Long non-coding RNAs (lncRNAs) are crucial in chronic liver diseases, but the specific molecular mechanism of lncRNAs in alcoholic fatty liver (AFL) remains unclear. In this study, we investigated the in-depth regulatory mechanism of mTOR affected by AIRN non-protein coding RNA (lncRNA-AIRN) in the development of AFL. LncRNA-AIRN was highly expressed in the liver tissues of AFL C57BL/6mice and oleic acid+alcohol (O+A)treated AML-12cells by using quantitative real-timePCR. RNA pull-down and RNA immunoprecipitation experiments demonstrated that there was an interaction between lncRNA-AIRN and mTOR, and that interference with lncRNA-AIRN could promote the mTOR protein level. Results ofcycloheximide-chase assay showed that the proteinlevel of mTOR was decreased with the treatment time after the knockdown of lncRNA-AIRN. Furthermore, the knockdown of lncRNA-AIRN reducedmTOR protein level by promoting the E3 ubiquitin ligase FBXW7-mediated ubiquitination.The lncRNA-AIRN/mTORaxis was involved in the regulation of the mitophagy of O+A treated hepatocytes, which was confirmed by the cell transfection and the MTT assay.SPSS 16.0 was used for analyzing data. The difference between the two groups was analyzed by performing Student's t-test, and ANOVA was used to analyze the difference when more than two groups. P values < 0.05 were considered to be significantly different.Our findings demonstrated that the knockdown of lncRNA-AIRN influencedmitophagy in AFL by promoting mTOR ubiquitination.
- MeSH
- alkoholická steatóza jater enzymologie genetika patologie MeSH
- buněčné linie MeSH
- down regulace MeSH
- F-Box a WD repetice obsahující protein 7 metabolismus MeSH
- hepatocyty enzymologie patologie MeSH
- jaterní mitochondrie enzymologie genetika patologie MeSH
- játra enzymologie patologie MeSH
- mitofagie * MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- RNA dlouhá nekódující genetika metabolismus MeSH
- signální transdukce MeSH
- TOR serin-threoninkinasy metabolismus MeSH
- ubikvitinace MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Flavonoids are abundant polyphenols in nature. They are extensively biotransformed in enterocytes and hepatocytes, where conjugated (methyl, sulfate, and glucuronide) metabolites are formed. However, bacterial microflora in the human intestines also metabolize flavonoids, resulting in the production of smaller phenolic fragments (e.g., hydroxybenzoic, hydroxyacetic and hydroxycinnamic acids, and hydroxybenzenes). Despite the fact that several colonic metabolites appear in the circulation at high concentrations, we have only limited information regarding their pharmacodynamic effects and pharmacokinetic interactions. Therefore, in this in vitro study, we investigated the interactions of 24 microbial flavonoid metabolites with human serum albumin and cytochrome P450 (CYP2C9, 2C19, and 3A4) enzymes. Our results demonstrated that some metabolites (e.g., 2,4-dihydroxyacetophenone, pyrogallol, O-desmethylangolensin, and 2-hydroxy-4-methoxybenzoic acid) form stable complexes with albumin. However, the compounds tested did not considerably displace Site I and II marker drugs from albumin. All CYP isoforms examined were significantly inhibited by O-desmethylangolensin; nevertheless, only its effect on CYP2C9 seems to be relevant. Furthermore, resorcinol and phloroglucinol showed strong inhibitory effects on CYP3A4. Our results demonstrate that, besides flavonoid aglycones and their conjugated derivatives, some colonic metabolites are also able to interact with proteins involved in the pharmacokinetics of drugs.
- MeSH
- erytrocyty enzymologie MeSH
- flavonoidy * chemie metabolismus MeSH
- hepatocyty enzymologie MeSH
- lidé MeSH
- lidský sérový albumin * chemie metabolismus MeSH
- systém (enzymů) cytochromů P-450 * chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mono-methylindoles (MMI) were described as agonists and/or antagonists of the human aryl hydrocarbon receptor (AhR). Here, we investigated the effects of MMI on AhR-CYP1A pathway in human hepatocytes and HepaRG cells derived from human progenitor hepatic cells. All MMI, except of 2-methylindole, strongly induced CYP1A1 and CYP1A2 mRNAs in HepaRG cells. Induction of CYP1A genes was absent in AhR-knock-out HepaRG cells. Consistently, CYP1A1 and CYP1A2 mRNAs and proteins were induced by all MMIs (except 2-methylindole), in human hepatocytes. The enzyme activity of CYP1A1 was inhibited by MMIs in human hepatocytes and LS180 colon cancer cells in a concentration-dependent manner (IC50 values from 1.2 μM to 23.8 μM and from 3.4 μM to 11.4 μM, respectively). Inhibition of CYP1A1 activity by MMI in human liver microsomes was much weaker as compared to that in intact cells. Incubation of parental MMI with human hepatocytes either diminished (4-methylindole, 6-methylindole) or enhanced (7-methylindole) their agonist effects on AhR in AZ-AHR reporter cells. In conclusion, overall effects of MMI on AhR-CYP1A pathway in human cells comprise the induction of CYP1A genes through AhR, the inhibition of CYP1A catalytic activity and possibly the metabolic transformation causing loss or gain of AhR agonist activity of parental compounds.
- MeSH
- cytochrom P-450 CYP1A1 antagonisté a inhibitory biosyntéza genetika MeSH
- enzymová indukce MeSH
- hepatocyty účinky léků enzymologie MeSH
- indoly farmakologie MeSH
- induktory cytochromu P450 farmakologie MeSH
- inhibitory cytochromu P450 farmakologie MeSH
- jaterní mikrozomy účinky léků enzymologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- nádorové buněčné linie MeSH
- nádory tračníku enzymologie MeSH
- receptory aromatických uhlovodíků agonisté genetika metabolismus MeSH
- senioři MeSH
- transkripční faktory bHLH agonisté genetika metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.
- MeSH
- adukty DNA metabolismus MeSH
- antitumorózní látky metabolismus farmakologie MeSH
- aromatické hydroxylasy metabolismus MeSH
- cytochrom-B(5)-reduktasa nedostatek genetika MeSH
- cytochromy b5 nedostatek genetika MeSH
- elipticiny metabolismus farmakologie MeSH
- fenotyp MeSH
- genotyp MeSH
- hepatocyty enzymologie MeSH
- jaterní mikrozomy enzymologie MeSH
- játra enzymologie MeSH
- metabolická aktivace MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- NADPH-cytochrom c-reduktasa metabolismus MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- diabetes mellitus 1. typu farmakoterapie MeSH
- diabetes mellitus 2. typu farmakoterapie MeSH
- enterocyty metabolismus účinky léků MeSH
- hepatocyty enzymologie metabolismus účinky léků MeSH
- játra enzymologie metabolismus účinky léků MeSH
- léková kontraindikace MeSH
- lidé MeSH
- metformin farmakokinetika farmakologie terapeutické užití MeSH
- nežádoucí účinky léčiv MeSH
- těhotenství účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- těhotenství účinky léků MeSH
Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 , which can also act as an electron donor from cytochrome b 5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b 5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.
- MeSH
- adukty DNA metabolismus MeSH
- benzopyren metabolismus MeSH
- cytochrom-B(5)-reduktasa metabolismus MeSH
- hepatocyty enzymologie MeSH
- jaterní mikrozomy enzymologie MeSH
- myši knockoutované MeSH
- myši MeSH
- NADPH-cytochrom c-reduktasa metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- ateroskleróza etiologie metabolismus prevence a kontrola MeSH
- cholesterol metabolismus sekrece MeSH
- hepatocyty enzymologie metabolismus MeSH
- interakce mezi receptory a ligandy MeSH
- karcinogeneze * metabolismus účinky léků MeSH
- kyselina chenodeoxycholová analýza metabolismus sekrece MeSH
- kyselina cholová analýza metabolismus sekrece MeSH
- lidé MeSH
- metabolické sítě a dráhy MeSH
- metabolismus lipidů * fyziologie imunologie MeSH
- neuroprotektivní látky metabolismus MeSH
- pankreas metabolismus sekrece MeSH
- regenerace jater MeSH
- statistika jako téma MeSH
- střevní mikroflóra fyziologie imunologie MeSH
- žluč enzymologie metabolismus sekrece MeSH
- žlučové kyseliny a soli * analýza metabolismus sekrece MeSH
- ztučnělá játra MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
The Pregnane X (PXR), Vitamin D (VDR) and Farnesoid X (FXR) nuclear receptors have been shown to be receptors of bile acids controlling their detoxification or synthesis. Chenodeoxycholic (CDCA) and lithocholic (LCA) acids are ligands of FXR and VDR, respectively, whereas 3-keto and acetylated derivates of LCA have been described as ligands for all three receptors. In this study, we hypothesized that oxidation or acetylation at position 3, 7 and 12 of bile acids DCA (deoxycholic acid), LCA, CA (cholic acid), and CDCA by detoxification enzymes or microbiome may have an effect on the interactions with bile acid nuclear receptors. We employed reporter gene assays in HepG2 cells, the TR-FRET assay with recombinant PXR and RT-PCR to study the effects of acetylated and keto bile acids on the nuclear receptors activation and their target gene expression in differentiated hepatic HepaRG cells. We demonstrate that the DCA 3,12-diacetate and CA 3,7,12-triacetate derivatives are ligands of PXR and DCA 3,12-diacetate induces PXR target genes such as CYP3A4, CYP2B6 and ABCB1/MDR1. In conclusion, we found that acetylated DCA and CA are potent ligands of PXR. Whether the acetylated bile acid derivatives are novel endogenous ligands of PXR with detoxification or physiological functions should be further studied in ongoing experiments.
- MeSH
- acetylace MeSH
- buněčné kultury MeSH
- buňky Hep G2 MeSH
- cytochrom P-450 CYP3A genetika MeSH
- cytochrom P450 CYP2B6 genetika MeSH
- hepatocyty účinky léků enzymologie metabolismus MeSH
- kyselina cholová chemie metabolismus farmakologie MeSH
- kyselina deoxycholová chemie metabolismus farmakologie MeSH
- lidé MeSH
- ligandy MeSH
- myši MeSH
- oxidace-redukce MeSH
- P-glykoprotein genetika MeSH
- plazmidy MeSH
- receptory cytoplazmatické a nukleární chemie genetika metabolismus MeSH
- receptory kalcitriolu chemie genetika metabolismus MeSH
- reportérové geny MeSH
- simulace molekulového dockingu MeSH
- steroidní receptory chemie genetika metabolismus MeSH
- techniky dvojhybridového systému MeSH
- transfekce MeSH
- vazba proteinů MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, exerts many beneficial effects on human health such as antioxidant, anti-inflammatory, and anticancer effects. The effect of SFN alone on drug-metabolizing enzymes (DMEs) has been investigated in numerous in vitro and in vivo models, but little is known about the effect of SFN in combination with cytochrome P450 (CYP) inducer. The aim of our study was to evaluate the effect of SFN on the activity and gene expression of selected DMEs in primary cultures of rat hepatocytes treated or non-treated with β-naphthoflavone (BNF), the model CYP1A inducer. In our study, SFN alone did not significantly alter the activity and expression of the studied DMEs, except for the glutathione S-transferase (GSTA1) mRNA level, which was significantly enhanced. Co-treatment of hepatocytes with SFN and BNF led to a substantial increase in sulfotransferase, aldoketoreductase 1C, carbonylreductase 1 and NAD(P)H:quinone oxidoreductase 1 activity and a marked decrease in cytochrome P450 (CYP) Cyp1a1, Cyp2b and Cyp3a4 expression in comparison to the treatment with BNF alone. Sulforaphane is able to modulate the activity and/or expression of DMEs, thus shifting the balance of carcinogen metabolism toward deactivation, which could represent an important mechanism of its chemopreventive activity.
Dihydropyridine calcium channel blockers (CCBs) are used as anti-hypertensives and in the treatment of angina pectoris. Structurally, CCBs have at least one chiral center in the molecule, thereby existing in two or more different enantiomers. In the current paper we examined effects of benidipine, felodipine and isradipine enantiomers on the expression and enzyme activities of human xenobiotics-metabolizing cytochromes P450. All CCBs dose-dependently activated aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), as revealed by gene reporter assays. Activation of AhR, but not PXR, was enantiospecific. Consistently, CCBs induced CYP1A1 and CYP1A2 mRNAs, but not protein, in human hepatocytes and HepG2 cells, with following pattern: benidipine (-)>(+), isradipine (-)>(+) and felodipine (+)>(-). All CCBs induced CYP2A6, CYP2B6 and CYP3A4 mRNA and protein in human hepatocytes, and there were not differences between the enantiomers. All CCBs transformed AhR in its DNA-binding form, as revealed by electromobility shift assay. Tested CCBs inhibited enzyme activities of CYP3A4 (benidipine (+)>(-); felodipine (-)>(+); isradipine (-)-(+)) and CYP2C9 (benidipine (-)>(+); felodipine (+)>(-); isradipine (-)>(+)). The data presented here might be of toxicological and clinical importance.
- MeSH
- blokátory kalciových kanálů farmakologie MeSH
- buněčné linie MeSH
- dihydropyridiny chemie farmakologie MeSH
- hepatocyty účinky léků enzymologie MeSH
- izoenzymy metabolismus MeSH
- lidé MeSH
- messenger RNA biosyntéza genetika MeSH
- nádorové buněčné linie MeSH
- receptory aromatických uhlovodíků účinky léků MeSH
- stereoizomerie MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- xenobiotika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
