GC). Genotyping was performed using the custom-designed 533,631 SNP Illumina OncoArray with imputation to the 1,000 Genomes Phase 3 v5 reference set in 755 EOC cases, including 537 high-grade serous (HGSOC), and 1,235 controls. All subjects are of African ancestry (AA). Logistic regression was performed to estimate odds ratios (OR) and 95% confidence intervals (CI). We further evaluated statistical significance of selected SNPs using the Bayesian False Discovery Probability (BFDP). A significant association with EOC was identified in the UGT2A1/2 region for the SNP rs10017134 (per allele OR = 1.4, 95% CI = 1.2-1.7, P = 1.2 × 10-6 , BFDP = 0.02); and an association with HGSOC was identified in the EGFR region for the SNP rs114972508 (per allele OR = 2.3, 95% CI = 1.6-3.4, P = 1.6 × 10-5 , BFDP = 0.29) and in the UGT2A1/2 region again for rs1017134 (per allele OR = 1.4, 95% CI = 1.2-1.7, P = 2.3 × 10-5 , BFDP = 0.23). Genetic variants in the EGFR and UGT2A1/2 may increase susceptibility of EOC in AA women. Future studies to validate these findings are warranted. Alterations in EGFR and UGT2A1/2 could perturb enzyme efficacy, proliferation in ovaries, impact and mark susceptibility to EOC.

The attachment of a sugar to a hydrophobic lipid carrier is the first step in the biosynthesis of many glycoconjugates. In the halophilic archaeon Haloarcula hispanica, HAH_1206, renamed AepG, is a predicted glycosyltransferase belonging to the CAZy Group 2 family that shares a conserved amino acid sequence with dolichol phosphate mannose synthases. In this study, the function of AepG was investigated by genetic and biochemical approaches. We found that aepG deletion led to the disappearance of dolichol phosphate-glucuronic acid. Our biochemical assays revealed that recombinant cellulosebinding, domain-tagged AepG could catalyze the formation of dolichol phosphate-glucuronic acid in time- and dosedependent manners. Based on the in vivo and in vitro analyses, AepG was confirmed to be a dolichol phosphate glucuronosyltransferase involved in the synthesis of the acidic exopolysaccharide produced by H. hispanica. Furthermore, lack of aepG resulted in hindered growth and cell aggregation in high salt medium, indicating that AepG is vital for the adaptation of H. hispanica to a high salt environment. In conclusion, AepG is the first dolichol phosphate glucuronosyltransferase identified in any of the three domains of life and, moreover, offers a starting point for further investigation into the diverse pathways used for extracellular polysaccharide biosynthesis in archaea. [ABSTRACT FROM AUTHOR]

Gypensapogenin C (GPC) is one of the important aglycones of Gynostemma pentaphyllum (GP), which is structurally glucuronidated and is highly likely to bind to UGT enzymes in vivo. Due to the important role of glucuronidation in the metabolism of GPC, the UDP-glucuronosyltransferase metabolic pathway of GPC in human and other species' liver microsomes is investigated in this study. In the present study, metabolites were detected using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The results show that GPC could generate a metabolite through glucuronidation in the human liver microsomes (HLMs). Additionally, chemical inhibitors combined with recombinant human UGT enzymes clarified that UGT1A4 is the primary metabolic enzyme for GPC glucuronidation in HLMs according to the kinetic analysis of the enzyme. Metabolic differential analysis in seven other species indicated that rats exhibited the most similar metabolic rate to that of humans. In conclusion, UGT1A4 is a major enzyme responsible for the glucuronidation of GPC in HLMs, and rats may be an appropriate animal model to evaluate the GPC metabolism. [ABSTRACT FROM AUTHOR]

Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays. In the present study, we characterised UGT activity in liver microsomes of green turtles (Chelonia mydas), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4. The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij®58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest Vmax (1203 pmol·min−1mg·protein−1). Lithocholic acid and diclofenac inhibited UGT activity in green turtles. This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species. [ABSTRACT FROM AUTHOR]

Tizoxanide (TZX) is an active metabolite of nitazoxanide (NTZ) originally developed as an antiparasitic agent, and is predominantly metabolized into TZX glucuronide. In the present study, TZX glucuronidation by the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice, and recombinant human UDP-glucuronosyltransferase (UGT) were examined. The kinetics of TZX glucuronidation by the liver and intestinal microsomes followed the Michaelis–Menten or biphasic model, with species-specific variations in the intrinsic clearance (CL int). Rats and mice exhibited the highest CL int values for liver microsomes, while mice and rats were the highest for intestinal microsomes. Among human UGTs, UGT1A1 and UGT1A8 demonstrated significant glucuronidation activity. Estradiol and emodin inhibited TZX glucuronidation activities in the human liver and intestinal microsomes in a dose-dependent manner, with emodin showing stronger inhibition in the intestinal microsomes. These results suggest that the roles of UGT enzymes in TZX glucuronidation in the liver and small intestine differ extensively across species and that UGT1A1 and/or UGT1A8 mainly contribute to the metabolism and elimination of TZX in humans. This study presents the relevant and novel-appreciative report on TZX metabolism catalyzed by UGT enzymes, which may aid in the assessment of the antiparasitic, antibacterial, and antiviral activities of NTZ for the treatment of various infections. [Display omitted] • TZX glucuronidation abilities in the liver and small intestine differ extensively across species. • TZX glucuronidation activities in the liver microsomes were rats > mice ≥ monkeys > dogs > humans. • TZX glucuronidation activities in the intestinal microsomes were mice ≥ rats > monkeys > dogs > humans. • UGT1A1 and/or UGT1A8 mainly contribute to the metabolism and elimination of TZX in humans. [ABSTRACT FROM AUTHOR]

Neonatal hyperbilirubinemia and the onset of bilirubin encephalopathy and kernicterus result in part from delayed expression of UDP-glucuronosyltransferase 1A1 (UGT1A1) and the ability to metabolize bilirubin. It is generally believed that acute neonatal forms of hyperbilirubinemia develop due to an inability of hepatic UGT1A1 to metabolize efficiently bilirubin for clearance through the hepatobiliary tract. Newly developed mouse models designed to study bilirubin metabolism have led to new insight into the role of the intestinal tract in controlling neonatal hyperbilirubinemia. Humanization of mice with the UGT1 locus (hUGT1 mice) and the UGT1A1 gene provide a unique tool to study the onset of hyperbilirubinemia since the human UGT1A1 gene is developmentally regulated during the neonatal period in hUGT1 mice. A new mechanism outlying developmental expression of intestinal UGT1A1 is presented and its implications in the control of neonatal hyperbilirubinemia discussed. New findings linking breast milk protection against necrotizing enterocolitis and intestinal control of UGT1A1 may help explain the contribution of breast milk toward the development of neonatal hyperbilirubinemia. Our findings outline a new model that includes an active intestinal ROS /IκB kinase/nuclear receptor corepressor 1 loop that can be applied to an understanding of breast milk-induced jaundice.

Simple Summary: In this study, we clarified the evolutional features of the UGT gene family in Carnivora. We firstly analyzed the gene synteny of UGT1As, 2Bs, ana 2Es and further demonstrated the phylogenetic analysis to reveal the evolutional gene duplication and loss event in Carnivora. We found specific UGT1A duplication in Canidae, brown bear and black bear, and UGT2Bs duplication in Canidae, some Mustelidae, and Ursidae. In addition, we observed gene contraction of UGT1A7–12 in Phocidae, Otariidae, and Felidae. This study strongly suggested closely related Carnivorans also showed significant evolutional differences of UGTs, and further imply the importance of appropriate approaches to assess pharmacokinetics and toxicokinetic from experimental animals. UDP-glucuronosyltransferases (UGTs) are one of the most important enzymes for xenobiotic metabolism or detoxification. Through duplication and loss of genes, mammals evolved the species-specific variety of UGT isoforms. Among mammals, Carnivora is one of the orders that includes various carnivorous species, yet there is huge variation of food habitat. Recently, lower activity of UGT1A and 2B were shown in Felidae and pinnipeds, suggesting evolutional loss of these isoforms. However, comprehensive analysis for genetic or evolutional features are still missing. This study was conducted to reveal evolutional history of UGTs in Carnivoran species. We found specific gene expansion of UGT1As in Canidae, brown bear and black bear. We also found similar genetic duplication in UGT2Bs in Canidae, and some Mustelidae and Ursidae. In addition, we discovered contraction or complete loss of UGT1A7–12 in phocids, some otariids, felids, and some Mustelids. These studies indicate that even closely related species have completely different evolution of UGTs and further imply the difficulty of extrapolation of the pharmacokinetics and toxicokinetic result of experimental animals into wildlife carnivorans. [ABSTRACT FROM AUTHOR]

Ethyl glucuronide (EtG) is a non-volatile, non-oxidative, hydrophilic, and stable ethanol phase II metabolite. EtG is produced through ethanol glucuronidation by UDP-glucuronosyltransferase (UGT), a phase II enzyme. EtG can be extracted from different biological matrices, including keratin ones, such as hair or nails. The purpose of this scoping review is to describe the relationship between EtG levels in hair and some of the most common and frequent pathological conditions and verify whether different reference cut-offs in relation to various pathologies have been identified in the scientific literature. In fact, in-depth knowledge of the influence of pathologies, such as diabetes mellitus, hepatic and renal dysfunction, on EtG production and its storage in keratin matrices would allow a more appropriate interpretation of obtained data and rule out false positives or false negatives. This scoping review is based on bibliographic research carried out on PubMed regarding the quantification of EtG in hair of subjects affected by different pathological conditions. According to the scientific literature, the main and most common pathologies that can affect the concentration of EtG in hair are liver and kidney diseases and diabetes. The EtG quantification analytical data should be interpreted carefully as they may have a great impact in both forensic and clinical contexts. [ABSTRACT FROM AUTHOR]

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.

BackgroundUridine 5'-diphosphate-glucuronosyltransferase 2B (UGT2B) genes code for enzymes that catalyze the clearance of testosterone, dihydrotestosterone (DHT), and DHT metabolites in the prostate basal and luminal tissue. The expression of the UGT2B15, UGT2B17, and UGT2B28 enzymes has not been evaluated in prostate tissue samples from hormone therapy-naïve patients.MethodsWe determined the expression of UGT2B15, UGT2B17, and UGT2B28 enzymes in 190 prostate tissue samples from surgical specimens of a multiethnic cohort of patients undergoing radical prostatectomy at the Durham Veterans Affairs Medical Center. The association between each protein's percent positive and H-score, a weighted score of staining intensity, and the risk of biochemical recurrence (BCR) was tested using separate Cox proportional hazards models. In an exploratory analysis, UGT2B17 total positive and H-score were divided at the median and we tested the association between UGT2B17 group and risk of BCR.ResultsThe median follow-up for all patients was 118 months (IQR: 85-144). Of 190, 83 (44%) patients developed BCR. We found no association between UGT2B15 or UGT2B28 and risk of BCR. However, there was a trend for an association between UGT2B17 and BCR (HR = 1.01, 95% CI 1.00-1.02, p = 0.11), though not statistically significant. Upon further investigation, we found that patients with UGT2B17 higher levels of expression had a significant increased risk of BCR on univariable analysis (HR = 1.57, 95% CI 1.02-2.43, p = 0.041), although this association was attenuated in the multivariable model (HR = 1.50, 95% CI 0.94-2.40, p = 0.088).ConclusionsOur findings suggest that UGT2B17 overexpression may be associated with a significant increased risk of BCR. These results are consistent with previous reports which showed UGT2B17 significantly expressed in advanced prostate cancer including prostate tumor metastases.

Human UDP-glucuronosyltransferase (UGT) 2B7 is a crucial phase II metabolic enzyme that transfers glucuronic acid from UDP-glucuronic acid (UDPGA) to endobiotic and xenobiotic substrates. Biophysical and biochemical investigations of UGT2B7 are hampered by the challenge of the integral membrane protein purification. This study focused on the expression and purification of recombinant UGT2B7 by optimizing the insertion sites for the thermostabilized fusion protein apocytochrome b562RIL (BRIL) and various mutations to improve the protein yields and homogeneity. Preparation of the recombinant proteins with high purity accelerated the measurement of pharmacokinetic parameters of UGT2B7. The dissociation constants (KD) of two classical substrates (zidovudine and androsterone) and two inhibitors (schisanhenol and hesperetin) of UGT2B7 were determined using the surface plasmon resonance spectroscopy (SPR) for the first time. Using negativestaining transmission electron microscopy (TEM), UGT2B7 protein particles were characterized, which could be useful for further exploring its threedimensional structure. The methods described in this study could be broadly applied to other UGTs and are expected to provide the basis for the exploration of metabolic enzyme kinetics, the mechanisms of drug metabolisms and drug interactions, changes in pharmacokinetics, and pharmacodynamics studies in vitro. [ABSTRACT FROM AUTHOR]

In the Asian general population, at least six single‐nucleotide variants (SNVs) in the UDP‐glucuronosyltransferase (UGT) 1A1 gene have been identified: −3279T>G, −53A(TA)6TAA>A(TA)7TAA, 211G>A, 686C>A, 1091C>T, and 1456T>G. Each of these six SNVs was observed in at least four ethnic groups of the 12 Asian populations studied. In East Asian populations, the descending frequency of these six SNVs was as follows: −3279G>[−53A(TA)7TAA, 211A]>(686A, 1091T)>1456G. Because of the presence of linkage disequilibrium and the expulsion phenomenon, when the SNVs −3279G, −53A(TA)7TAA, 211A, and 686A were simultaneously involved, 15 instead of the estimated 81 genotypes were observed. Those carrying 686AA or 1456GG developed Gilbert's syndrome or Crigler–Najjar syndrome type 2. Both −53A(TA)7TAA/A(TA)7TAA and 211AA are the main causes of Gilbert's syndrome in East Asian populations. In East Asian populations, the 211AA genotype is the main cause of neonatal hyperbilirubinemia, whereas −53A(TA)7TAA/A(TA)7TAA exerts a protective effect on hyperbilirubinemia development in neonates fed with breast milk. Both 211A and −53A(TA)7TAA are significantly associated with adverse drug reactions induced by irinotecan (one of the most widely used anticancer agents) in Asians. However, at least three common SNVs (−3279G, −53A(TA)7TAA, and 211A) should be comprehensively analyzed. This study investigated the clinical significance of these six SNVs and demonstrated that examining UGT1A1 variants in Asian populations is considerably challenging. [ABSTRACT FROM AUTHOR]

IPUT1 is a glycosyltransferase capable of synthesizing the glycosyl inositol phosphorylceramide (GIPC) sphingolipid. The GIPC sphingolipid is a Na+ receptor on cell membranes which can sense extracellular Na+ concentrations, promote the increase in intracellular Ca2+ concentrations, and plays critical roles in maintaining intracellular Na+ balance. Therefore, the IPUT1 gene plays an important role in the genetic improvement of crop salt tolerance. Herein, the IbIPUT1 gene, which encodes an ortholog of Arabidopsis AtIPUT1, from sweet potato was cloned. Agrobacterium rhizogenes-mediated in vivo transgenic technology, non-invasive micro-measuring technology (NMT) and Na+ fluorescence imaging technology were then combined to quickly study the potential function of IbIPUT1 in salt tolerance. The data showed that IbIPUT1 was involved in the regulation of root cell Na+ balance, and the overexpression of IbIPUT1 could not promote sweet potato root cell Na+ efflux under salt stress, but it could significantly inhibit the Na+ absorption of root cells, thereby reducing the accumulation of Na+ in root cells under salt stress. Additionally, Ca2+ efflux in transgenic root cells was slightly higher than that in control roots under salt stress. Collectively, an efficient transgenic method for gene function studies was established, and our results suggested that IbIPUT1 acts as a candidate gene for the genetic enhancement of sweet potato salt tolerance. [ABSTRACT FROM AUTHOR]

Hyperbilirubinemia, caused by the accumulation of unconjugated bilirubin, is one of the most common clinical diagnoses in both premature and term newborns. Owing to the fact that bilirubin is metabolized solely through glucuronidation by UDP-glucuronosyltransferase (UGT) 1A1, it is now known that immaturity of UGT1A1, in combination with the overproduction of bilirubin during the developmental stage, acts as a bottleneck to bilirubin elimination and predisposes the infant to high total serum bilirubin levels. Although neonatal jaundice is mostly benign, excessively high levels of serum bilirubin in a small percentage of newborns can cause bilirubin-induced neurologic dysfunction, potentially leading to permanent brain damage, a condition known as kernicterus Although a large portion of hyperbilirubinemia cases in newborns are associated with hemolytic diseases, we emphasize here the impaired ability of UGT1A1 to eliminate bilirubin that contributes to hyperbilirubinemia-induced neurotoxicity in the developmental stage. As a series of hereditary UGT1A1 mutations have been identified that are associated with UGT1A1 deficiency, new evidence has verified that delayed expression of UGT1A1 during the early stages of neonatal development is a tightly controlled event involving coordinated intrahepatic and extrahepatic regulation. This review recapitulates the progress that has been made in recent years in understanding the causes and physiopathology of severe hyperbilirubinemia, investigating molecular mechanisms underlying bilirubin-induced encephalopathy, and searching for potential therapies for treating pathologic hyperbilirubinemia. Several animal models have been developed to make it possible to examine bilirubin-induced neurotoxicity from multiple directions. Moreover, environmental factors that may alleviate or worsen the condition of hyperbilirubinemia are discussed.

Isothiocyanates, such as phenethyl isothiocyanate (PEITC), are formed following the consumption of cruciferous vegetables and generate reactive oxygen species (ROS) that lead to the induction of cytoprotective genes such as the UDP-glucuronosyltransferases (UGTs). The induction of ROS activates the Nrf2-Keap 1 pathway leading to the induction of genes through antioxidant response elements (AREs). UGT1A1, the sole enzyme responsible for the metabolism of bilirubin, can be induced following activation of Nrf2. When neonatal humanized UGT1 (hUGT1) mice, which exhibit severe levels of total serum bilirubin (TSB) because of a developmental delay in expression of the UGT1A1 gene, were treated with PEITC, TSB levels were reduced. Liver and intestinal UGT1A1 were induced, along with murine CYP2B10, a consensus CAR target gene. In both neonatal and adult hUGT1/Car-/- mice, PEITC was unable to induce CYP2B10. A similar result was observed following analysis of UGT1A1 expression in liver. However, TSB levels were still reduced in hUGT1/Car-/- neonatal mice because of ROS induction of intestinal UGT1A1. When oxidative stress was blocked by exposing mice to N-acetylcysteine, induction of liver UGT1A1 and CYP2B10 by PEITC was prevented. Thus, new findings in this report link an important role in CAR activation that is dependent upon oxidative stress.

ObjectivesNicotine metabolism rates differ considerably among individuals, even after controlling for variation in the major nicotine-metabolizing enzyme, CYP2A6. In this study, the impact of genetic variation in alternative metabolic enzymes and transporters on nicotine and cotinine (COT) pharmacokinetics and smoking was investigated.MethodsWe examined the impact of UGT2B10, UGT2B17, FMO3, NAT1, and OCT2 variation on pharmacokinetics and smoking (total nicotine equivalents and topography) before and after stratifying by CYP2A6 genotype in 60 African American (AA) smokers who received a simultaneous intravenous infusion of deuterium-labeled nicotine and COT.ResultsVariants in UGT2B10 and UGT2B17 were associated with urinary glucuronidation ratios (glucuronide/free substrate). UGT2B10 rs116294140 was associated with significant alterations in COT and modest alterations in nicotine pharmacokinetics. These alterations, however, were not sufficient to change nicotine intake or topography. Neither UGT2B10 rs61750900, UGT2B17*2, FMO3 rs2266782, nor NAT1 rs13253389 altered nicotine or COT pharmacokinetics among all individuals (n=60) or among individuals with reduced CYP2A6 activity (n=23). The organic cation transporter OCT2 rs316019 significantly increased nicotine and COT Cmax (P=0.005, 0.02, respectively) and decreased nicotine clearance (P=0.05). UGT2B10 rs116294140 had no significant impact on the plasma or urinary trans-3'-hydroxycotinine/COT ratio, commonly used as a biomarker of CYP2A6 activity.ConclusionWe found that polymorphisms in genes other than CYP2A6 represent minor sources of variation in nicotine pharmacokinetics, insufficient to alter smoking in AAs. The change in COT pharmacokinetics with UGT2B10 rs116294140 highlights the UGT2B10 gene as a source of variability in COT as a biomarker of tobacco exposure among AA smokers.

Severe neonatal hyperbilirubinemia (SNH) and the onset of bilirubin encephalopathy and kernicterus result in part from delayed expression of UDP-glucuronosyltransferase 1A1 (UGT1A1) and the inability to metabolize bilirubin. Although there is a good understanding of the early events after birth that lead to the rapid increase in serum bilirubin, the events that control delayed expression of UGT1A1 during development remain a mystery. Humanized UGT1 (hUGT1) mice develop SNH spontaneously, which is linked to repression of both liver and intestinal UGT1A1. In this study, we report that deletion of intestinal nuclear receptor corepressor 1 (NCoR1) completely diminishes hyperbilirubinemia in hUGT1 neonates because of intestinal UGT1A1 gene derepression. Transcriptomic studies and immunohistochemistry analysis demonstrate that NCoR1 plays a major role in repressing developmental maturation of the intestines. Derepression is marked by accelerated metabolic and oxidative phosphorylation, drug metabolism, fatty acid metabolism, and intestinal maturation, events that are controlled predominantly by H3K27 acetylation. The control of NCoR1 function and derepression is linked to IKKβ function, as validated in hUGT1 mice with targeted deletion of intestinal IKKβ. Physiological events during neonatal development that target activation of an IKKβ/NCoR1 loop in intestinal epithelial cells lead to derepression of genes involved in intestinal maturation and bilirubin detoxification. These findings provide a mechanism of NCoR1 in intestinal homeostasis during development and provide a key link to those events that control developmental repression of UGT1A1 and hyperbilirubinemia.