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Leydig cells (LCs) in the testes produce the male sex hormone testosterone (T). Several xenobiotics, including clinical drugs, supplements, and environmental chemicals, are known to disrupt T homeostasis. Notably, some of these xenobiotics are known to activate the pregnane X receptor (PXR), a ligand-dependent nuclear receptor. However, it is currently unknown whether PXR is expressed in LCs and whether PXR activation alters T synthesis in rodent LCs. Therefore, in this study, we sought to determine whether PXR is expressed in rodent LCs and whether pregnenolone 16-alpha carbonitrile (PCN), the prototype agonist of rodent PXR, regulates T biosynthesis in rodent LCs. Hormonal as well as protein and gene expression analyses were conducted in rat primary LCs and MA-10 mouse Leydig cells. Results showed that PXR was expressed at the mRNA and protein level in both rat primary LCs and MA-10 cells. Incubation of rat primary LCs with PCN resulted in a significant decrease in T secretion. This PCN-induced decrease in T secretion was associated with decreased protein expression of key steroidogenic enzymes such as 3β-HSD and CYP17A1. RNA-seq results from MA-10 cells showed that PCN down-regulated the transcripts of steroidogenic enzymes and proteins involved in the T synthesis pathway. Together, these results suggest that PCN, an agonist of rodent PXR, can regulate T biosynthesis in rodent LCs by down-regulating the expression of the steroidogenic enzymes involved in T biosynthesis. Our results are significant as they provide a potential novel mechanism for disruption of testosterone homeostasis by a variety of xenobiotics. [ABSTRACT FROM AUTHOR]

Leydig cells (LCs) in the testes produce the male sex hormone testosterone (T). Several xenobiotics, including clinical drugs, supplements, and environmental chemicals, are known to disrupt T homeostasis. Notably, some of these xenobiotics are known to activate the pregnane X receptor (PXR), a ligand-dependent nuclear receptor. However, it is currently unknown whether PXR is expressed in LCs and whether PXR activation alters T synthesis in rodent LCs. Therefore, in this study, we sought to determine whether PXR is expressed in rodent LCs and whether pregnenolone 16-alpha carbonitrile (PCN), the prototype agonist of rodent PXR, regulates T biosynthesis in rodent LCs. Hormonal as well as protein and gene expression analyses were conducted in rat primary LCs and MA-10 mouse Leydig cells. Results showed that PXR was expressed at the mRNA and protein level in both rat primary LCs and MA-10 cells. Incubation of rat primary LCs with PCN resulted in a significant decrease in T secretion. This PCN-induced decrease in T secretion was associated with decreased protein expression of key steroidogenic enzymes such as 3β-HSD and CYP17A1. RNA-seq results from MA-10 cells showed that PCN down-regulated the transcripts of steroidogenic enzymes and proteins involved in the T synthesis pathway. Together, these results suggest that PCN, an agonist of rodent PXR, can regulate T biosynthesis in rodent LCs by down-regulating the expression of the steroidogenic enzymes involved in T biosynthesis. Our results are significant as they provide a potential novel mechanism for disruption of testosterone homeostasis by a variety of xenobiotics.

Introduction: As a devastating neurological disease, spinal cord injury (SCI) results in severe tissue loss and neurological dysfunction. Pregnane X receptor (PXR) is a ligand‐activated nuclear receptor with a major regulatory role in xenobiotic and endobiotic metabolism and recently has been implicated in the central nervous system. In the present study, we aimed to investigate the role and mechanism of PXR in SCI. Methods: The clip‐compressive SCI model was performed in male wild‐type C57BL/6 (PXR+/+) and PXR‐knockout (PXR−/−) mice. The N2a H2O2‐induced injury model mimicked the pathological process of SCI in vitro. Pregnenolone 16α‐carbonitrile (PCN), a mouse‐specific PXR agonist, was used to activate PXR in vivo and in vitro. The siRNA was applied to knock down the PXR expression in vitro. Transcriptome sequencing analysis was performed to discover the relevant mechanism, and the NRF2 inhibitor ML385 was used to validate the involvement of PXR in influencing the NRF2/HO‐1 pathway in the SCI process. Results: The expression of PXR decreased after SCI and reached a minimum on the third day. In vivo, PXR knockout significantly improved the motor function of mice after SCI, meanwhile, inhibited apoptosis, inflammation, and oxidative stress induced by SCI. On the contrary, activation of PXR by PCN negatively influenced the recovery of SCI. Mechanistically, transcriptome sequencing analysis revealed that PXR activation downregulated the mRNA level of heme oxygenase‐1 (HO‐1) after SCI. We further verified that PXR deficiency activated the NRF2/HO‐1 pathway and PXR activation inhibited this pathway in vitro. Conclusion: PXR is involved in the recovery of motor function after SCI by regulating NRF2/HO‐1 pathway. [ABSTRACT FROM AUTHOR]

Background and aims: The herbal supplement Gancao, also known as licorice, belongs to the genus Glycyrrhiza and has been used worldwide for its hepatoprotective effect. Recent studies have raised concerns about potential herb-drug interactions associated with Gancao via pregnane X receptor (PXR)-mediated induction of hepatic cytochrome P450 3A4 (CYP3A4). The current work aimed to determine the phytochemicals in Gancao that activate PXR and induce CYP3A4. Methods: DPX2 cells were used for cell-based PXR reporter assays. The phytochemicals in Gancao extract were identified using a metabolomics approach. The effects of PXR activators identified from in vitro studies were further investigated in PXR- and CYP3A4-humanized mouse models. Results: Gancao was verified to be a PXR-activating herb. Two major phytochemicals in Gancao, glycyrrhizin (GZ) and glycyrrhetinic acid (GA), did not activate PXR in the cell-based reporter assays. However, glabridin was shown to activate PXR in a dose-dependent manner. In vivo studies confirmed that GZ is not a PXR activator and glabridin is a weak PXR activator. Although GA did not active PXR in vitro, it induced CYP3A4 expression in a PXR-dependent manner in the PXR- and CYP3A4-humanized mice. Conclusions: GZ is not a PXR activator. GA could not activate PXR in cell-based reporter assays but it could activate PXR in vivo. Glabridin is a weak PXR activator. This work provides novel insights into the underlying mechanisms of Gancao-related herb-drug interactions via PXR.

As a member of the nuclear receptor (NR) superfamily, pregnane X receptor (PXR; NR1I2) is a ligand-activated transcription factor that plays a crucial role in the metabolism of xenobiotics and endobiotics in mammals. The tissue distribution of PXR is parallel to its function with high expression in the liver and small intestine and moderate expression in the kidney, stomach, skin, and blood-brain barrier, which are organs and tissues in frequent contact with xenobiotics. PXR was first recognized as an exogenous substance receptor regulating metabolizing enzymes and transporters and functioning in detoxification and drug metabolism in the liver. However, further research revealed that PXR acts as an equally important endogenous substance receptor in the metabolism and homeostasis of endogenous substances. In this review, we summarized the functions of PXR in metabolism of different substances such as glucose, lipid, bile acid, vitamin, minerals, and endocrines, and also included insights of the application of PXR ligands (drugs) in specific diseases. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease in adults and children worldwide. The symptoms of NAFLD range from simple steatosis and non-alcoholic steatohepatitis (NASH) to hepatic fibrosis or cirrhosis, even ultimately develops to hepatocellular carcinoma. Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors, most of which are ligand-activated that control cellular homeostasis in the liver and other tissues. A growing number of studies demonstrated the important role of NRs in NAFLD. In this review, the current findings on the role of NRs in NAFLD are summarized and future perspectives to target NRs for NAFLD are discussed.

Background Tacrolimus, a drug for prevention of rejection after kidney transplantation, has a narrow therapeutic window and is metabolized by the cytochrome P540 3A (CYP3A) system. Tacrolimus exposure increases after steroid tapering in many patients. The pregnane X receptor (PXR)—a mediator for CYP3A —has a steroid receptor and might regulate CYP3A5 activity depending on single nucleotide polymorphisms (SNPs) of CYP3A5 or PXR. This may contribute to differences in tacrolimus exposure after steroid tapering. Methods In a cohort of renal transplant recipients, the influence of CYP3A5 and PXR SNPs (A7635G, C8055T and C25385T) on the dose-normalized Tacrolimus trough concentration (DnC0) and their potential interaction with each other after steroid taper were analysed by linear regression. Eligible were all 83 outpatient renal transplant patients on tacrolimus and steroids in a pharmacokinetic steady state at least 6 weeks after transplantation and whose blood was available for genetic analysis. Results Compared with the CYP3A5*1/*3 genotype, the CYP3A5*3/*3 SNP showed a significantly stronger increase in DnC0 after steroid taper (+0.29 µg/L/mg; P = 0.002). Of the tested PXR SNPs, PXR G7635G individuals had a significantly stronger increase in DnC0 (compared with A7635A, +0.31 µg/L/mg; P = 0.02), with a weaker increase in A7635G heterozygotes (+0.17 µg/L/mg; P = 0.124). There was neither interaction nor association between CYP3A5 and PXR SNPs. Conclusions The magnitude of the DnC0 increase due to steroid taper after renal transplantation is related to CYP3A5 SNPs. Independently, the PXR G7635G SNP is related to this increase, proving the role of PXR in tacrolimus metabolism. [ABSTRACT FROM AUTHOR]

Pregnane X receptor (PXR) is an important member of the nuclear receptor superfamily that copes with various endobiotic and xenobiotic stimuli, such as carcinogens by regulating an array of environmental response genes. Low PXR expression has been shown to promote tumor initiation and metastasis. The aim of the current study was to investigate whether the single nucleotide polymorphisms (SNPs) of PXR could alter lung cancer susceptibility in Chinese by affecting the function or expression of PXR. We genotyped three putatively functional SNPs of PXR (i.e., rs3814055C>T, rs3732360C>T, and rs3814058C>T) and analyzed their associations with lung cancer risk in a two-stage case-control study with a total of 1559 lung cancer cases and 1679 controls in the southern and eastern Chinese population. We found that in comparison to the rs3814058CC common genotype, the rs3814058T variants (TC/TT) which is located in the 3'-untranslated region (3'-UTR) of PXR conferred a consistently increased risk of lung cancer in both the southern Chinese (odd ratios (OR) = 1.24, 95% confidence interval (CI) = 1.03−1.49) and the eastern Chinese (OR = 1.33, 95% CI = 1.02−1.75). The variants also significantly interacted with smoking on increasing cancer risk (p = 0.023). Moreover, lung cancer tissues with the rs3814058T variants showed significantly lower PXR expression than those with rs3814058CC genotype in the smokers (p = 0.041). These results suggested that the rs3814058C>T polymorphism of PXR interacts with smoking on increasing lung cancer risk in Chinese smokers, which might be a functional genetic biomarker for lung cancer.

In recent years many sterols with unusual structures and promising biological profiles have been identified from marine sources. Here we report the isolation of a series of 24-alkylated-hydroxysteroids from the soft coral Sinularia kavarattiensis, acting as pregnane X receptor (PXR) modulators. Starting from this scaffold a number of derivatives were prepared and evaluated for their ability to activate the PXR by assessing transactivation and quantifying gene expression. Our study reveals that ergost-5-en-3β-ol (4) induces PXR transactivation in HepG2 cells and stimulates the expression of the PXR target gene CYP3A4. To shed light on the molecular basis of the interaction between these ligands and PXR, we investigated, through docking simulations, the binding mechanism of the most potent compound of the series, 4, to the PXR. Our findings provide useful functional and structural information to guide further investigations and drug design.

Vitamin K is a fat-soluble vitamin that was originally found as an essential factor for blood coagulation. With the discovery of its role as a co-factor for γ-glutamyl carboxylase (GGCX), its function for blood coagulation was understood as the activation of several blood coagulation factors by their γ-carboxylation. Over the last two decades, other modes of vitamin K actions have been discovered, such as the regulation of transcription by activating the steroid and xenobiotic receptor (SXR), physical association to 17β-Hydroxysteroid dehydrogenase type 4 (17β-HSD4), covalent modification of Bcl-2 antagonist killer 1 (Bak), and the modulation of protein kinase A (PKA) activity. In addition, several epidemiological studies have revealed that vitamin K status is associated with some aging-related diseases including osteoporosis, osteoarthritis, and sarcopenia. Clinical studies on single nucleotide polymorphisms of GGCX suggested an association between higher GGCX activity and bone protective effect, while recent findings using conditional knockout mice implied that a contribution in protective effect for bone loss by GGCX in osteoblastic lineage was unclear. GGCX in other cell lineages or in other tissues might play a protective role for osteoporosis. Meanwhile, animal experiments by our groups among others revealed that SXR, a putative receptor for vitamin K, could be important in the bone metabolism. In terms of the cartilage protective effect of vitamin K, both GGCX- and SXR-dependent mechanisms have been suggested. In clinical studies on osteoarthritis, the γ-carboxylation of matrix Gla protein (MGP) and gla-rich protein (GRP) may have a protective role for the disease. It is also suggested that SXR signaling has protective role for cartilage by inducing family with sequence similarity 20a (Fam20a) expression in chondrocytes. In the case of sarcopenia, a high vitamin K status in plasma was associated with muscle strength, large muscle mass, and high physical performance in some observational studies. However, the basic studies explaining the effects of vitamin K on muscular tissue are limited. Further research on vitamin K will clarify new biological mechanisms which contribute to human longevity and health through the prevention and treatment of aging-related musculoskeletal disorders.

In the present study we provide evidence that solomonsterol A, a selective pregnane X receptor (PXR) agonist isolated from the marine sponge Theonella swinhoei, exerts anti-inflammatory activity and attenuates systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. Solomonsterol A was effective in protecting against the development of arthritis induced by injecting transgenic mice harboring a humanized PXR, with anti-collagen antibodies (CAIA) with beneficial effects on joint histopathology and local inflammatory response reducing the expression of inflammatory markers (TNFα, IFNγ and IL-17 and chemokines MIP1α and RANTES) in draining lymph nodes. Solomonsterol A rescued mice from systemic inflammation were assessed by measuring arthritis score, CRP and cytokines in the blood. In summary, the present study provides a molecular basis for the regulation of systemic local and systemic immunity by PXR agonists.

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug–drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time. More recent mechanistic studies have shown that PXR is modulated by multiple PTMs. Herein we provide the first investigation of the role of acetylation in modulating PXR activity. Through LC–MS/MS analysis, we identified lysine 109 (K109) in the hinge as PXR's major acetylation site. Using various biochemical and cell-based assays, we show that PXR's acetylation status and transcriptional activity are modulated by E1A binding protein (p300) and sirtuin 1 (SIRT1). Based on analysis of acetylation site mutants, we found that acetylation at K109 represses PXR transcriptional activity. The mechanism involves loss of RXRα dimerization and reduced binding to cognate DNA response elements. This mechanism may represent a promising therapeutic target using modulators of PXR acetylation levels. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. [ABSTRACT FROM AUTHOR]

Highlights: [•] Clotrimazole concentrates in the plasma and liver of common carp exposed via water. [•] The calculated BCF for clotrimazole in the liver was between 144 and 1691. [•] Measurable levels of clotrimazole remained in the liver after 4 days depuration. [•] Clotrimazole exposure induced expression of a number of biotransformation genes. [•] Expression of drug transporter genes was altered at an environmental relevant concentration. [Copyright &y& Elsevier]

Highlights: [•] DINP-exposed toxicity is associated with the overproduction of ROS. [•] Oral exposure to DINP is likely to present hepatic and renal tissue injury. [•] ROS accumulation maybe further causes IL-1 and TNF-α production at increased levels. [ABSTRACT FROM AUTHOR]

Highlights: [•] We isolated cDNAs of AHR2, Sult1 st1, Ugt2a1 and Ugt2b6 in rare minnow. [•] Sexually dimorphic in hepatic transcripts of Cyp1a was found in adult rare minnow. [•] AHR2 and PXR are vital in regulating biotransformation enzymes in MT catabolism. [•] UGT2A1 is more effective than SULT1 ST4 and SULT1 ST6 in MT catabolism in males. [Copyright &y& Elsevier]

Abstract: Introduction: Computational methods have been widely applied to toxicology across pharmaceutical, consumer product and environmental fields over the past decade. Progress in computational toxicology is now reviewed. Methods: A literature review was performed on computational models for hepatotoxicity (e.g. for drug-induced liver injury (DILI)), cardiotoxicity, renal toxicity and genotoxicity. In addition various publications have been highlighted that use machine learning methods. Several computational toxicology model datasets from past publications were used to compare Bayesian and Support Vector Machine (SVM) learning methods. Results: The increasing amounts of data for defined toxicology endpoints have enabled machine learning models that have been increasingly used for predictions. It is shown that across many different models Bayesian and SVM perform similarly based on cross validation data. Discussion: Considerable progress has been made in computational toxicology in a decade in both model development and availability of larger scale or ‘big data’ models. The future efforts in toxicology data generation will likely provide us with hundreds of thousands of compounds that are readily accessible for machine learning models. These models will cover relevant chemistry space for pharmaceutical, consumer product and environmental applications. [Copyright &y& Elsevier]

Highlights: [•] Integrated analysis of multi-omics datasets yields insight into EE liver toxicity. [•] Cholestasis and oxidative stress are involved in EE-induced liver injury. [•] Anti-cholestatic adaptive mechanism is mediated through ABC transporters pathway. [ABSTRACT FROM AUTHOR]

Abstract: In pigs the endogenously produced compound androstenone is metabolised in the liver in two steps by 3β-hydroxysteroid dehydrogenase (3β-HSD) and sulphotransferase 2A1 (SULT2A1). The present study investigated the effect of selected sex-steroids (0.01–1μM androstenone, testosterone and estradiol), skatole (1–100μM) and secondary plant metabolites (1–100μM) on the expression of 3β-HSD and SULT2A1 mRNA. Additionally the effect of a global methanolic extract of dried chicory root was investigated and compared to previous obtained in vivo effects. Primary hepatocytes were isolated from the livers of piglets (crossbreed: Landrace×Yorkshire and Duroc) and cultured for 24h before treatment for an additionally 24h. RNA was isolated from the hepatocytes and specific gene expression determined by RT-PCR using TaqMan probes. The investigated sex-steroids had no effect on the mRNA expression of 3β-HSD and SULT2A1, while skatole decreased the content of SULT2A1 30% compared to control. Of the investigated secondary plant metabolites artemisinin and scoparone (found in Artemisia sp.) lowered the content of SULT2A1 by 20 and 30% compared to control, respectively. Moreover, we tested three secondary plant metabolites (lactucin, esculetin and esculin) found in chicory root. Lactucin increased the mRNA content of both 3β-HSD and SULT2A1 by 200% compared to control. An extract of chicory root was shown to decrease the expression of both 3β-HSD and SULT2A1. It is concluded that the gene expression of enzymes with importance for androstenone metabolism is regulated by secondary plant metabolites in a complex manner. [Copyright &y& Elsevier]

Highlights: [•] We investigated cytotoxicity of enantiopure drugs in cancer cell lines. [•] We examined effects of enantiopure drugs on AhR, GR and PXR receptors. [•] We measured the effects of enantiopure drugs on expression of CYP3A4. [•] Tolterodine, tamsulosin, modafinil, citalopram and zopiclone were tested. [•] No significant differences between effects of enantiomers were observed. [ABSTRACT FROM AUTHOR]

In the present study we provide evidence that solomonsterol A, a selective pregnane X receptor (PXR) agonist isolated from the marine sponge Theonella swinhoei, exerts anti-inflammatory activity and attenuates systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. Solomonsterol A was effective in protecting against the development of arthritis induced by injecting transgenic mice harboring a humanized PXR, with anti-collagen antibodies (CAIA) with beneficial effects on joint histopathology and local inflammatory response reducing the expression of inflammatory markers (TNFa, IFN? and IL-17 and chemokines MIP1a and RANTES) in draining lymph nodes. Solomonsterol A rescued mice from systemic inflammation were assessed by measuring arthritis score, CRP and cytokines in the blood. In summary, the present study provides a molecular basis for the regulation of systemic local and systemic immunity by PXR agonists. [ABSTRACT FROM AUTHOR]

Highlights: [•] Full-length pxr has been cloned from zebrafish. [•] Alleles of pxr were identified in zebrafish. [•] Full length Pxr was activated less strongly than ligand binding domain in cell-based reporter assays. [•] High levels of pxr expression were found in eye and brain as well as in liver. [•] TCPOBOP and PB did not significantly alter expression of pxr in liver. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Tissue samples are routinely formalin-fixed and paraffin-embedded (FFPE) for long term preservation. Gene expression analysis of archival FFPE tissues may advance knowledge of the molecular perturbations contributing to disease. However, formalin causes extensive degradation of RNA. Methods: We compared RNA quality/yield from FFPE samples using six commercial FFPE RNA extraction kits. In addition we compared four DNA microarray protocols for the Agilent 8×60K platform using 16year old FFPE mouse liver samples treated with phenobarbital or vehicle. Results: Despite low quality RNA, archival phenobarbital samples exhibited strong induction of the positive control genes Cyp2b9 and Cyp2b10 by quantitative real-time PCR (qPCR). We tested one- and two-color microarray designs and evaluated the effects of increasing the amount of hybridized cDNA. Canonical gene responders to phenobarbital were measurably induced under each experimental condition. Increasing the amount of labeled cDNA did not improve the overall signal intensity. One-color experiments yielded larger fold changes than two-color and the number of differentially expressed genes varied between protocols. Gene expression changes were validated by qPCR and literature searches. Individual protocols exhibited high rates of false positives; however, pathway analysis revealed that nine of the top ten canonical pathways were consistent across experiments. Genes that were differentially expressed in more than one experiment were more likely to be validated. Thus, we recommend that experiments on FFPE samples be done in duplicate to reduce false positives. Discussion: In this analysis of archival FFPE samples we were able to identify pathways that are consistent with phenobarbital's mechanism of action. Therefore, we conclude that FFPE samples can be used for meaningful microarray gene expression analyses. [Copyright &y& Elsevier]

Abstract: Background: Variability in MDR1 and PXR has been associated with differences in drug plasma levels and response to antiretroviral therapy. We investigated whether polymorphisms in MDR1 (T-129C, C1236T and C3435T) and PXR (C63396T) affect lopinavir plasma concentration and the virological or immunological response to HAART in HIV-1-infected children. Methods: Genotypes were identified in 100 blood donors and 38 HIV-1-infected children. All children received HAART with lopinavir boosted with ritonavir (LPV/r) at the time of LPV plasma level quantification, before (Ctrough) and between 1 and 2h after (Cpost-dose) the administration of the next dose of drug. CD4+ T-cell counts and plasma viral load were analyzed before and after the initiation of LPV/r. Results: MDR1 1236T, MDR1 3435T and PXR 63396T alleles showed a frequency of ~50% while the MDR1 -129C allele only reached 5%. Children heterozygotes 1236CT showed a significantly lower LPV Cpost-dose than homozygotes 1236TT (median Cpost-dose =3.04μg/ml and 6.50μg/ml, respectively; p =0.016). Children heterozygotes 1236CT also had a lower decrease of viral load after 36weeks of LPV/r exposure compared with homozygotes 1236CC (median viral load changes=−0.50log10 copies/ml and −2.08log10 copies/ml, respectively; p =0.047). No effect on the immunological response was observed for polymorphisms of MDR1 or PXR. Conclusions: Our results suggest that the MDR1 C1236T SNP significantly reduces LPV plasma concentration affecting the virological response to HAART. Heterozygotes 1236CT might have an altered level of P-gp expression/activity in enterocytes and CD4+ T lymphocytes that limits the absorption of LPV leading to an impaired virological suppression. [Copyright &y& Elsevier]

As a member of the nuclear receptor (NR) superfamily, pregnane X receptor (PXR; NR1I2) is a ligand-activated transcription factor that plays a crucial role in the metabolism of xenobiotics and endobiotics in mammals. The tissue distribution of PXR is parallel to its function with high expression in the liver and small intestine and moderate expression in the kidney, stomach, skin, and blood-brain barrier, which are organs and tissues in frequent contact with xenobiotics. PXR was first recognized as an exogenous substance receptor regulating metabolizing enzymes and transporters and functioning in detoxification and drug metabolism in the liver. However, further research revealed that PXR acts as an equally important endogenous substance receptor in the metabolism and homeostasis of endogenous substances. In this review, we summarized the functions of PXR in metabolism of different substances such as glucose, lipid, bile acid, vitamin, minerals, and endocrines, and also included insights of the application of PXR ligands (drugs) in specific diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lv, Luo, Ren, Li, Xiang and Luan.)

Abstract: Perfluorooctanoic acid (PFOA), a potentially toxic perfluorinated compound (PFC), has been widely disseminated in the environment. In the present study, rare minnows (Gobiocypris rarus) exposed to PFOA exhibited histopathological gill damage, including epithelial hyperplasia of the lamellae, inflammatory cell infiltration, and lamellar fusion. Cytochrome P450s (CYPs) play a central role in the metabolism and biotransformation of a wide range of endogenous substrates and foreign compounds. Thus, we studied the CYPs and the effects of waterborne PFOA on their corresponding mRNA levels in the gills of rare minnows. Two novel CYP cDNAs (CYP1A and CYP3A) were identified in rare minnow and their mRNAs were ubiquitously expressed in all tissues examined. Upregulation of CYP3A mRNA was observed in the gills of male rare minnows exposed to 30mg/L PFOA, while no significant changes occurred in exposed females. In contrast, down regulation of CYP1A mRNA was detected in the gills of male and female minnows exposed to PFOA. However, the effect of PFOA on gill mRNA levels of their potential regulators, aryl hydrocarbon receptor (AhR) for CYP1A, and pregnane X receptor (PXR) for CYP3A, were not consistent with the observed effects of PFOA on the corresponding CYP mRNA concentrations. This suggests a different or more complex transcriptional regulation of CYP expression following PFOA exposure. [Copyright &y& Elsevier]

Abstract: The CYP3A gene cluster consists of four members, CYP3A4, CYP3A5, CYP3A7 and CYP3A43. Especially the CYP3A4 and CYP3A5 enzymes play a significant role in the metabolism of numerous exogenous (drugs, pollutants, procarcinogens) and endogenous (steroids, bile acids) compounds. CYP3A5 protein is present in the liver and some extrahepatic tissues, such as the gut wall, kidney, adrenal gland, prostate and many cell types in the lung. In the lung, the highest amounts of CYP3A5 protein are present in bronchial and alveolar epithelial cells, bronchial glands and alveolar macrophages. The same cells types have little or no CYP3A4 expression. Cigarette smoking markedly represses CYP3A5 content in alveolar macrophages. CYP3A5 is upregulated by glucocorticoids via the glucocorticoid receptor (GR) in lung adenocarcinoma derived A549 cells. Tissue selective distribution of CYP3A4 is controlled by tissue enriched transcription factors, such as hepatic nuclear factor 4α (HNF4α), and ligand dependent nuclear receptors, most notably pregnane X receptor (PXR) and constitutive androstane receptor (CAR). The selective expression of CYP3A5 over CYP3A4 in specific lung cells is likely to be the sum of the effects of tissue-specific upregulating and downregulating transcription factors in these cells. Since the CYP3A4/5 enzymes mediate the metabolism of many exogenous and endogenous compounds with direct relevance to pulmonary physiology and pathology, the functions of these enzymes and factors controlling them should be elucidated in much more detail. [Copyright &y& Elsevier]

Alkylphenols are continuously released into the ocean as a result of offshore oil production. Alkylphenols, including 4-tert-butylphenol (C4), 4n-pentylphenol (C5), 4n-hexylphenol (C6), and 4n-heptylphenol (C7), up to 237 ppb concentrations, have been detected in produced water from oil platforms. Previous studies have shown that alkylphenols induce vitellogenesis in fish. Atlantic cod (Gadus morhua) of both sexes were force-fed with various doses ranging between 0.02 and 80 ppm of a mixture of alkylphenols (C4:C5:C6:C7 ratio 1:1:1:1) or 5 ppm 17β-estradiol. We investigated effects on hepatic CYP1A and CYP3A protein expression in protein blots, using antibodies against scup (Stenotomus chrysops) CYP1A1 and rainbow trout (Oncorhynchus mykiss) CYP3A. There was a sexually dimorphic expression of CYP1A and CYP3A protein levels, with females expressing higher levels than males. Treatment of male Atlantic cod with 17β-estradiol resulted in increased CYP1A and CYP3A protein levels. Exposure to alkylphenols resulted in a dose-dependent increase of CYP1A and CYP3A protein expression in males, but not in females. However, this increase of CYP1A protein levels was not reflected on the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activity, implying that alkylphenols inhibited the CYP1A enzyme activity in vivo. In vitro inhibition studies with pooled liver microsomes from Atlantic cod confirmed that the alkylphenols mixture efficiently inhibited the CYP1A activity (IC50=10 μM), although the inhibitory effect of each individual alkylphenol varied. The IC50 values for each individual alkylphenol on the CYP1A activity were, in a descending order of magnitude: [C7>C6>C5≫C4], ranging from 12 to 300 μM with decreased length of the 4-alkyl chain. The effect of alkylphenols on the CYP3A activity in vitro in liver microsomes also was investigated, using the fluorescent 7-benzyloxy-4-[trifluoromethyl]-coumarin (BFC) as a diagnostic CYP3A substrate. The alkylphenol mixture inhibited CYP3A activity with IC50 value at 100 μM. The IC50 values for each individual alkylphenol on CYP3A activity were, in a descending order of magnitude: [C5>C6>C7>C4], ranging between 60 and 250 μM. Taken together, our results show that the alkylphenol mixture and 17β-estradiol resulted in elevated hepatic CYP1A and CYP3A expression in male Atlantic cod. The alkylphenol mixture strongly inhibited CYP1A activities, whereas it weakly inhibited CYP3A activity in Atlantic cod liver microsomes in vitro. In addition, 17β-estradiol was a weak inhibitor of CYP3A activity (IC50=75 μM) and did not notably inhibit the CYP1A activity in vitro. [Copyright &y& Elsevier]

Background/Aims: Estrogen-mediated cholestasis is an important clinical entity, but its molecular pathophysiology is still not fully understood. Impaired sodium-dependent uptake of bile acids has been associated with diminished expression of a basolateral Na+/bile acid cotransporter (Ntcp), whereas sodium-independent uptake is maintained despite a down-regulation of the organic anion transporter Oatp1. Thus, expression of the two other rat Oatps (Oatps2 and -4) was determined in estrogen-induced cholestasis. In addition, known transactivators of Oatp2 and Ntcp were studied to further characterize transcriptional regulation of these transporter genes. Methods: Hepatic protein and mRNA expression of various Oatps (1, 2, 4) in comparison to Ntcp were analyzed after 0.5, 1, 3 and 5 days of ethinylestradiol (EE) treatment (5 mg/kg) in rats. Binding activities of Oatp2 and Ntcp transactivators were assessed by electrophoretic mobility shift assays. Results: All basolateral Oatps (1, 2 and 4) were specifically down-regulated at the protein level by 30–40% of controls, but less pronounced than Ntcp (minus 70–80%). In contrast to unaltered Oatp4 mRNA levels, Oatp1 and Oatp2 mRNAs were reduced to various extents (minus 40–90% of controls). Binding activity of known transactivators of Ntcp and Oatp2 such as hepatocyte nuclear factor 1 (HNF1), CAAT enhancer binding protein α (C/EBPα) and pregnane X receptor (PXR) were also diminished during the time of cholestasis. Conclusions: Estrogen-induced cholestasis results in a down-regulation of all basolateral organic anion transporters. The moderate decline in expression of Oatp1, -2 and -4 may explain the unchanged sodium-independent transport of bile acids due to overlapping substrate specificity. Reduction in transporter gene expression seems to be mediated by a diminished nuclear binding activity of transactivators such as HNF1, C/EBP and PXR by estrogens. [Copyright &y& Elsevier]

Introduction: Enzyme induction is undesirable in new drug discovery process, with consequences spanning from auto-induction to toxicity. Cytochrome P450 (CYP) 1A1 has long been known to be one of the metabolic enzymes involved in activating many procarcinogens, the first step toward tumor formation during chemical carcinogenesis. Induction of CYP1A1 during drug treatment may predispose the patients to some risk of chemical carcinogenesis. Methods: Based on the signal-transduction mechanism of CYP1A1 induction, a high-throughput reporter-gene system was established by stable transformation of H4IIE cells to incorporate the luciferase gene under control of CYP1A1 promoter. This stable cell line was validated with known CYP1A1 inducers, such as 3-methylcholanthrene (3-MC), β-naphthoflavone (β-NF), α-naphthoflavone (α-NF) and 3-indocarbinol. Thirty in-house new chemical entities (NCEs) were then screened with this reporter-gene system, and also administered to rats to evaluate in vivo CYP1A1 induction. Results: CYP1A1 reporter gene system can be used to identify strong inducers, such as 3-MC, β-NF and α-NF, and weak inducers, such as 3-indocarbinol. In vitro induction of 30 in-house compounds in reporter gene system did not correlate with in vivo induction in rat liver microsome measured by ethoxyresorufin-O-dealkylation (EROD) activity, but had a reasonable correlation with Western blot signals. Discussion: This reporter-gene system may be useful in eliminating compounds that can cause CYP1A1 induction at an early stage of drug discovery. [Copyright &y& Elsevier]

Background: Aminophylline is widely used for the treatment of asthma, but the therapeutic dose is very close to the toxic dose, which makes this drug prone to accumulation poisoning. In the present study, we explored whether the Chinese herbal component, Praeruptorin E (PE), enhances anti-asthma efficacy and prevents the toxicity of aminophylline., Methods: First, an ovalbumin (OVA)-induced mouse model of asthma, immunohistochemistry, pathological staining, and bronchoalveolar lavage fluid (BALF) were used to detect the lung condition of asthmatic mice. The content of Th2 cytokines in serum was measured by enzyme-linked immunosorbent assay (ELISA), and the expression of related proteins was detected by Western blotting and immunofluorescence. Concentrations of theophylline and its metabolites in rat serum were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). siRNA transfection and chromatin immunoprecipitation (ChIP) were used to investigate the mechanism of PE., Results: PE was found to synergize with aminophylline to reduce the infiltration of inflammatory cells, collagen deposition, and mucus hyperplasia in the lungs of asthmatic mice. It inhibited the expression of Th2 cytokines, interleukin (IL)-4, IL-5, and IL-13; promoted lung tissue repair; and reduced the toxic effect of aminophylline on the heart. Moreover, LC-MS/MS analysis showed that PE reduced the plasma concentration of the parent theophylline and its metabolite 1,3-dimethyluric acid (1,3-DMU). PE facilitated aminophylline's suppression of nuclear factor-κB ( NF-κB ), and increased the expression of the xenobiotic nuclear receptor pregnane X receptor ( PXR ) and its primary target gene, CYP3A11 [this is the mouse homolog of cytochrome P450 3A ( CYP3A )] in the asthmatic mouse liver and in the L-02 human fetal hepatocyte cell culture model. In addition, the ChIP assay revealed that PE attenuated the binding of NF-κB to the promoter region of the PXR gene and prevented the suppression of PXR gene expression by NF-κB ., Conclusions: PE has a dual function in enhancing the immune regulation and anti-inflammatory effects of theophylline, as well as preventing theophylline toxicity by targeting the NF-κB/PXR/CYP3A4 axis. PE is a promising herbal medicine that will benefit asthmatics taking theophylline., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-386/coif). The authors have no conflicts of interest to declare., (2022 Annals of Translational Medicine. All rights reserved.)

• First to study xenobiotic genes polymorphisms and CBZ response in epilepsy patients. • CAR rs2502815 may be related to CBZ responsiveness in patients with epilepsy. • Patients with GG genotype of rs2502815 are less likely to develop CBZ resistance. Drug-resistant epilepsy is a problem worldwide. Xenobiotic receptors may play a significant role in the establishment of resistance to antiepileptic agents. Previous studies have confirmed that the metabolism and efficacy of carbamazepine (CBZ) can be influenced by xenobiotic receptors, especially pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AHR). Therefore, this study intends to elucidate the pharmacogenomic associations of polymorphisms of these xenobiotic receptors with the CBZ response in epilepsy patients, and these genetic data may be useful for the treatment of clinical prophylaxis and individualized treatment of intractable epilepsy. Adult patients with epilepsy who were on CBZ-based monotherapy and combination therapy (n = 257) were genotyped, and the patients were divided into drug-responsive and drug-resistant groups according to the International League Against Epilepsy criteria. We sought to tag single-nucleotide polymorphisms (SNPs) of PXR , CAR and AHR that principally represent alleles associated with drug resistance risk; in addition, a gene interaction analysis reference panel was constructed for SNP-based imputation. No significant effects of PXR or AHR polymorphisms were observed. However, an interaction between the CAR rs2502815 variant and CBZ response was observed: in CBZ-based monotherapy and combination therapy patients, the GG genotype of the CAR rs2502815 variant (vs. wild-type homozygous) was independently associated with CBZ response after adjusting for variables [odds ratio (OR) = 0.389, 95% confidence interval (CI) 0.203–0.743, p = 0.004]. The results of the haplotype and gene interaction case-control analyses of the CBZ response were negative. Our results provide clinical data regarding the genetic possibilities of drug responses related to CAR variation in epilepsy patients. This study is the first to indicate a potentially relevant interaction between the CAR rs2502815 polymorphism and the CBZ response in epilepsy patients. [ABSTRACT FROM AUTHOR]

Garcinoic acid (GA or δ-T3-13'COOH), is a natural vitamin E metabolite that has preliminarily been identified as a modulator of nuclear receptors involved in β-amyloid (Aβ) metabolism and progression of Alzheimer's disease (AD). In this study, we investigated GA's effects on Aβ oligomer formation and deposition. Specifically, we compared them with those of other vitamin E analogs and the soy isoflavone genistein, a natural agonist of peroxisome proliferator-activated receptor γ (PPARγ) that has therapeutic potential for managing AD. GA significantly reduced Aβ aggregation and accumulation in mouse cortical astrocytes. Similarly to genistein, GA up-regulated PPARγ expression and apolipoprotein E (ApoE) efflux in these cells with an efficacy that was comparable with that of its metabolic precursor δ-tocotrienol and higher than those of α-tocopherol metabolites. Unlike for genistein and the other vitamin E compounds, the GA-induced restoration of ApoE efflux was not affected by pharmacological inhibition of PPARγ activity, and specific activation of pregnane X receptor (PXR) was observed together with ApoE and multidrug resistance protein 1 (MDR1) membrane transporter up-regulation in both the mouse astrocytes and brain tissue. These effects of GA were associated with reduced Aβ deposition in the brain of TgCRND8 mice, a transgenic AD model. In conclusion, GA holds potential for preventing Aβ oligomerization and deposition in the brain. The mechanistic aspects of GA's properties appear to be distinct from those of other vitamin E metabolites and of genistein., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest., (© 2020 Marinelli et al.)

Vitamin K is a fat-soluble vitamin that was originally found as an essential factor for blood coagulation. With the discovery of its role as a co-factor for γ-glutamyl carboxylase (GGCX), its function for blood coagulation was understood as the activation of several blood coagulation factors by their γ-carboxylation. Over the last two decades, other modes of vitamin K actions have been discovered, such as the regulation of transcription by activating the steroid and xenobiotic receptor (SXR), physical association to 17β-Hydroxysteroid dehydrogenase type 4 (17β-HSD4), covalent modification of Bcl-2 antagonist killer 1 (Bak), and the modulation of protein kinase A (PKA) activity. In addition, several epidemiological studies have revealed that vitamin K status is associated with some aging-related diseases including osteoporosis, osteoarthritis, and sarcopenia. Clinical studies on single nucleotide polymorphisms of GGCX suggested an association between higher GGCX activity and bone protective effect, while recent findings using conditional knockout mice implied that a contribution in protective effect for bone loss by GGCX in osteoblastic lineage was unclear. GGCX in other cell lineages or in other tissues might play a protective role for osteoporosis. Meanwhile, animal experiments by our groups among others revealed that SXR, a putative receptor for vitamin K, could be important in the bone metabolism. In terms of the cartilage protective effect of vitamin K, both GGCX- and SXR-dependent mechanisms have been suggested. In clinical studies on osteoarthritis, the γ-carboxylation of matrix Gla protein (MGP) and gla-rich protein (GRP) may have a protective role for the disease. It is also suggested that SXR signaling has protective role for cartilage by inducing family with sequence similarity 20a (Fam20a) expression in chondrocytes. In the case of sarcopenia, a high vitamin K status in plasma was associated with muscle strength, large muscle mass, and high physical performance in some observational studies. However, the basic studies explaining the effects of vitamin K on muscular tissue are limited. Further research on vitamin K will clarify new biological mechanisms which contribute to human longevity and health through the prevention and treatment of aging-related musculoskeletal disorders. [ABSTRACT FROM AUTHOR]

Despite the major advances in developing diagnostic techniques and effective treatments, atherosclerotic cardiovascular disease (CVD) is still the leading cause of mortality and morbidity worldwide. While considerable progress has been achieved to identify gene variations and environmental factors that contribute to CVD, much less is known about the role of "gene-environment interactions" in predisposing individuals to CVD. Our chemical environment has significantly changed in the last few decades, and there are more than 100,000 synthetic chemicals in the market. Recent large-scale human population studies have associated exposure to certain chemicals including many endocrine disrupting chemicals (EDCs) with increased CVD risk, and animal studies have also confirmed that some EDCs can cause aberrant lipid homeostasis and increase atherosclerosis. However, the underlying mechanisms of how exposure to those EDCs influences CVD risk remain elusive. Numerous EDCs can activate the nuclear receptor pregnane X receptor (PXR) that functions as a xenobiotic sensor to regulate host xenobiotic metabolism. Recent studies have demonstrated the novel functions of PXR in lipid homeostasis and atherosclerosis. In addition to directly regulating transcription, PXR has been implicated in the epigenetic regulation of gene transcription. Exposure to many EDCs can also induce epigenetic modifications, but little is known about how the changes relate to the onset or progression of CVD. In this review, we will discuss recent research on PXR and EDCs in the context of CVD and propose that PXR may play a previously unrealized role in EDC-mediated epigenetic modifications that affect lipid homeostasis and atherosclerosis., Competing Interests: Conflict of interest statement None declared.

Background & Aims: Rifaximin is used to treat patients with functional gastrointestinal disorders, but little is known about its therapeutic mechanism. We propose that rifaximin modulates the ileal bacterial community, reduces subclinical inflammation of the intestinal mucosa, and improves gut barrier function to reduce visceral hypersensitivity. Methods: We induced visceral hyperalgesia in rats, via chronic water avoidance or repeat restraint stressors, and investigated whether rifaximin altered the gut microbiota, prevented intestinal inflammation, and improved gut barrier function. Quantitative polymerase chain reaction (PCR) and 454 pyrosequencing were used to analyze bacterial 16S ribosomal RNA in ileal contents from the rats. Reverse transcription, immunoblot, and histologic analyses were used to evaluate levels of cytokines, the tight junction protein occludin, and mucosal inflammation, respectively. Intestinal permeability and rectal sensitivity were measured. Results: Water avoidance and repeat restraint stress each led to visceral hyperalgesia, accompanied by mucosal inflammation and impaired mucosal barrier function. Oral rifaximin altered the composition of bacterial communities in the ileum (Lactobacillus species became the most abundant) and prevented mucosal inflammation, impairment to intestinal barrier function, and visceral hyperalgesia in response to chronic stress. Neomycin also changed the composition of the ileal bacterial community (Proteobacteria became the most abundant species). Neomycin did not prevent intestinal inflammation or induction of visceral hyperalgesia induced by water avoidance stress. Conclusions: Rifaximin alters the bacterial population in the ileum of rats, leading to a relative abundance of Lactobacillus. These changes prevent intestinal abnormalities and visceral hyperalgesia in response to chronic psychological stress. [Copyright &y& Elsevier]

The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90β (Hsp90β) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90β at the Ser-225/254 residues. Phosphorylated Hsp90β then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90β enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)