The hepatic cytochrome p450 enzymes 1 A, 2A19 and 2E1 is very important for the elimination of skatole from the body of pigs. Impaired skatole metabolism, results in skatole accumulation, which give rise to off flavor of the meat. Several metabolites of skatole has been identified, however the role of these metabolites in the inhibition of the skatole metabolizing enzymes are not documented. Using microsomes from pigs and fish, we determined the ability of several skatole metabolites to inhibit CYP1 A, CYP2A19 and CYP2E1 dependent activity. Our results show that 2-aminoacetophenone is an inhibitor of porcine CYP2A19 and CYP2E1 activity, but not the piscine orthologues. In conclusion, there is species specific differences in the inhibition of CYP1 A and CYP2A19 dependent metabolism of probe substrates. This is relevant to the evaluation of different model systems and to the reduction of off flavor of meat.
- MeSH
- acetofenony toxicita MeSH
- červené maso analýza MeSH
- cytochrom P-450 CYP1A1 antagonisté a inhibitory metabolismus MeSH
- cytochrom P-450 CYP2E1 metabolismus MeSH
- druhová specificita MeSH
- inhibitory cytochromu P450 CYP2E1 toxicita MeSH
- jaterní mikrozomy účinky léků metabolismus MeSH
- játra účinky léků metabolismus MeSH
- kumariny toxicita MeSH
- nitrofenoly toxicita MeSH
- oxaziny toxicita MeSH
- potrava z moře (živočišná) analýza MeSH
- prasata MeSH
- ryby MeSH
- skatol toxicita MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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
Naturally- and anthropogenically-produced cresols could pose serious risks to fish health. In this study, three piscine CYP isoforms were investigated for their abilities to interact with p-cresol. Therefore, the activity of 7-ethoxyresorufin-O-deethylase (EROD), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD), and p-nitrophenol hydroxylase (PNPH) were evaluated in the hepatic microsomes of juvenile rainbow trout. Results showed that EROD activity was inhibited in a competitive manner, BFCOD activity was inhibited in presence the highest tested p-cresol concentration and PNPH activity was not affected. These results indicate that p-cresol might affect the ability of fish to metabolize numerous aromatic hydrocarbons and dioxin compounds, which are present in the aquatic environment.
1. Humans and animals are commonly exposed to indole-3-carbinol (I3C) and resveratrol (RES) via food or beverages. Moreover, these compounds have been demonstrated to potentially cause food-drug interactions. However, information about their combined effects is limited. Therefore, we investigated the effects of I3C and RES, both as single compounds and in combination, on cytochrome P450 1A and 3A activity and gene expression. 2. Using porcine microsomes, we demonstrated that RES caused non-competitive inhibition of CYP1A activity and un-competitive inhibition of CYP3A activity. Compared to the effect of single compounds, co-treatment with I3C and RES increased a degree of inhibition of CYP1A activity. 3. In porcine primary hepatocytes, treatment with I3C and RES resulted in induction of CYP1A1, CYP1A2 and CYP3A29 mRNA expression. 4. In conclusion, we demonstrated that both RES and I3C could cause food-drug interactions and that the combined effect could be more potent in doing so.
- MeSH
- cytochrom P-450 CYP1A1 antagonisté a inhibitory metabolismus MeSH
- cytochrom P-450 CYP3A genetika metabolismus MeSH
- hepatocyty účinky léků MeSH
- indoly farmakologie MeSH
- inhibitory cytochromu P450 CYP3A farmakologie MeSH
- interakce mezi potravou a léky MeSH
- jaterní mikrozomy účinky léků enzymologie MeSH
- prasata MeSH
- receptory aromatických uhlovodíků genetika MeSH
- regulace genové exprese enzymů účinky léků MeSH
- steroidní receptory genetika MeSH
- stilbeny farmakologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Deregulation of Wnt/β-catenin signalling plays an important role in the pathogenesis of colorectal cancer. Interestingly, this pathway has been recently implicated in transcriptional control of cytochrome P450 (CYP) family 1 enzymes, which are responsible for bioactivation of a number of dietary carcinogens. In the present study, we investigated the impact of inhibition of Wnt/β-catenin pathway on metabolism and genotoxicity of benzo[a]pyrene (BaP), a highly mutagenic polycyclic aromatic hydrocarbon and an efficient ligand of the aryl hydrocarbon receptor, which is known as a primary regulator of CYP1 expression, in cellular models derived from colorectal tumours. We observed that a synthetic inhibitor of β-catenin, JW74, significantly increased formation of BaP-induced DNA adducts in both colorectal adenoma and carcinoma-derived cell lines. Using the short interfering RNA (siRNA) targeting β-catenin, we then found that β-catenin knockdown in HCT116 colon carcinoma cells significantly enhanced formation of covalent DNA adducts by BaP and histone H2AX phosphorylation, as detected by (32)P-postlabelling technique and immunocytochemistry, respectively, and it also induced expression of DNA damage response genes, such as CDKN1A or DDB2. The increased formation of DNA adducts formed by BaP upon β-catenin knockdown corresponded with enhanced production of major BaP metabolites, as well as with an increased expression/activity of CYP1 enzymes. Finally, using siRNA-mediated knockdown of CYP1A1, we confirmed that this enzyme plays a major role in formation of BaP-induced DNA adducts in HCT116 cells. Taken together, the present results indicated that the siRNA-mediated inhibition of β-catenin signalling, which is aberrantly activated in a majority of colorectal cancers, modulated genotoxicity of dietary carcinogen BaP in colon cell model in vitro, via a mechanism involving up-regulation of CYP1 expression and activity.
- MeSH
- adukty DNA škodlivé účinky MeSH
- apoptóza MeSH
- benzopyren škodlivé účinky MeSH
- beta-katenin antagonisté a inhibitory genetika metabolismus MeSH
- cytochrom P-450 CYP1A1 antagonisté a inhibitory genetika metabolismus MeSH
- imunoenzymatické techniky MeSH
- karcinogeny životního prostředí škodlivé účinky MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- malá interferující RNA genetika MeSH
- messenger RNA genetika MeSH
- nádorové buňky kultivované MeSH
- nádory tračníku farmakoterapie etiologie patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- poškození DNA * MeSH
- proliferace buněk MeSH
- receptory aromatických uhlovodíků genetika metabolismus MeSH
- regulace genové exprese enzymů účinky léků MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The effects of clotrimazole (CLO) and dexamethasone (DEX), both detected in the aquatic environment, were assessed on inhibition of cytochrome P450 (CYP450) in hepatic microsomes of rainbow trout. Activity of three CYP450 isoforms: ethoxyresorufin O-deethylase (EROD; CYP1A), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD; CYP3A) and p-nitrophenol hydroxylase (PNPH; CYP2E1-like protein) was investigated in the presence of four concentrations of CLO and DEX. Clotrimazole in a concentration range of 1-100μM decreased the activity of EROD and BFCOD. The inhibition was reversible, as pre-incubation of the microsomes with CLO, before addition of the substrate, had no effect. EROD activity was non-competitively inhibited with a Ki of 0.5μM, and BFCOD activity revealed competitive inhibition with a Ki of 0.04μM. The relatively low Ki for CLO inhibition of EROD and BFCOD activity may indicate that the ability of CYP1A and CYP3A to metabolize xenobiotics is reduced in the presence of CLO. PNPH activity was not affected by CLO. DEX showed no inhibitory potency on any investigated reaction. CLO, but not DEX, inhibited EROD and BFCOD activity by different mechanisms.
- MeSH
- cytochrom P-450 CYP1A1 antagonisté a inhibitory metabolismus MeSH
- cytochrom P-450 CYP2E1 antagonisté a inhibitory metabolismus MeSH
- cytochrom P-450 CYP3A antagonisté a inhibitory metabolismus MeSH
- dexamethason chemie farmakologie MeSH
- jaterní mikrozomy účinky léků enzymologie MeSH
- kinetika MeSH
- klotrimazol chemie farmakologie MeSH
- Oncorhynchus mykiss metabolismus MeSH
- systém (enzymů) cytochromů P-450 antagonisté a inhibitory metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The JEG-3 choriocarcinoma cell line has been proposed as a model cell line of human placental trophoblast for induction studies via aryl hydrocarbon receptor (AHR). We examined whether glucocorticoid dexamethasone influences AHR-mediated induction of CYP1A1 enzyme in the JEG-3 cell line. We found that dexamethasone dose- and time-dependently suppresses CYP1A1 transactivation in gene reporter assays, CYP1A1 mRNA induction, and upregulation of 7-ethoxyresorufin-O-deethylase (EROD) activity by 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in JEG-3 cells. Co-transfection of JEG-3 cells with glucocorticoid receptor (GR) expression construct and treatment with dexamethasone abolished the effect of MC on CYP1A1 promoter construct in transient transfection gene reporter assays. RU486, a GR antagonist, suppressed the effect of dexamethasone on MC-induced transactivation of AHR responsive reporter constructs. We also found that dexamethasone stimulates both ligand-dependent and ligand-independent degradation of AHR but not of aryl hydrocarbon receptor nuclear translocator (ARNT) protein in JEG-3 cells. In experiments with proteasome inhibitors MG132 and bortezomib, we found that the degradation is not sensitive to proteasome inhibition in JEG-3. We can conclude that dexamethasone suppresses AHR-mediated CYP1A1 induction in JEG-3 cells through the unique mechanism of AHR-GR crosstalk, which involves accelerated degradation of AHR.
- MeSH
- aktivace transkripce MeSH
- cytochrom P-450 CYP1A1 antagonisté a inhibitory genetika metabolismus MeSH
- dexamethason farmakologie MeSH
- glukokortikoidy farmakologie MeSH
- interakce mezi receptory a ligandy MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- methylcholanthren toxicita MeSH
- nádorové buněčné linie MeSH
- placenta účinky léků metabolismus MeSH
- polychlorované dibenzodioxiny toxicita MeSH
- receptory aromatických uhlovodíků - jaderný translokátor genetika metabolismus MeSH
- receptory aromatických uhlovodíků metabolismus MeSH
- receptory glukokortikoidů antagonisté a inhibitory genetika metabolismus MeSH
- reportérové geny MeSH
- těhotenství MeSH
- upregulace MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. Individual susceptibility to AA-induced disease likely reflects individual differences in enzymes that metabolize AA. Herein, we evaluated AAI metabolism by human cytochrome P450 (CYP) 1A1 and 1A2 in two CYP1A-humanized mouse lines that carry functional human CYP1A1 and CYP1A2 genes in the absence of the mouse Cyp1a1/1a2 orthologs. Human and mouse hepatic microsomes and human CYPs were also studied. Human CYP1A1 and 1A2 were found to be principally responsible for reductive activation of AAI to form AAI-DNA adducts and for oxidative detoxication to 8-hydroxyaristolochic acid (AAIa), both in the intact mouse and in microsomes. Overall, AAI-DNA adduct levels were higher in CYP1A-humanized mice relative to wild-type mice, indicating that expression of human CYP1A1 and 1A2 in mice leads to higher AAI bioactivation than in mice containing the mouse CYP1A1 and 1A2 orthologs. Furthermore, an exclusive role of human CYP1A1 and 1A2 in AAI oxidation to AAIa was observed in human liver microsomes under the aerobic (i.e., oxidative) conditions. Because CYP1A2 levels in human liver are at least 100-fold greater than those of CYP1A1 and there exists a > 60-fold genetic variation in CYP1A2 levels in human populations, the role of CYP1A2 in AAI metabolism is clinically relevant. The results suggest that, in addition to CYP1A1 and 1A2 expression levels, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.
- MeSH
- adukty DNA metabolismus MeSH
- cytochrom P-450 CYP1A1 antagonisté a inhibitory genetika metabolismus MeSH
- cytochrom P-450 CYP1A2 antagonisté a inhibitory genetika metabolismus MeSH
- cytosol enzymologie MeSH
- dealkylace MeSH
- inhibitory enzymů farmakologie MeSH
- jaterní mikrozomy enzymologie MeSH
- játra účinky léků enzymologie MeSH
- karcinogeny metabolismus toxicita MeSH
- kyseliny aristolochové metabolismus toxicita moč MeSH
- lidé MeSH
- metabolická inaktivace MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši transgenní MeSH
- myši MeSH
- oxidace-redukce MeSH
- rekombinantní proteiny metabolismus MeSH
- urologické nádory chemicky indukované MeSH
- urotel účinky léků MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
OBJECTIVES: Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogens, which frequently occur in the human diet. Their metabolic activation to reactive species binding to DNA is mediated by cytochromes P450 (CYPs) 1A1 and 1A2. Thus, levels and activities of these CYPs are crucial for initiation of BaP- and PhPI-mediated carcinogenesis. Here, the effect of CYP1A1/2 induction due to their prototype flavonoid inducer, β-naphthoflavone (BNF), on BaP- and PhPI-derived DNA adduct formation in rats was examined. METHODS: Male rats pretreated with BNF were treated with a single dose of either carcinogen by oral gavage. Nuclease P1 version of 32P-postlabeling assay and online column-switching liquid chromatography-electrospray ionization-tandem mass spectrometry were used to detect and quantify covalent DNA adducts formed by BaP and PhIP in-vivo, respectively. Expression of CYP1A1/2 enzymes was examined by Western blot. Enzymatic activities of CYP1A1/2 were assessed using their marker substrates (ethoxyresorufin and methoxyresorufin). RESULTS: Treatment of rats with a single dose of BNF produced an increase in levels CYP1A1/2 and CYP1A1 proteins in liver and small intestine, respectively. An increase in CYP1A1 protein expression found in both organs correlated well with specific activities of these CYPs. The CYP1A1 expression levels and its specific activity in small intestine decreased along the length of the organ, being highest in its proximal part and lowest in its distal part. The BNF induction of CYP1A1/2 resulted in a significant increase in the formation of BaP- and PhIP-DNA adducts in liver and in the distal part of the small intestine, respectively. Thus, pretreatment of rats with BNF did not prevent the PhIP and BaP activation, but vice versa, enhanced their genotoxicity. CONCLUSIONS: The results of this study demonstrate that the administration of only a single dose of CYP-inducing flavonoid prior to the intake of food carcinogens may increase the risk of a tumor formation.
- MeSH
- beta-naftoflavon farmakologie MeSH
- cytochrom P-450 CYP1A1 antagonisté a inhibitory genetika metabolismus MeSH
- enzymová indukce účinky léků MeSH
- inhibitory enzymů farmakologie MeSH
- játra účinky léků enzymologie metabolismus MeSH
- karcinogeny životního prostředí izolace a purifikace toxicita MeSH
- kontaminace potravin analýza MeSH
- krysa rodu rattus MeSH
- poškození DNA účinky léků MeSH
- potkani Wistar MeSH
- potraviny toxicita MeSH
- preklinické hodnocení léčiv MeSH
- tenké střevo účinky léků enzymologie metabolismus MeSH
- testy genotoxicity MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The flavonolignan silybin and its derivative dehydrosilybin have been proposed as candidate UV-protective agents in skin care products. This study addressed the effect of silybin and dehydrosilybin on the activity of cytochrome P450 isoform CYP1A1 in human keratinocytes (HaCaT) and human hepatoma cells (HepG2). CYP1A1 catalytic activity was assessed as O-deethylation of 7-ethoxyresorufin using fluorescence detection. Silybin and dehydrosylibin inhibited basal and dioxin-inducible CYP1A1 catalytic activity in both cell lines used. The inhibitory effect of tested compounds was more pronounced in HaCaT cells than in HepG2 cells, and dehydrosilybin was a much stronger inhibitor than silybin. Analyses on CYP1A1 human recombinant protein yielded IC(50) values of 22.9 +/- 4.7 micromol/L and 0.43 +/- 0.04 micromol/L for silybin and dehydrosilybin, respectively. Since CYP1A enzymes are some of the most prominent actors in the process of chemically induced carcinogenesis, the inhibitory activity of the flavonolignans tested against CYP1A1 favors their use as cytoprotective agents in terms of skin and hepatic metabolism. In addition, the capability of dehydrosilybin to inhibit CYP1A1 in submicromolar concentrations makes this compound a potential biological probe in CYP1A1 analyses.
- MeSH
- buněčné linie MeSH
- časové faktory MeSH
- cytochrom P-450 CYP1A1 antagonisté a inhibitory metabolismus MeSH
- dioxiny MeSH
- enzymová indukce MeSH
- financování organizované MeSH
- hepatocelulární karcinom enzymologie MeSH
- keratinocyty enzymologie MeSH
- lidé MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- nádory jater enzymologie MeSH
- silymarin farmakologie MeSH
- viabilita buněk MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH