Article pubs.acs.org/JAFC
Effects of Anthocyanidins and Anthocyanins on the Expression and Catalytic Activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in Primary Human Hepatocytes and Human Liver Microsomes Alzbeta Srovnalova,†,# Michaela Svecarova,†,# Michaela Kopecna Zapletalova,‡,# Pavel Anzenbacher,‡ Petr Bachleda,§ Eva Anzenbacherova,*,∥ and Zdenek Dvorak*,† †
Department of Cell Biology and Genetics, Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacky University, Slechtitelu 11, 783 71 Olomouc, Czech Republic ‡ Institute of Pharmacology, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic § Second Department of Surgery, University Hospital Olomouc, I. P. Pavlova 6, 775 20 Olomouc, Czech Republic ∥ Institute of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic ABSTRACT: Anthocyanidins and anthocyanins are pharmacologically active constituents of various berry fruits, such as blueberry and cranberry. These compounds are also contained in massively used nutritional supplements based on extracts or dry matter from berry fruits. The current study evaluated the effects of anthocyanidins and anthocyanins on the expression and catalytic activity of major drug-metabolizing enzymes CYP2C9, CYP2A6, CYP2B6, and CYP3A4 in primary cultures of human hepatocytes and human liver microsomes. Expression of mRNA was quantified by qRT-PCR. Expression of proteins was evaluated by Western blotting and immunochemiluminescence. The catalytic activity of CYP enzymes was measured by HPLC using specific enzyme substrates. Tested anthocyanidins (6) and anthocyanins (21) did not induce the expression of mRNA and protein of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 genes in human hepatocytes. Catalytic activities of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 enzymes were inhibited by all anthocyanidins to different extents (e.g., delphinidin inhibits CYP3A4 by >90% at 100 μM with IC50 = 32 μM). Of 21 anthocyanins tested, only cyanidin-3-O-rhamnoside (CYP3A4 by >75% at 100 μM with IC50 = 44 μM) and two glycosides of delphinidin significantly inhibited examined cytochromes P450. It may be concluded that in the ranges of common ingestion of either food or dietary supplement an induction or significant inhibition of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 activity is most probably not expected. KEYWORDS: anthocyans, cytochrome P450, food−drug interactions, human hepatocytes, xenobiotics
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biological effects, including antiproliferative,7 antiapoptotic,8 antitumor,9 antimutagenic,10 antioxidant,11 antiradical,12 and nitric oxide inhibitory effects.13 Despite numerous studies of anthocyan biological activities, a systematic study focused on the interactions between anthocyans and drug-metabolizing cytochromes P450 was not carried out yet. Because common foods, beverages, and food supplements contain various natural or synthetic xenobiotics, including anthocyans, a phenomenon of food−drug interactions emerged. Dietary xenobiotics can interfere with drugmetabolizing enzymes and may cause their inhibition or induction. Indeed, we recently described the activation of the aryl hydrocarbon receptor (AhR) and induction of drugmetabolizing CYP1A1 in human cancer cell lines and human hepatocytes by anthocyanidin pelargonidin14 and by anthocyanins pelargonidin-3-O-rutinoside and cyanidin-3,5-O-diglucoside.15 In the current study, we have evaluated the effects of 21 anthocyanins and 6 anthocyanidins on the main drugmetabolizing cytochromes P450, those being CYP2A6,
INTRODUCTION The anthocyans (in Greek anthos means flower and kyanos means blue) are widespread natural flavonoids that occur in all tissues of higher plants (flowers, leaves, roots, stems, fruits, and vegetables) as water-soluble vacuolar pigments. They have significant roles in the animal kingdom in pollination and seed dispersal.1 The stability and color of anthocyans depend on the pH and the occurrence of chelating metal ions.2 The structural differences between anthocyans are the number of the hydroxyl groups; the degree of methylation of −OH groups; the number, nature, and position of sugar attachments; and the number and nature of aliphatic or aromatic acids fixed to sugars in the molecule.3 The most common sugars are glucose, galactose, and rhamnose.4 Anthocyans comprise two types of compounds, that is, anthocyanins and anthocyanidins, which are aglycon (sugar-free) backbones of anthocyanins (for structures see Table 1). Anthocyanins are found in plant foods including cereals, tubers, roots, pulses, etc., and also in beverages and dietary supplements.5 The most common anthocyanidins in higher plants include cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin.1,5 The anthocyans are important for their various health benefits.4,6 They play an important role in obesity and diabetes control, neuronal and cardiovascular disease prevention, and improvement of visual and brain functions.5 Anthocyans display a plethora of © 2014 American Chemical Society
Received: Revised: Accepted: Published: 789
October 16, 2013 December 20, 2013 January 5, 2014 January 6, 2014 dx.doi.org/10.1021/jf404643w | J. Agric. Food Chem. 2014, 62, 789−797
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Table 1. Chemical Structures of Anthocyanins and Anthocyanidins
R1 PEL-1 PEL-2 DEL-1 DEL-2 DEL-3 DEL-4 DEL-5 MAL-1 MAL-2 MAL-3 CYA-1 CYA-2 CYA-3 CYA-4 CYA-5 CYA-6 CYA-7 CYA-8 CYA-9
pelargonidin-3,5-di-O-glucoside chloride pelargonidin-3-O-rutinoside chloride delphinidin-3-O-glucoside chloride delphinidin-3-O-rutinoside chloride delphinidin-3,5-di-O-glucoside chloride delphinidin-3-O-sambubioside chloride delphinidin-3-O-rhamnoside chloride malvidin-3-O-glucoside chloride malvidin-3,5-di-O-glucoside chloride malvidin-3-O-galactoside chloride cyanidin-3-O-glucoside chloride cyanidin-3-O-rutinoside chloride cyanidin-3,5-di-O-glucoside chloride cyanidin-3-O-sophoroside chloride cyanidin-3-O-arabinoside chloride cyanidin-3-O-rhamnoside chloride cyanidin-3-O-galactoside chloride cyanidin-3-O-sambubioside chloride cyanidin-3-O-lathyroside chloride pelargonidin chloride cyanidin chloride delphinidin chloride petunidin chloride malvidin chloride peonidin chloride
Anthocyanins H H OH OH OH OH OH OCH3 OCH3 OCH3 OH OH OH OH OH OH OH OH OH Anthocyanidins H OH OH OCH3 OCH3 OCH3
R3
R4
R5
H H H H H H H H H H H H H H H H H H H
H H OH OH OH OH OH OCH3 OCH3 OCH3 H H H H H H H H H
glucoside rutinoside glucoside rutinoside glucoside sambubioside rhamnoside glucoside glucoside galactoside glucoside rutinoside glucoside sophoroside arabinoside rhamnoside galactoside sambubioside lathyroside
glucose H H H glucose H H H glucose H H H glucose H H H H H H
H H H H H H
H H OH OH OCH3 H
H H H H H H
H H H H H H
sambubioside chloride (DEL-4), delphinidin-3-O-rhamnoside chloride (DEL-5), malvidin-3-O-glucoside chloride (MAL-1), malvidin-3,5-diO-glucoside chloride (MAL-2), malvidin-3-O-galactoside chloride (MAL-3), cyanidin-3-O-glucoside chloride (CYA-1), cyanidin-3-Orutinoside chloride (CYA-2), cyanidin-3,5-di-O-glucoside chloride (CYA-3), cyanidin-3-O-sophoroside chloride (CYA-4), cyanidin-3-Oarabinoside chloride (CYA-5), cyanidin-3-O-rhamnoside chloride (CYA-6), cyanidin-3-O-galactoside chloride (CYA-7), cyanidin-3-Osambubioside chloride (CYA-8), cyanidin-3-O-lathyroside chloride (CYA-9), cyanidin chloride (CYA), delphinidin chloride (DEL), malvidin chloride (MAL), peonidin chloride (PEO), petunidin chloride (PET), and pelargonidin chloride (PEL). Luciferase lysis buffer and P450-Glo CYP1A1 assay were from Promega (www. promega.com; Hercules, CA, USA). Oligonucleotide primers used in RT-PCR reactions were from Invitrogen. LightCycler FastStart DNA MasterPLUS SYBR Green I was from Roche Diagnostic Corp. (Intes Bohemia, Czech Republic). Cryopreserved human liver microsomes (pooled) were purchased from Biopredic International (Rennes, France). Microsomes were obtained under approval of the local ethics committee and in accordance with the ethic regulations of the country of origin (France). They were from 20 males and 6 females with a protein content of 25 mg/mL; the CYP1A1/2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, and CYP3A4/5 enzyme activities were verified prior to the experiment. For determination of CYP activities, testosterone (CYP3A4), coumarin (CYP2A6), 7-ethoxy-4-(trifluoromethyl)coumarin (CYP2B6), and diclofenac (CYP2C9) as well as respective metabolites
CYP2B6, CYP2C9, and CYP3A4. We measured (i) induction = expression of CYPs at the levels of mRNA and protein in primary cultures of human hepatocytes and (ii) inhibition = catalytic activity of CYPs in human liver microsomes. Tested anthocyanins did not induce CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in primary human hepatocytes. On the other hand, CYP3A4 activity was inhibited by all anthocyanidins tested (with delphinidin at a concentration of 100 μM down to 8% of the original activity). The inhibition of CYP2C9 by anthocyanidins was prominent with pelargonidin and peonidin only, whereas CYP2A6 and CYP2B6 were not inhibited. Of the anthocyanins tested, only cyanidin-3-O-rhamnoside inhibited activities of all CYP isoforms examined, with the strongest effect on CYP3A4 activity (inhibition down to 25% of the control).
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R2
MATERIALS AND METHODS
Compounds and Reagents. Dimethyl sulfoxide (DMSO), rifampicin (RIF), and hygromycin B were purchased from SigmaAldrich (Prague, Czech Republic). The following anthocyanins and anthocyanidins were purchased from Extrasynthese (Lyon, France): peonidin-3-O-glucoside chloride (PEO-1), peonidin-3-O-rutinoside chloride (PEO-2), pelargonidin-3,5-di-O-glucoside chloride (PEL-1), pelargonidin-3-O-rutinoside chloride (PEL-2), delphinidin-3-O-glucoside chloride (DEL-1), delphinidin-3-O-rutinoside chloride (DEL-2), delphinidin-3,5-di-O-glucoside chloride (DEL-3), delphinidin-3-O790
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7-hydroxy-4-(trifluoromethyl)coumarin and 7-hydroxycoumarin were supplied by Sigma-Aldrich; 6β-hydroxytestosterone was from Ultrafine (Manchester, UK), and 4-hydroxydiclofenac was supplied by BD Biosciences (San Jose, CA, USA). All other chemicals were of the highest quality commercially available. Human Hepatocytes. Human hepatocytes (six different primary cultures) were obtained from two sources: (i) human liver obtained from multiorgan donors LH44 (F, 57 years), LH45 (M, 46 years), LH46 (M, 37 years), LH47 (M, 47 years), and LH49 (M, 38 years); tissue acquisition protocol was in accordance with the requirements issued by the local ethical commission in the Czech Republic; (ii) long-term human hepatocytes in monolayer batch HEP220670 (F, 64 years) (Biopredic International, Rennes, France). Hepatocytes were treated in a serum-free medium for 24 or 48 h with the tested compounds, RIF (10 μM), and/or vehicle (DMSO; 0.1% v/v). Cultures were maintained at 37 °C and 5% CO2 in a humidified incubator. Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). Total RNA was isolated using TRI reagent, and cDNA was synthesized according to the common protocol, using M-MLV reverse transcriptase F-572 (Finnzymes) and random hexamers 3801 (Takara). qRT-PCR was carried out on a Light Cycler apparatus 480 II (Roche Diagnostic Corp., Prague, Czech Republic). The levels of CYP2A6, CYP2B6, CYP2C9, CYP3A4, and GAPDH mRNAs were determined as described elsewhere.16 The measurements were performed in triplicates. Gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene. Data were processed according to the delta−delta method. Protein Detection and Western Blotting. Total protein extracts were prepared, and they were subjected to SDS-PAGE separation and Western blot transfer. The blots were probed with antibodies against CYP2A6 (mouse monoclonal; sc-53615, F16 P2 D8), CYP2B6 (rabbit polyclonal; sc-67224, H-110), CYP2C9 (goat polyclonal; sc-23436, C-21), CYP3A4 (mouse monoclonal; sc-53850, HL3), and β-actin (goat polyclonal; sc-1616, I-19), all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Chemiluminescent detection was performed using horseradish peroxidase-conjugated secondary antibody and an Amersham (GE Healthcare) ECL kit. Enzyme Activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in Human Liver Microsomes. Activities of individual CYP isoforms were measured according to published protocols.17 The following microsomal CYP activities were tested: CYP3A4, testosterone 6β-hydroxylation; CYP2A6, coumarin 7-hydroxylation; CYP2B6, 7-ethoxy-4-(trifluoromethyl)coumarin 7-deethylation; and CYP2C9 activity, diclofenac 4′-hydroxylation. Reaction mixtures of individual determinations contained, for determination of CYP2A6, CYP2B6, or CYP2C9 activity, 35 pmol P450 and for determination of CYP3A4 activity, the amount of microsomal fraction with 100 pmol of P450. All reaction mixtures were buffered by 100 mM K/PO4 (pH 7.4) and contained a NADPH generating system consisting of isocitrate dehydrogenase (9 U/mlL), NADP+ (0.5 mM), isocitric acid (4 mM), and MgSO4 (5 mM). Final concentrations of substrates in the corresponding reaction mixtures were as follows: CYP2A6, coumarin (10 μM); CYP2B6, 7-ethoxy-4(trifluoromethyl)coumarin (15 μM); CYP2C9, diclofenac (16 μM); CYP3A4, testosterone (100 μM). Aqueous stock solutions of anthocyanins were 1 mM (pH 6.7); anthocyanidins of the same concentration were dissolved in aqueous solution of pH 3.5. For detailed description of the incubations, see the previous papers.14,15 For each enzyme assay, a preliminary experiment was done to determine the KM and Vmax for a given enzyme reaction and to obtain the values of substrate concentrations suitable for the inhibition experiments. Inhibition experiments were routinely performed with six concentrations of the tested compound (0, 10, 20, 40, 80, and 100 μM in the reaction mixture). All determinations were done in triplicates. The effects of anthocyans on CYP activities were determined according to methods indicated above by HPLC using the Prominence system (Shimadzu, Kyoto, Japan). Sigma Plot v. 10.0 (Systat, Chicago, IL, USA) was used for plotting the results. The inhibition was expressed as the relative activity (control, 100%) remaining after addition of the respective inhibitor.
Article
RESULTS
Effects of Anthocyanidins on the Expression of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in Human Hepatocytes. Six major anthocyanidins (i.e., cyanidin, peonidin, petunidin, pelargonidin, delphinidin, malvidin) were examined in three different primary cultures of human hepatocytes (i.e., LH44, LH45, HEP220670). Cells were incubated with tested compounds (50 μM), model panel CYP2/3 inducer rifampicin (RIF; 10 μM), and DMSO (0.1% v/v) as vehicle for control. The duration of the treatments was 24 and 48 h for mRNA and protein analyses, respectively. Rifampicin strongly induced CYP3A4 mRNA in all human hepatocyte cultures, LH44 (117-fold), LH45 (55-fold), and HEP220670 (45-fold). Consistently, CYP3A4 protein was strongly induced by rifampicin in all human hepatocyte cultures. None of the anthocyanidins tested induced the expression of CYP3A4 mRNA (Figure 1) or protein (Figure 2) in any of the three cultures used. Similarly, no induction of CYP2B6 mRNA and protein was observed for tested anthocyanidins. Strong induction of CYP2B6 protein and mRNA was achieved by rifampicin, yielding the inductions of mRNA by factors of 9-, 15-, and 22-fold, in cultures LH44, LH45, and HEP220670, respectively (Figures 1 and 2). The expression of CYP2C9 mRNA was increased by rifampicin by factors of 3-, 2-, and 48-fold, in cultures LH44, LH45, and HEP220670 (Figure 1), respectively, whereas the level of CYP2C9 protein was not significantly increased by rifampicin (Figure 2). The lack of CYP2C9 protein induction and weak CYP2C9 mRNA induction (LH44, LH45) by rifampicin is probably due to high constitutive levels of CYP2C9 in human hepatocytes. None of the anthocyanidins tested induced the expression of CYP2C9 mRNA (Figure 1) or protein (Figure 2) in any human hepatocyte culture. Rifampicin induced CYP2A6 mRNA in all human hepatocyte cultures, LH44 (3-fold), LH45 (4-fold), and HEP220670 (18-fold). Cyanidin, malvidin, delphinidin, and petunidin increased CYP2A6 mRNA levels, but the effects were not systematic in all hepatocyte cultures and occurred rather randomly, perhaps due to interindividual metabolic profiles (Figure 1). Rifampicin also induced CYP2A6 protein, whereas the tested anthocyanins had no systematic effects on CYP2A6 protein level in human hepatocyte cultures (Figure 2). Effects of Anthocyanins on the Expression of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in Human Hepatocytes. In the next series of experiments, the effects of 21 anthocyanins (for details see Materials and Methods) were tested in four different human hepatocyte cultures (LH45, LH46, LH47, LH49). For this purpose, hepatocytes were incubated for 24 and 48 h with tested compounds (50 μM), rifampicin (RIF; 10 μM), and vehicle (DMSO; 0.1% v/v). Rifampicin significantly induced CYP2A6 (3-, 4-, 8-, 5-fold), CYP2B6 (9-, 15-, 25-, 14-fold), CYP2C9 (3-, 2-, 5-, 2-fold), and CYP3A4 (117-, 55-, 121-, 13-fold) mRNAs in all human hepatocyte cultures. None of the anthocyanins tested induced CYP2B6, CYP2C9, or CYP3A4 mRNAs. Similarly as in the case of anthocyanidins, the expression of CYP2A6 mRNA was increased by some anthocyanins, but these effects were not reproducible between individual human hepatocyte cultures (Figure 3). The data from Western blot analyses of CYP3A4 and CYP2B6 proteins were consistent with those at mRNA level. Whereas rifampicin strongly induced CYP3A4 and CYP2B6 proteins, the tested anthocyanins had no effects (Figure 4). Rifampicin strongly induced CYP2A6 protein, but the tested anthocyanins had no
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Figure 1. Effects of anthocyanidins on the expression of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 mRNAs in human hepatocytes. Primary human hepatocyte cultures (LH44, LH45, HEP220670) were incubated for 24 h with cyanidin (50 μM), peonidin (50 μM), petunidin (50 μM), pelargonidin (50 μM), delphinidin (50 μM), malvidin (50 μM), rifampicin (10 μM), and DMSO (0.1% v/v) as a vehicle for control. Bar graphs show RT-PCR analyses of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 mRNAs. The data are the mean from triplicate measurements and are expressed as a fold-induction as compared to DMSO-treated cells. The data were normalized per GAPDH mRNA levels. ∗, value is significantly different from DMSO-treated cells (p < 0.05) as determined by Student’s t test.
Effects of Anthocyanidins and Anthocyanins on Catalytic Activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in Human Liver Microsomes. Enzyme activities were examined with anthocyanidins (and anthocyanins) in
effects, and CYP2A6 protein was not detectable in cells incubated with anthocyanins. The levels of CYP2C9 protein were not significantly and systematically altered either by rifampicin or by tested anthocyanins (Figure 4). 792
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activity (Figure 5; top right). All anthocyanidins tested inhibited the CYP2A6 enzyme activity (coumarin 4-hydroxylation), albeit to different extents. The strongest inhibition was attained by delphinidin, where the activity was inhibited down to 75% at the 100 μM concentration (Figure 5; bottom left). All other anthocyanidins exhibited concentration-dependent, however much lower, degrees of inhibition. Concentration-dependent inhibition of CYP2B6 activity (7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation) was observed with all anthocyanidins. On the other hand, the extent of inhibition was not greater than 20% only (Figure 5; bottom right). Interestingly, among the anthocyanins (i.e., the respective glycosides of anthocyanidins) CYA-6 (cyanidin-3-O-rhamnoside chloride) inhibited activities of all CYP isoforms examined with the strongest effects on the CYP3A4 activity (down to 25% of the control at 100 μM with IC50 = 44 μM). The CYP2C9, CYP2A6, and CYP2B6 activities were inhibited by CYA-6 down to 40% of the initial enzyme activity (Figure 6). Besides CYA-6, the CYP3A4 activity was inhibited also by DEL-2 (delphinidin3-O-rutinoside; down to 35% at 100 μM, IC50 = 67 μM), whereas other anthocyanins inhibited the CYP activities to lower extents; a more prominent decrease in the enzyme activity was observed for CYP2B6 and CYA-2 (to 45% at 100 μM) and for CYP2C9 with DEL-1 (to 55% at 100 μM concentration).
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DISCUSSION Metabolism of xenobiotics, including drugs, may be influenced by the presence of second xenobiotic, producing a phenomenon of drug−drug interaction. Because many natural and synthetic xenobiotics originate from food, a phenomenon of food−drug interactions was established. A mechanistic base for food−drug interaction may be either inhibition or induction of drugmetabolizing enzymes by xenobiotics. Therefore, in the current study, we have examined the effects of common dietary anthocyanidins (6) and anthocyanins (21) on the expression and catalytic activity of cytochromes P450 CYP2A6, CYP2B6, CYP2C9, and CYP3A4. The major findings of the study are that (i) tested anthocyans (i.e., both the anthocyanidins and anthocyanins) do not induce CYP2A6, CYP2B6, CYP2C9, and CYP3A4 mRNAs and proteins in primary human hepatocytes and that (ii) all of the anthocyanidins did inhibit the CYP3A4 activity; among anthocyanins, the enzyme activity of this isoform was also inhibited the most, although not so largely as with the anthocyanidin aglycons. Xenobiotic-mediated induction of cytochromes P450 CYP2A6, CYP2B6, CYP2C9, and CYP3A4 is controlled by various transcriptional regulators, including xenoreceptors (e.g., pregnane X receptor, PXR; constitutive androstane receptor, CAR), nuclear receptors (e.g., retinoic acid receptors, RARs; retinoid X receptors, RXRs; liver X receptor, LXR; farnesoid X receptor, FXR; vitamin D receptor, VDR), and receptors for steroid hormones (e.g., glucocorticoid receptor, GR; estrogen receptor, ER).18 The activators of listed receptors are therefore usually inducers of CYP2 and CYP3 genes, and vice versa. Importantly, xenoreceptors and steroid and nuclear receptors are important transcriptional regulators of many genes, involved in energy metabolism, cells growth, differenciation, immunity, etc. Activation of these receptors by xenobiotics often leads to so-called endocrine disruption, besides from CYP gene induction. In addition, induction of drug-metabolizing CYPs by xenobiotics may cause chemically induced cell damage or carcinogenesis. Overall, the finding that 27 dietary anthocyans are not inducers of human CYP2A6, CYP2B6,
Figure 2. Effects of anthocyanidins on the expression of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 proteins in human hepatocytes. Primary human hepatocytes cultures (LH44, LH45, HEP220670) were incubated for 48 h with cyanidin (50 μM), peonidin (50 μM), petunidin (50 μM), pelargonidin (50 μM), delphinidin (50 μM), malvidin (50 μM), rifampicin (10 μM), and DMSO (0.1% v/v) as a vehicle for control. Western blots show analyses of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 proteins. As a loading control, the blots were probed to β-actin (data not shown).
concentrations of 0, 10, 20, 40, 80, and 100 μM. The most prominent inhibitions of CYP activities were observed for CYP3A4 (testosterone 6-β-hydroxylation) and CYP2C9 (diclofenac 4′-hydroxylation). The enzyme activity of CYP3A4 was inhibited by at least down to 50% with all six anthocyanidins tested. The most prominent inhibitory effects were observed with delphinidin (down to 9% of the control value by 100 μM delphinidin with IC50 = 32 μM) and petunidin and peonidin (down to about 20% of the control at 100 μM; the IC50 values reached 31 μM for petunidin and 36 μM for peonidin). Pelargonidin inhibited this activity down to 35% at 100 μM concentration with IC50 = 55 μM. Inhibition of CYP3A4 activity by malvidin and cyanidin did not reach 50% of control value (Figure 5; top left). Significant inhibition of CYP2C9 was attained by pelargonidin, peonidin (down to 25−30% of the control value with 100 μM, IC50 = 25 and 21 μM) and petunidin (100 μM, IC50 = 61 μM), whereas cyanidin did not inhibit CYP2C9 enzyme 793
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Figure 3. Effects of anthocyanins on the expression of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 mRNAs in human hepatocytes. Primary human hepatocytes cultures (LH44, LH45, LH47, LH49) were incubated for 24 h with 21 different anthocyanins (for details see Materials and Methods), each at a concentration of 50 μM, rifampicin (10 μM), and DMSO (0.1% v/v) as a vehicle for control. Bar graphs show RT-PCR analyses of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 mRNAs. The data are the mean from triplicate measurements and are expressed as a fold-induction as compared to DMSO-treated cells. The data were normalized per GAPDH mRNA levels. ∗, value is significantly different from DMSO-treated cells (p < 0.05) as determined by Student’s t test. 794
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Figure 4. Effects of anthocyanins on the expression of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 proteins in human hepatocytes. Primary human hepatocytes cultures (LH44, LH45, LH47, LH49) were incubated for 48 h with 21 different anthocyanins (for details see Materials and Methods), each at a concentration of 50 μM, rifampicin (10 μM), and DMSO (0.1% v/v) as a vehicle for control. Representative Western blots show analyses of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 proteins in human hepatocytes cultures LH45 and LH49. Similar profiles were observed in cultures LH44 and LH47. As a loading control, the blots were probed to β-actin (data not shown).
Figure 5. Effects of anthocyanidins on catalytic activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 proteins in human liver microsomes. Inhibition of catalytic activities is expressed as the activity remaining relative to control (100%, without anthocyanidin) in percent. Concentration of respective anthocyanidins in the reaction mixture was 0, 10, 20, 40, 80, and 100 μM. For experimental details, see Materials and Methods. ∗, value significantly different from control (p < 0.05).
CYP2C9, and CYP3A4 is of positive value, with regard to massive use of anthocyan-containing foods and products in alimentation and herbal medicine. However, the anthocyans were able to inhibit the marker activities of CYP enzymes to a significant extent. In particular, CYP3A4 activity was inhibited by all anthocyanidins tested and at least with two anthocyanins (CYA-6 and DEL-2) to 25 and 35% at 100 μM, respectively. In other cases, the decrease of enzyme activity has exceed 50% only exceptionally, as in case of CYP2C9 with pelargonidin and peonidin (25−30% at 100 μM) and to 40% with cyanidin glycoside (CYA-6). Interestingly, the ability of anthocyanidins to inhibit an enzyme activity is not directly reflected in properties of corresponding anthocyanins. For example, it is the delphinidin that inhibits the CYP3A4 enzyme activity the most (Figure 5), but the most prominent inhibition of the same activity was
observed with a cyanidin glycoside (CYA-6). However, cyanidin aglycon (i.e., the anthocyanidin) inhibited the CYP to a much lesser extent (Figure 5). Hence, it seems that the ability of an anthocyanin to inhibit a particular CYP enzyme is given by the molecule as a whole; in other words, it cannot be simply derived from the property of the respective aglycon (anthocyanidin). The fact that it is the activity of CYP3A4 that is influenced the most by an interaction with an anthocyan molecule is clearly in line with the relative openness, accessibility, and flexibility of the active site of this enzyme, which is known to accommodate molecules with different sizes and properties.19−21 The most important question is the relevance of these interactions to human nutrition. The concentrations of anthocyans and anthocyanidins as well as anthocyanins in plasma may reach the micromolar range after ingestion of tens of milligrams of fruit. However, the level may 795
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Figure 6. Effects of anthocyanins on catalytic activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 proteins in human liver microsomes. Inhibition is expressed as in Figure 5; for abbreviations, see Compounds and Reagents. Anthocyanin concentrations were 0, 10, 20, 40, 80, and 100 μM. ∗, value significantly different from control (p < 0.05).
*(E.A.) E-mail:
[email protected]. Phone: +420-585632321. Fax: +420-58-5632302.
be much higher when a dietary supplement is taken or, more generally, at the site of action. However, it may be concluded that, in the ranges of common ingestion of either food or a dietary supplement, an induction or significant inhibition of, for example, CYP3A4 activity is most probably not expected.
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Author Contributions #
A.S., M.S., and M.K.Z. contributed equally.
Funding
Our laboratories are supported by Grant GACR 303/12/G163 from the Grant Agency of the Czech Republic, by a grant from the Czech Ministry of Health, IGA NT/13591, Operational Program Research and Development for Innovations - European Regional Development Fund (CZ.1.05/2.1.00/03.0058), and by student grants from Palacky University Olomouc, PrF-2013-002
AUTHOR INFORMATION
Corresponding Authors
*(Z.D.) E-mail:
[email protected]. Phone: +420-58-5634903. Fax: +420-58-5634901. 796
dx.doi.org/10.1021/jf404643w | J. Agric. Food Chem. 2014, 62, 789−797
Journal of Agricultural and Food Chemistry
Article
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and LF-2013-007. The infrastructural part of this work has been supported by Project CZ.1.05/2.1.00/01.0030 (BIOMEDREG). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED AhR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; CYP, cytochrome P450; ER, estrogen receptor; FXR, farnesoid X receptor; GR, glucocorticoid receptor; LXR, liver X receptor; PXR, pregnane X receptor; RARs, retinoic acid receptors; RXRs, retinoid X receptors; VDR, vitamin D receptor
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