The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.

• MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• konstitutivní androstanový receptor * agonisté   chemie   MeSH
• lidé MeSH
• myši MeSH
• pyridiny farmakologie   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• steroidní receptory * agonisté   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A combination of several pharmacophores in one molecule has been successfully used for multi-target-directed ligands (MTDL) design. New propargylamine substituted derivatives combined with salicylic and cinnamic scaffolds were designed and synthesized as potential cholinesterases and monoamine oxidases (MAOs) inhibitors. They were evaluated invitro for inhibition of acetyl- (AChE) and butyrylcholinesterase (BuChE) using Ellman's method. All the compounds act as dual inhibitors. Most of the derivatives are stronger inhibitors of AChE, the best activity showed 5-bromo-N-(prop-2-yn-1-yl)salicylamide 1e (IC50 = 8.05 μM). Carbamates (4-bromo-2-[(prop-2-yn-1-yl)carbamoyl]phenyl ethyl(methyl)carbamate 2d and 2,4-dibromo-6-[(prop-2-yn-1-yl)carbamoyl]phenyl ethyl(methyl)carbamate 2e were selective and the most active for BuChE (25.10 and 26.09 μM). 4-Bromo-2-[(prop-2-yn-1-ylimino)methyl]phenol 4a was the most potent inhibitor of MAOs (IC50 of 3.95 and ≈10 μM for MAO-B and MAO-A, respectively) along with a balanced inhibition of both cholinesterases being a real MTDL. The mechanism of action was proposed, and binding modes of the hits were studied by molecular docking on human enzymes. Some of the derivatives also exhibited antioxidant properties. Insilico prediction of physicochemical parameters affirm that the molecules would be active after oral administration and able to reach brain tissue.

• MeSH
• antioxidancia chemická syntéza   chemie   farmakologie   MeSH
• butyrylcholinesterasa metabolismus   MeSH
• cholinesterasové inhibitory chemická syntéza   chemie   farmakologie   MeSH
• cholinesterasy metabolismus   MeSH
• Electrophorus MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• inhibitory MAO chemická syntéza   chemie   farmakologie   MeSH
• koně MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární struktura MeSH
• monoaminoxidasa metabolismus   MeSH
• pargylin analogy a deriváty   chemická syntéza   chemie   farmakologie   MeSH
• potkani Wistar MeSH
• propylaminy chemická syntéza   chemie   farmakologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• simulace molekulového dockingu * MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• antipsychotika aplikace a dávkování   škodlivé účinky   terapeutické užití   MeSH
• dospělí MeSH
• hepatocyty účinky léků   MeSH
• hyperamonemie chemicky indukované   diagnóza   etiologie   MeSH
• klozapin aplikace a dávkování   škodlivé účinky   terapeutické užití   MeSH
• kyselina valproová * škodlivé účinky   MeSH
• lékové postižení jater * diagnóza   etiologie   prevence a kontrola   MeSH
• lidé MeSH
• paranoidní schizofrenie farmakoterapie   komplikace   MeSH
• Reyeův syndrom etiologie   MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

Metformin, an oral antidiabetic drug, recently demonstrated a reducing effect on bile acids (BA) plasma concentrations in one patient with intrahepatic cholestasis of pregnancy (ICP) by unknown mechanism. Therefore, the aim of the present study was to examine the effect of metformin on BA homeostasis and related molecular pathways in the liver and intestine using a mouse model of ICP. The cholestasis was induced in female C57BL/6 mice by repeated administration of ethinylestradiol (10 mg/kg BW s.c.) and/or metformin (150 mg/kg BW orally) over 5 consecutive days with subsequent bile collection and molecular analysis of samples. We demonstrated that metformin significantly increased the rate of bile secretion in control mice. This increase was BA dependent and was produced both by increased liver BA synthesis via induced cholesterol 7α-hydroxylase (Cyp7a1) and by increased BA reabsorption in the ileum via induction of the apical sodium-dependent BA transporter (Asbt). In contrast, metformin further worsened ethinylestradiol-induced impairment of bile secretion. This reduction was also BA dependent and corresponded with significant downregulation of Bsep, and Ntcp, major excretory and uptake transporters for BA in hepatocytes, respectively. The plasma concentrations of BA were consequently significantly increased in the metformin-treated mice. Altogether, our data indicate positive stimulation of bile secretion by metformin in the intact liver, but this drug also induces serious impairment of BA biliary secretion, with a marked increase in plasma concentrations in estrogen-induced cholestasis. Our results imply that metformin should be used with caution in situations with hormone-dependent cholestasis, such as ICP.

• MeSH
• cholestáza chemicky indukované   metabolismus   patologie   MeSH
• ethinylestradiol škodlivé účinky   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• homeostáza účinky léků   MeSH
• intestinální absorpce účinky léků   MeSH
• metformin farmakologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• žlučové kyseliny a soli metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Metabolic deactivation by cytochrome P450 (CYP) is considered a potential mechanism of anticancer drug resistance. However, this hypothesis is predominantly based on indirect pieces of evidence and/or is influenced by interfering factors such as the use of multienzymatic models. Thus, an experimental approach for its verification is needed. In the present work, we employed HepG2 cells transduced with CYP enzymes involved in docetaxel, paclitaxel and vincristine metabolism to provide mechanistic evidence on their possible roles in resistance to these chemotherapeutic agents. Using MTT proliferation tests, we showed that overexpression of CYP3A4 resulted in decreased antiproliferative activity of 1 μM docetaxel (by 11.2, 23.2 and 22.9% at 24, 48 and 72 h intervals, respectively), while the sensitivity of CYP3A4-transduced cells was restored by co-administration of ketoconazole. Paclitaxel exhibited differential efficacy in CYP2C8- and empty vector-transduced cells (significant differences between 10.9 and 24.4% for 0.01, 0.1 and 1 μM concentrations), but neither montelukast nor clotrimazole was capable of affecting this asymmetry. Finally, the pharmacological activity of vincristine was not influenced by CYP3A4 or CYP3A5 overexpression. In the follow-up caspase activation assays, docetaxel was confirmed to be a victim of CYP3A4-mediated resistance, which is, at least partly, brought by impaired activation of caspases 3/7, 8 and 9. In summary, our data demonstrate that CYP3A4-mediated metabolic deactivation of docetaxel might represent a significant mechanism of pharmacokinetic resistance to this drug. In contrast, the possible role of CYPs in resistance to paclitaxel and vincristine has been disconfirmed. Importantly, the expression of CYP3A4 in HepG2_CYP3A4 cells is comparable to that in primary hepatocytes and HepaRG cells, which suggests that our results might be relevant for in vivo conditions, e.g., for hepatocellular carcinoma. Thus, our data may serve as a valuable in vitro background for future in vivo studies exploring the area of intratumoural metabolism-based drug resistance.

• MeSH
• antitumorózní látky farmakologie   MeSH
• buňky Hep G2 MeSH
• chemorezistence fyziologie   MeSH
• cytochrom P-450 CYP3A metabolismus   MeSH
• cytochrom P450 CYP2C8 metabolismus   MeSH
• cytostatické látky farmakologie   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• lidé MeSH
• metabolická clearance účinky léků   MeSH
• metabolická inaktivace účinky léků   MeSH
• nádorové buněčné linie MeSH
• proliferace buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Polyphenols, secondary metabolites of plants, exhibit different anti-cancer and cytoprotective properties such as anti-radical, anti-angiogenic, anti-inflammation, or cardioprotective. Some of these activities could be linked to modulation of miRNAs expression. MiRNAs play an important role in posttranscriptional regulation of their target genes that could be important within cell signalling or preservation of cell homeostasis, e.g., cell survival/apoptosis. We evaluated the influence of a non-toxic concentration of taxifolin and quercetin on the expression of majority human miRNAs via Affymetrix GeneChip™ miRNA 3.0 Array. For the evaluation we used two cell models corresponding to liver tissue, Hep G2 and primary human hepatocytes. The array analysis identified four miRNAs, miR-153, miR-204, miR-211, and miR-377-3p, with reduced expression after taxifolin treatment. All of these miRNAs are linked to modulation of ZEB2 expression in various models. Indeed, ZEB2 protein displayed upregulation after taxifolin treatment in a dose dependent manner. However, the modulation did not lead to epithelial mesenchymal transition. Our data show that taxifolin inhibits Akt phosphorylation, thereby diminishing ZEB2 signalling that could trigger carcinogenesis. We conclude that biological activity of taxifolin may have ambiguous or even contradictory outcomes because of non-specific effect on the cell.

• MeSH
• apoptóza účinky léků   MeSH
• buňky Hep G2 metabolismus   MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• exprese genu účinky léků   genetika   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• lidé MeSH
• mikro RNA účinky léků   genetika   MeSH
• pohyb buněk účinky léků   MeSH
• polyfenoly farmakologie   MeSH
• primární buněčná kultura MeSH
• proliferace buněk účinky léků   MeSH
• quercetin analogy a deriváty   metabolismus   farmakologie   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• signální transdukce genetika   MeSH
• transkripční faktor Zeb2 účinky léků   metabolismus   MeSH
• transkriptom účinky léků   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Dihydromyricetin (DHM) is a natural flavonoid showing several health promoting effects such as protective activity during severe alcohol intoxication. The mechanism underlying the effects of DHM on alcohol metabolism is virtually unknown. The present paper is focused on clarifying the role of DHM in the liver alcohol elimination at its molecular level. First, impact of DHM on alcohol dehydrogenase (ADH) activity in vitro and the enzyme induction in vivo was examined. Neither the ADH activity nor the enzyme expression were influenced by DHM. Next, the effect of DHM during alcohol intoxication were studied on primary hepatocytes isolated from EtOH-premedicated and untreated rats. The viability of cells exposed to alcohol, estimated based on the released enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), was slightly affected by DHM. Although the expected hepatoprotective effect of DHM was not fully achieved, DHM (in a concentration manner) proved to reduce the level of ROS/RNS in hepatocytes. However, no change in the rate of alcohol metabolism in vivo was found when rats were administered with a single or repeated dose of ethanol supplemented with DHM. In conclusion, the proposed positive effect of DHM during alcohol intoxication has not been proven. Moreover, there is no effect of DHM on the alcohol metabolism. The "hoped-for" DHM hepatoprotective activity can be attributed to the reduction of ROS/RNS levels in cells.

• MeSH
• alkoholdehydrogenasa metabolismus   MeSH
• antioxidancia farmakologie   MeSH
• cytochrom P-450 CYP2E1 metabolismus   MeSH
• ethanol metabolismus   MeSH
• flavonoly farmakologie   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• játra účinky léků   metabolismus   MeSH
• kultivované buňky MeSH
• metabolická inaktivace MeSH
• nitrosativní stres účinky léků   MeSH
• oxidační stres účinky léků   MeSH
• potkani Wistar MeSH
• reaktivní formy dusíku metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

• MeSH
• buněčné linie MeSH
• diazepam farmakologie   MeSH
• dospělí MeSH
• hepatocyty účinky léků   MeSH
• játra účinky léků   MeSH
• lidé středního věku MeSH
• lidé MeSH
• ligandy MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• proteinové domény účinky léků   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• reportérové geny účinky léků   genetika   MeSH
• transport proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

• MeSH
• aflatoxin B1 toxicita   MeSH
• biologické modely MeSH
• buněčné kultury metody   MeSH
• buněčné linie MeSH
• buněčné sféroidy * MeSH
• buňky Hep G2 MeSH
• hepatocyty účinky léků   MeSH
• játra účinky léků   MeSH
• lidé MeSH
• methylmethansulfonát toxicita   MeSH
• mikrojaderné testy metody   MeSH
• mutageny toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Graphene oxide (GO) is an engineered nanomaterial which was demonstrated to have outstanding capacity for adsorption of organic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), the ligands and activators of the aryl hydrocarbon receptor (AhR). Due to the partially overlapping ligand capacity of AhR and pregnane X receptor (PXR), we tested the impact of GO particles on their signalling. While reporter gene assay revealed potentiating effect of GO on ligand-activated AhR-dependent luciferase activity, there was no effect for PXR. However, inducible target genes for AhR (CYP1A1) or PXR (ABCB1) were decreased at mRNA as well as protein levels by the presence of GO in HepG2 (for AhR), LS180 (for PXR) or primary human hepatocytes (both receptors). Moreover, the presence of GO diminished PXR and AhR protein levels in primary cultures of human hepatocytes. This was partially reversed by proteasome inhibitor MG132 for AhR but not for PXR. In conclusion, GO decreases ligand-stimulated activities of AhR and PXR in human cells.

• MeSH
• biotransformace MeSH
• cytochrom P-450 CYP1A1 metabolismus   MeSH
• grafit chemie   MeSH
• hepatocyty účinky léků   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• pregnanový X receptor MeSH
• receptory aromatických uhlovodíků metabolismus   MeSH
• reportérové geny MeSH
• signální transdukce MeSH
• steroidní receptory genetika   MeSH
• xenobiotika chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH