Panaxytriol upregulates CYP3A4 expression through the interaction between nuclear regulators and DNA response elements.

ETHNOPHARMACOLOGICAL RELEVANCE: Cytochrome P3A4 (CYP3A4) is a crucial drug-metabolizing enzyme, and its expression is regulated by the pregnane X receptor (PXR), constitutive androstane receptor (CAR), steroid receptor coactivator 1 (SRC-1), and acetyltransferase P300. Panaxytriol is a naturally derived active substance extracted from the roots of Panax ginseng C. A. Mey. which is widely used clinically. Our previous studies have shown that panaxytriol induces CYP3A4 expression through PXR activation, which is antagonized by high CAR expression. However, the underlying mechanism remains unclear. AIM OF THE STUDY: This study aimed to investigate the mechanism of panaxytriol in inducing CYP3A4 expression via interactions between nuclear regulators and DNA response elements. MATERIALS AND METHODS: Immunoprecipitation technique was used to assess the binding levels of PXR and CAR with the coactivators SRC-1 and P300 in HepG2 and Huh-7 cells. Furthermore, chromatin immunoprecipitation assay was used to investigate the PXR and CAR interaction with the CYP3A4 promoter response element ER-6/DR-3. RESULTS: The binding of PXR to SRC-1, P300, and the response elements ER-6 and DR-3 was improved with an increase in panaxytriol concentration (10-80 µM), and the binding affinity was further enhanced upon CAR silencing. The binding of CAR to SRC-1 and the response elements ER-6 and DR-3 was significantly higher at 80 µM panaxytriol, whereas no significant binding was observed between CAR and P300. CONCLUSION: Panaxytriol promoted the recruitment of PXR to SRC-1 and P300, binding to ER-6 and DR-3, and upregulating CYP3A4 expression. Furthermore, an interactive dialogue regulatory mechanism between PXR and CAR was observed.

Screening of medicinal plants for possible herb-drug interactions through modulating nuclear receptors, drug-metabolizing enzymes and transporters.

ETHNOPHARMACOLOGICAL RELEVANCE: The last three decades have witnessed a surge in popularity and consumption of herbal products. An unintended consequence of such popularity is that chronic consumption of these products can often modulate the functions of various proteins involved in drug disposition and may, in turn, impose risks for herb-drug interactions (HDIs), leading to serious adverse health outcomes. Identifying plants that may give rise to clinically relevant HDIs is essential, and proactive dissemination of such research outcomes is necessary for researchers, clinicians, and average consumers. AIM OF THE STUDY: The main objective of this study was to evaluate the HDI potential of plants commonly used as ingredients in many herbal products, including BDS. MATERIALS AND METHODS: The dried material of 123 plants selected from the NCNPR repository was extracted with 95% ethanol. The extracts were screened for agonistic effects on nuclear receptors (PXR and AhR) by reporter gene assays in PXR-transfected HepG2 and AhR-reporter cells. For cytochrome P450 enzyme (CYP) inhibition studies, CYP450 baculosomes were incubated with enzyme-specific probe substrates by varying concentrations of extracts. The inhibitory effect on the efflux transporter P-glycoprotein (P-gp) was investigated via rhodamine (Rh-123) uptake assay in P-gp overexpressing MDR1-MDCK cells. RESULTS: Out of 123 plants, 16 increased transcriptional activity of human PXR up to 4 to 7-fold at 60 µg/mL, while 18 plants were able to increase AhR activity up to 10 to 40-fold at 30 µg/mL. Thirteen plants inhibited the activity of CYP3A4, while 10 plants inhibited CYP1A2 activity with IC50 values in the range of 1.3-10 µg/mL. Eighteen plants (at 50 µg/mL) increased intracellular accumulation of Rh-123 (>150%) in MDR1-MDCK cells. Additionally, other plants tested in this study were able to activate PXR, AhR, or both to lesser extents, and several inhibited the catalytic activity of CYPs at higher concentrations (IC50 >10 µg/mL). CONCLUSIONS: The results indicate that prolonged or excessive consumption of herbal preparations rich in such plants (presented in Figs. 1a, 2a, 3a, 4a, and 5a) may pose a risk for CYP- and P-gp-mediated HDIs, leading to unwanted side effects due to the altered pharmacokinetics of concomitantly ingested medications.

Functional association of NR4A3 downregulation with impaired differentiation in myeloid leukemogenesis.

The coincident downregulation of NR4A1 and NR4A3 has been implicated in myeloid leukemogenesis, but it remains unknown how these two genes function in myeloid cells and how their combined downregulation promotes myeloid leukemogenesis. Since NR4A1 abrogation is thought to confer a survival and proliferation advantage to myeloid cells, we hypothesized that downregulation of NR4A3 may have a complementary effect on myeloid cell differentiation. First, we tested the association between differentiation status of leukemic cells and NR4A3 expression using two large clinical datasets from patients with different acute myeloid leukemia (AML) subtypes. The analysis revealed a close association between differentiation status and different subtypes of AML Then, we probed the effects of differentiation-inducing treatments on NR4A3 expression and NR4A3 knockdown on cell differentiation using two myeloid leukemia cell lines. Differentiation-inducing treatments caused upregulation of NR4A3, while NR4A3 knockdown prevented differentiation in both cell lines. The cell culture findings were validated using samples from chronic myeloid leukemia (CML) patients at chronic, accelerated and blastic phases, and in acute promyelocytic leukemia (APL) patients before and after all trans-retinoic acid (ATRA)-based differentiation therapy. Progressive NR4A3 downregulation was coincident with impairments in differentiation in patients during progression to blastic phase of CML, and NR4A3 expression was increased in APL patients treated with ATRA-based differentiating therapy. Together, our findings demonstrate a tight association between impaired differentiation status and NR4A3 downregulation in myeloid leukemias, providing a plausible mechanistic explanation of how myeloid leukemogenesis might occur upon concurrent downregulation of NR4A1 and NR4A3.

Panaxytriol upregulates CYP3A4 expression based on the interaction of PXR, CAR, HSP90α, and RXRα.

BACKGROUND: Cytochrome P450 3A4 (CYP3A4) is one of the most important drug-metabolizing enzymes in the human body, mainly existing in the liver, small intestine, and kidney. Panaxytriol is one of the key active components in red ginseng and Shenmai injection. Our previous study demonstrated that panaxytriol regulates CYP3A4 expression mainly by activating pregnancy X receptor (PXR). At a high concentration of panaxytriol (80 µM), the constitutive androstane receptor (CAR) is also involved in the upregulation of CYP3A4. PURPOSE: This study investigated how the cofactors heat shock protein 90 alpha (HSP90α) and retinoid X receptor alpha (RXRα) interact with PXR and CAR to participate in the regulation of CYP3A4 by panaxytriol from the perspective of the PXR and CAR interaction. METHODS: The mRNA and protein expressions of PXR, CAR, CYP3A4, RXRα, and HSP90α in HepG2 cells and Huh-7 cells were detected by quantitative PCR and western blot analysis, respectively. The binding levels of PXR and CAR to RXRα and HSP90α were determined by co-immunoprecipitation analysis. The nuclear translocation of PXR and RXRα into HepG2 cells and human (hCAR)-silenced HepG2 cells were measured by immunofluorescence. RESULTS: In HepG2 cells and Huh-7 cells, panaxytriol (10-80 µM) upregulated CYP3A4 expression in a concentration-dependent manner by decreasing PXR binding to HSP90α and increasing PXR binding to RXRα. When hCAR was silenced, panaxytriol further enhanced CYP3A4 expression by strengthening PXR binding to RXRα, but it had no significant effect on the binding level of PXR and HSP90α. Additionally, at the high concentration of 80 µM panaxytriol, CAR binding to HSP90α was weakened while binding to RXRα was enhanced. CONCLUSION: Panaxytriol can upregulate CYP3A4 expression by promoting PXR dissociation from HSP90α and enhancing PXR binding to RXRα in HepG2 cells and Huh-7 cells. At high concentrations of panaxytriol, CAR also participates in the induction of CYP3A4 through a similar mechanism. However, in general, CAR antagonizes PXR binding to RXRα, thereby attenuating the upregulation of CYP3A4 by panaxytriol.

Differential Effects of Components in Artemisia annua Extract on the Induction of Drug-Metabolizing Enzyme Expression Mediated by Nuclear Receptors.

Artemisia annua tea is a popular dosage form used to treat and prevent malaria in some developing countries. However, repeated drinking leads to an obviously decreased efficacy, which may be related to the induction of metabolizing enzymes by artemisinin. In the present study, the ability of different components in A. annua to activate the pregnane X receptor and constitutive androstane receptor was evaluated by the dual luciferase reporter gene system. The changes in mRNA and protein expression of CYP3A4 and CYP2B6 were determined by quantitative real-time PCR and Western blotting. Results showed that in the pregnane X receptor-mediated CYP3A4 reporter gene system, chrysosplenetin and arteannuin B exhibited a weak induction effect on pregnane X receptor wt, while arteannuin A had a strong induction effect on pregnane X receptor wt and pregnane X receptor 370 and a weak induction effect on pregnane X receptor 163. In the pregnane X receptor-mediated CYP2B6 reporter gene system, arteannuin A had a moderate induction effect on pregnane X receptor wt and pregnane X receptor 379, and a weak induction effect on pregnane X receptor 403, while arteannuin B had a weak induction effect on pregnane X receptor wt and pregnane X receptor 379. Arteannuin A had a strong induction effect on constitutive androstane receptor 3 in constitutive androstane receptor-mediated CYP3A4/2B6 reporter gene systems, while arteannuin B showed a weak induction effect on constitutive androstane receptor 3 in the constitutive androstane receptor-mediated CYP2B6 reporter gene system. The mRNA and protein expressions of CYP3A4 and CYP2B6 were increased when the pregnane X receptor or constitutive androstane receptor was activated. Various components present in A. annua differentially affect the activities of pregnane X receptor isoforms and the constitutive androstane receptor, which indicates the possibility of a drug-drug interaction. This partly explains the decline in efficacy after repeated drinking of A. annua tea.

Traditional Medicine (Mahuang-Tang) Improves Ovarian Dysfunction and the Regulation of Steroidogenic Genes in Letrozole-Induced PCOS Rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Mahuang-Tang (MHT) has traditionally been used in Asia to treat a variety of diseases, such as fever without sweating, joint pain, lower back pain, asthma, and gynecological conditions. Polycystic ovary syndrome (PCOS) is a kind of gynecological disease that causes amenorrhea, infertility, and menopausal and urogenital disorders that could benefit from MHT treatment. AIM OF THE STUDY: In this study, we examined the effects of MHT on ovarian hormones and steroidogenic enzymes in female PCOS rats. METHODS AND RESULTS: The PCOS rat model was induced by Letrozole, and an in vivo evaluation of whether the dietary consumption of MHT improved the PCOS-like symptoms was conducted. The luteinizing hormone (LH) level and luteinizing hormone/follicular-stimulating hormone (LH/FSH) ratio increased in PCOS rats but decreased following MHT treatment. In the PCOS rats, the reduced estrogen level was restored to that of normal controls with MHT treatment in serum. The transcription level(s) of gonadotropin receptors (Fshr and Lhr), steroid receptors (Pgr, and Esr1) and steroidogenic enzymes (Cyp19a1, Hsd3b1, Hsd17a1, and Cyp11a1) changed under the PCOS condition, and were regulated by MHT treatment in the ovaries of PCOS rats. The reproductive tissues of Letrozole-induced PCOS rats were restored into estrogenic condition from androgen environments. CONCLUSION: These results suggest that MHT ameliorates the symptoms of PCOS by improving the dysregulation of ovarian steroids and steroidogenic enzymes in PCOS rats.

Epigenetics Predicts Serum 25-Hydroxyvitamin D Response to Vitamin D3 Supplementation in African Americans.

SCOPE: The effects of vitamin D3 supplementations on circulating 25-hydroxyvitamin D [25(OH)D] are varied. The hypothesis that the baseline DNA methylation plays a role in the serum 25(OH)D response to vitamin D3 supplementation is tested. METHODS AND RESULTS: A randomized clinical trial is first conducted among 64 African Americans, who are randomly assigned to a placebo or a 16-week treatment of 600, 2000, and 4000 IU d-1 of vitamin D3 supplements. Expected serum 25(OH)D concentrations at posttest are estimated by intervention, age, gender, body mass index, baseline 25(OH)D concentrations, and seasonal variations. The 25(OH)D response is categorized into a high-response group when the actual 25(OH)D concentrations at posttest are higher than expected, and a low-response group otherwise. The 25(OH)D response is associated with baseline methylation levels of CYP family and VDR genes (raw p < 0.05). At a genome-wide level, the baseline methylation level of cg07873128 (OSBPL5) that regulates cholesterol balance and calcium homeostasis is higher in the low-response group (false discovery rate = 0.028). CONCLUSIONS: The baseline methylation levels of CYP family and VDR modulate 25(OH)D response. In addition, the hypermethylation of cg07873128 at the baseline, which is located in the imprinted gene OSBPL5, may reduce the serum 25(OH)D response to vitamin D3 supplementation.

Radiotherapy in combination with hyperthermia suppresses lung cancer progression via increased NR4A3 and KLF11 expression.

Purpose: Hyperthermia (HT), a clinical treatment involving delivery of heat to tumors, has been used in combination with traditional chemotherapy and radiotherapy to enhance their effects. However, the molecular mechanism underlying the high efficacy of combination therapy is not clear. This study was conducted to identify the molecular mechanism underlying the sensitization of lung cancer to radiotherapy by HT.Materials and methods: Nuclear receptor subfamily 4, group A, member 3 (NR4A3) and Krüppel-like factor 11 (KLF11) expression in non-small-cell lung cancer cells was confirmed by performing real-time quantitative reverse transcription-polymerase chain reaction. Tumor cell proliferation and apoptosis were assessed via a colony-forming assay and Annexin V/propidium iodide staining.Results and conclusions: Expression profile analysis revealed elevated levels of NR4A3 and KLF11 in A549 lung cancer cells after treatment with HT combined with radiation. We also confirmed that NR4A3 and KLF11 induced apoptosis and inhibited cell proliferation by elevating intracellular reactive oxygen species levels. Knockdown of NR4A3 or KLF11 using siRNA led to decreased effects of radiohyperthermia. Finally, the effect of these two factors on lung cancer progression was evaluated by in vivo xenograft studies. Taken together, the results suggest that NR4A3 and KLF11 are critical for increasing the efficacy of radiotherapy in combination with HT.

Genome-wide Identification of Tebufenozide Resistant Genes in the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae).

The smaller tea tortrix, Adoxophyes honmai, has developed strong resistance to tebufenozide, a diacylhydrazine-type (DAH) insecticide. Here, we investigated its mechanism by identifying genes responsible for the tebufenozide resistance using various next generation sequencing techniques. First, double-digest restriction site-associated DNA sequencing (ddRAD-seq) identified two candidate loci. Then, synteny analyses using A. honmai draft genome sequences revealed that one locus contained the ecdysone receptor gene (EcR) and the other multiple CYP9A subfamily P450 genes. RNA-seq and direct sequencing of EcR cDNAs found a single nucleotide polymorphism (SNP), which was tightly linked to tebufenozide resistance and generated an amino acid substitution in the ligand-binding domain. The binding affinity to tebufenozide was about 4 times lower in in vitro translated EcR of the resistant strain than in the susceptible strain. RNA-seq analyses identified commonly up-regulated genes in resistant strains, including CYP9A and choline/carboxylesterase (CCE) genes. RT-qPCR analysis and bioassays showed that the expression levels of several CYP9A and CCE genes were moderately correlated with tebufenozide resistance. Collectively, these results suggest that the reduced binding affinity of EcR is the main factor and the enhanced detoxification activity by some CYP9As and CCEs plays a supplementary role in tebufenozide resistance in A. honmai.

Transgenic potato lines expressing hairpin RNAi construct of molting-associated EcR gene exhibit enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata, Say).

Most of the commercialized insect resistant transgenic crops express cry gene(s) isolated from Bacillus thuringiensis; however, intensive cultivation of Bt crops over almost two decades has been questioned regarding its sustainability and durability in pest management. The present study focused on silencing of highly specific molting-associated Ecdysone receptor (EcR) gene of Colorado potato beetle (CPB) using RNA interference (RNAi) approach. The partial cDNA of EcR gene of CPB was amplified using specific primers in sense and anti-sense orientations, and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Agria and Lady Olympia potato cultivars were infected with Agrobacterium strain LBA4404 harboring constructs under the control of CaMV 35S promoter. Standard molecular analysis of primary transformants showed proper integration of T-DNA in plant genome. The transgenic plants of both cultivars were evaluated for their efficacy against first, second and third instar CPB larvae. The leaf biotoxicity assays revealed 15-80% of CPB mortality. A significantly lower fold-change (0.87-4.14×) in larval weight was observed in insects fed on transgenic plants compared to the ones fed on control plants (1.87-6.53×). Furthermore, CPB larvae fed on transgenic plants exhibited reduced EcR transcripts, indicating the functionality of dsRNA EcR in silencing EcR gene expression. This study is an excellent example of the integration of an alternative, effective and reliable method to cope with potato insect pests that incur significant losses to potato production in the world.

Stable cellular models of nuclear receptor PXR for high-throughput evaluation of small molecules.

Pregnane & Xenobiotic Receptor (PXR) is one of the 48 members of the ligand-modulated transcription factors belonging to nuclear receptor superfamily. Though PXR is now well-established as a 'xenosensor', regulating the central detoxification and drug metabolizing machinery, it has also emerged as a key player in several metabolic disorders. This makes PXR attractive to both, researchers and pharmaceutical industry since clinical success of small drug molecules can be pre-evaluated on PXR platform. At the early stages of drug discovery, cell-based assays are used for high-throughput screening of small molecules. The future success or failure of a drug can be predicted by this approach saving expensive resources and time. In view of this, we have developed human liver cell line-based, dual-level screening and validation protocol on PXR platform having application to assess small molecules. We have generated two different stably transfected cell lines, (i) a stable promoter-reporter cell line (HepXREM) expressing PXR and a commonly used CYP3A4 promoter-reporter i.e. XREM-luciferase; and (ii) two stable cell lines integrated with proximal PXR-promoter-reporter (Hepx-1096/+43 and Hepx-497/+43). Employing HepXREM, Hepx-1096/+43 and Hepx-497/+43 stable cell lines > 25 anti-cancer herbal drug ingredients were screened for examining their modulatory effects on a) PXR transcriptional activity and, b) PXR-promoter activity. In conclusion, the present report provides a convenient and economical, dual-level screening system to facilitate the identification of superior therapeutic small molecules.

Pregnane X receptor mediates sorafenib resistance in advanced hepatocellular carcinoma.

BACKGROUND: Kinase inhibitor sorafenib is the most widely used drug for advanced HCC clinical treatment nowadays. However, sorafenib administration is only effective for a small portion of HCC patients, and the majority develop sorafenib-resistance during treatment. Thus, it is urgent to discover the endogenous mechanism and identify new pharmaceutical targets of sorafenib-resistance. METHODS: Pregnane X receptor (PXR) was detected by immunohistochemistry and quantitative PCR. GST-pull down and LC-MS/MS was used to detect the interaction of PXR and Sorafenib. To test the properties of HCC tumor growth and metastasis, in vivo tumor explant model, FACS, trans-well assay, cell-survival inhibitory assay and Western blot were performed. In terms of mechanistic study, additional assays such as ChIP and luciferase reporter gene assay were applied. RESULTS: In the present work, we found high PXR level in clinical specimens is related to the poor prognosis of Sorafenib treated patients. By the mechanistic studies, we show that sorafenib binds to PXR and activates PXR pathway, and by which HCC cells develop sorafenib-resistance via activating. Moreover, PXR overexpression helps HCC cells to persist to sorafenib treatment. CONCLUSION: This study reports the endogenous sorafenib-resistance mechanism in HCC cells, which offers an opportunity to design new therapeutic approaches for HCC treatment. GENERAL SIGNIFICANCE: PXR mediates sorafenib-resistance in HCC cells and targeting PXR can be a useful approach to facilitate HCC treatment.

Atrazine-induced environmental nephrosis was mitigated by lycopene via modulating nuclear xenobiotic receptors-mediated response.

The burden and morbidity of environmental nephrosis is increasing globally. Atrazine (ATR) and degradation products in the environment are considered key determinants of nephrosis. However, the lack of highly effective treatments for environmental nephrosis creates an urgent need to better understand the preventive strategies and mechanisms. This study aimed to highlight the mechanism of ATR-induced environmental nephrosis and the chemoprotective potential of lycopene (LYC) against the renal injury and nephrosis. Male mice were treated with LYC (5 mg/kg) and/or ATR (50 mg/kg or 200 mg/kg) by gavage administration for 21 days. Histopathological changes and biochemical function, cytochrome P450 enzymes system (CYP450s), nuclear xenobiotic receptors (NXRs) response and the transcription of CYP isoforms (CYPs) were detected. ATR exposure caused the changes of the histopathological and biochemical function, activated the NXR response and disturbed the CYP450s homeostasis. Supplementary LYC significantly prevented ATR-induced nephrotoxicity and alleviated the alternation of histopathological and biochemical function via modulating the CYP450s homeostasis and the NXR response. The results demonstrated AHR, CAR, PXR, PPAR (α, γ), CYP1, CYP2, CYP3 and CYP4 superfamily play a vital role in LYC-ATR interaction. Our findings provide new evidence that ATR exposure can cause the environmental nephrosis via inducing the kidney injury. Supplementary LYC showed significant chemoprotective potential against ATR-induced renal injury and environmental nephrosis via regulating the NXR response and the CYP450s homeostasis.

Modulation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) activation by ursolic acid (UA) attenuates rifampin-isoniazid cytotoxicity.

BACKGROUND: Interactions between transcriptional inducers of cytochrome P450 (CYP450) enzymes and therapeutic drugs may be prevented by antagonizing the activation of a nuclear receptor (NR), pregnane X receptor (PXR, NR1I2), thus improving therapeutic efficacy. PURPOSE: In the present study, we aim to identify that ursolic acid (UA), a widely distributed pentacyclic triterpene, may act as an effective antagonist of PXR and its sister NR receptor, constitutive androstane receptor (CAR, NR1I3). METHODS: The hepatocellular carcinoma cell line, HepG2, was used to evaluate the promoter activity of PXR and CAR target genes, CYP3A4 and CYP2B6, respectively. Catalytic activities, mRNA, and protein expression of CYP3A4 and CYP2B6 were evaluated in a differentiated HepaRG cell line. Coregulation of PXR with coregulators on CYP3A4 promoter response elements was also been characterized. RESULTS: Transient transfection assays showed that UA effectively attenuated CYP3A4 and CYP2B6 promoter activities mediated by rifampin (RIF, human PXR agonist) and CITCO (human CAR agonist). These inhibitory effects were well correlated with the expression and catalytic activities of CYP3A4 and CYP2B6. Furthermore, the interaction of co-regulators with PXR and the transcriptional complexes in the CYP3A4 promoter activity and CYP3A4 promoter xenobiotic response element (everted repeat 6, ER6), respectively, were disrupted in the presence of UA. UA showed an antagonistic effect against PXR, and reversed the cytotoxic effects of isoniazid (INH) induced by RIF. Taken together, these results show that UA inhibits the transactivation effects of PXR and CAR, and reduces the expression and function of CYP3A4 and CYP2B6. CONCLUSION: The present study suggests that UA could be a powerful agent for reducing potentially dangerous interactions between transcriptional inducers of CYP enzymes and therapeutic drugs.

Steviol, an aglycone of steviol glycoside sweeteners, interacts with the pregnane X (PXR) and aryl hydrocarbon (AHR) receptors in detoxification regulation.

Stevia rebaudiana Bertoni is a herb known for the high content of natural sweeteners in its leaves. Its main secondary metabolite stevioside is used as non-caloric sweetener. No information, however, is available on whether stevioside or steviol interact with drug-metabolizing enzymes and pose the potential risk of food-drug interactions. Similarly, data are lacking on the interactions of steviol and stevioside with key nuclear receptors controlling the expression of the main drug metabolizing enzymes. We studied the interactions of steviol and stevioside with the pregnane X (PXR), vitamin D (VDR), constitutive androstane (CAR), farnesoid X (FXR), glucocorticoid (GR) and aryl hydrocarbon (AHR) receptors, which control expression of genes of xenobiotic metabolism. In addition, the inhibitory activities of steviol and stevioside towards the major cytochrome P450 enzymes CYP3A4, CYP2C9, CYP2D6, CYP1A2 and CYP2B6 were evaluated in vitro. We found that steviol moderately activated the PXR and AHR, resulting in the induction of their target genes including CYP3A4 and CYP1A2 in primary human hepatocytes. A weak inhibition of CYP3A4 and CYP2C9 with steviol was also found. Our results provide mechanistic data indicating that stevioside and stevia sweeteners may have the potential to induce food-drug interactions, a finding that warrants future prospective clinical investigation.

Development of a common carp (Cyprinus carpio) pregnane X receptor (cPXR) transactivation reporter assay and its activation by azole fungicides and pharmaceutical chemicals.

In mammals, the pregnane X receptor (PXR) is a transcription factor with a key role in regulating expression of several genes involved in drug biotransformation. PXR is present in fish and some genes known to be under its control can be up-regulated by mammalian PXR ligands. Despite this, direct involvement of PXR in drug biotransformation in fish has yet to be established. Here, the full length PXR sequence was cloned from carp (Cyprinus carpio) and used in a luciferase reporter assay to elucidate its role in xenobiotic metabolism in fish. A reporter assay for human PXR (hPXR) was also established to compare transactivation between human and carp (cPXR) isoforms. Rifampicin activated hPXR as expected, but not cPXR. Conversely, clotrimazole (CTZ) activated both isoforms and was more potent on cPXR, with an EC50 within the range of concentrations of CTZ measured in the aquatic environment. Responses to other azoles tested were similar between both isoforms. A range of pharmaceuticals tested either failed to activate, or were very weakly active, on the cPXR or hPXR. Overall, these results indicate that the cPXR may differ from the hPXR in its responses and/or sensitivity to induction by different environmental chemicals, with implications for risk assessment because of species differences.

Geniposide attenuates ANIT-induced cholestasis through regulation of transporters and enzymes involved in bile acids homeostasis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Geniposide (GE) is one of the major iridoid glycosides isolated from the fruit of Gardenia jasminoides Ellis that has been used to treat hepatic disorders including cholestasis. However, the underlying mechanisms for GE ameliorating the reduction in bile acids accumulation by α-naphthylisothiocyanate (ANIT) remain unclear. AIM OF THE STUDY: The purpose of this study is to characterize the efficacy of GE in regulation of bile acids uptake, synthesis, metabolism, and transport in ANIT-induced rats. MATERIALS AND METHODS: Sprague-Dawley rats were orally administrated with vehicle, GE (25, 50, and 100mg/kg), and ursodeoxycholic acid (UDCA) (60mg/kg) once daily for seven days. On the fifth day, a single dose of ANIT (75mg/kg) was administrated via oral gavage. Blood biochemical determination, bile flow rate and liver histopathology were measured to evaluate the protective effect of GE. The mRNA expressions and protein levels of transporters and enzymes involved in bile acids homeostasis were determined by quantitative real-time polymerase chain reaction (PCR) and western blot to study the underlying mechanism of GE against ANIT-induced rats. RESULTS: GE (25, 50, and 100mg/kg, po) dose-dependently prevented ANIT-induced changes in serum markers for liver injury. GE treatment reduced basolateral bile acids uptake via repression of OATP2 (P<0.05). Bile acids biosynthesis was decreased through down-regulation of CYP7A1, CYP8B1, and CYP27A1 (P<0.05). GE significantly increased canalicular bile acids secretion via BSEP (P<0.05), subsequently stimulating bile flow during cholestasis. GE also markedly enhanced mRNA level of basolateral transporter OSTß (P<0.01). Bile acids transported to the plasma were cleared into the urine, resulting in down-regulation of plasma bile acids. However, GE did not alter the mRNA levels of CYP3A2, UGT1A1 and SULT2A1. Furthermore, the gene and protein expression analysis demonstrated activation of FXR, PXR, and SHP after GE administration. CONCLUSION: GE attenuates ANIT-induced hepatotoxicity and cholestasis in rats, due to regulation enzymes and transporters responsible for bile acids homeostasis.

Transactivation Assays to Assess Canine and Rodent Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) Activation.

The pregnane X receptor (PXR/SXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are nuclear receptors (NRs) involved in the regulation of many genes including cytochrome P450 enzymes (CYPs) and transporters important in metabolism and uptake of both endogenous substrates and xenobiotics. Activation of these receptors can lead to adverse drug effects as well as drug-drug interactions. Depending on which nuclear receptor is activated will determine which adverse effect could occur, making identification important. Screening for NR activation by New Molecular Entities (NMEs) using cell-based transactivation assays is the singular high throughput method currently available for identifying the activation of a particular NR. Moreover, screening for species-specific NR activation can minimize the use of animals in drug development and toxicology studies. With this in mind, we have developed in vitro transactivation assays to identify compounds that activate canine and rat PXR and CAR3. We found differences in specificity for canine and rat PXR, with the best activator for canine PXR being 10 µM SR12813 (60.1 ± 3.1-fold) and for rat PXR, 10 µM dexamethasone (60.9 ± 8.4 fold). Of the 19 test agents examined, 10 and 9 significantly activated rat and canine PXR at varying degrees, respectively. In contrast, 5 compounds exhibited statistically significant activation of rat CAR3 and 4 activated the canine receptor. For canine CAR3, 50 µM artemisinin proved to be the best activator (7.3 ± 1.8 and 10.5 ± 2.2 fold) while clotrimazole (10 µM) was the primary activator of the rat variant (13.7 ± 0.8 and 26.9 ± 1.3 fold). Results from these studies demonstrated that cell-based transactivation assays can detect species-specific activators and revealed that PXR was activated by at least twice as many compounds as was CAR3, suggesting that there are many more agonists for PXR than CAR.

Cloning and Expression of Ecdysone Receptor and Retinoid X Receptor from Procambarus clarkii: Induction by Eyestalk Ablation.

Ecdysone receptor and retinoid X receptor are key regulators in molting. Here, full length ecdysone receptor (PcEcR) and retinoid X receptor (PcRXR) cDNAs from Procambarus clarkii were cloned. Full length cDNA of PcEcR has 2500 bp, encoding 576 amino acid proteins, and full length cDNA of PcRXR has 2593 bp, in which a 15 bp and a 204 bp insert/deletion splice variant regions in DNA binding domain and hinge domain were identified. The two splice variant regions in PcRXR result four isoforms: PcRXR1-4, encoding 525, 520, 457 and 452 amino acids respectively. PcEcR was highly expressed in the hepatopancreas and eyestalk and PcRXR was highly expressed in the eyestalk among eight examined tissues. Both PcEcR and PcRXR had induced expression after eyestalk ablation (ESA) in the three examined tissues. In muscle, PcEcR and PcRXR were upregulated after ESA, PcEcR reached the highest level on day 3 after ESA and increased 33.5-fold relative to day 0, and PcRXR reached highest the level on day 1 after ESA and increased 2.7-fold relative to day 0. In the hepatopancreas, PcEcR and PcRXR dEcReased continuously after ESA, and the expression levels of PcEcR and PcRXR were only 0.7% and 1.7% on day 7 after ESA relative to day 0, respectively. In the ovaries, PcEcR was upregulated after ESA, reached the highest level on day 3 after ESA, increased 3.0-fold relative to day 0, and the expression level of PcRXR changed insignificantly after ESA (p > 0.05). The different responses of PcEcR and PcRXR after ESA indicates that different tissues play different roles (and coordinates their functions) in molting.

The liquorice root derivative glycyrrhetinic acid can ameliorate ionoregulatory disturbance in rainbow trout (Oncorhynchus mykiss) abruptly exposed to ion-poor water.

To consider the idea that a dietary botanical supplement could act as an adaptogen in a teleost fish, the effect of a liquorice root derivative (18ß-glycyrrhetinic acid, 18ßGA) on rainbow trout following an acute ionoregulatory stressor was examined. Freshwater (FW) trout were fed a control or 18ßGA supplemented diet (0, 5, or 50µg 18ßGA/g diet) for 2weeks, then abruptly exposed to ion-poor water (IPW) for 24h. Following IPW exposure, muscle moisture content and serum cortisol levels elevated and serum [Na(+)] and/or [Cl(-)] reduced in control and 50µg/g 18ßGA-fed fish. However, these endpoints were unaltered in 5µg/g 18ßGA-fed fish. Gill tissue was investigated for potential mechanisms of 18ßGA action by examining mRNA abundance of genes encoding corticosteroid receptors (CRs), 11ß-hydroxysteroid dehydrogenase 2 (11ß-hsd2), and tight junction (TJ) proteins, as well as Na(+)-K(+)-ATPase and H(+)-ATPase activity, and mitochondrion-rich cell (MRC) morphometrics. Following IPW exposure, CR and 11ß-hsd2 mRNA, MRC fractional surface, Na(+)-K(+)-ATPase and H(+)-ATPase activity were unaltered or decreased in 50µg 18ßGA fish, as was mRNA encoding select TJ proteins. In contrast, 5µg 18ßGA-fed fish exhibited elevated 11ß-hsd2 and CR mRNA abundance versus 50µg 18ßGA-fed, and reduced MRC apical area as well as some differences in TJ protein mRNA abundance versus control fish. Data suggest that 18ßGA, at low levels, may be adaptogenic in trout and might help to ameliorate ionoregulatory perturbation following IPW exposure. This seems to occur, in part, through 18ßGA-induced alterations in the biochemistry and physiology of the gill.