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The effect of the flavonoid myricetin on the transport of the pro-carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through differentiated Caco-2 monolayers, a model for the intestinal epithelium, is described. Myricetin causes an increase of the transport of PhIP from the apical to the basolateral compartment. This effect was observed at physiologically relevant concentrations of PhIP and myricetin. Cyclosporin A (MRP2 inhibitor) but not PSC833 (P-gp inhibitor) showed a similar effect on PhIP transport. The results indicate that myricetin induces an increased basolateral uptake of the pro-carcinogen PhIP, in part through inhibition of the MRP2 mediated excretion of PhIP from the intestinal cells back to the lumen. © 2005 Elsevier Ireland Ltd. All rights reserved.

In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC50 values for cellular vincristine transport inhibition by myricetin were 30.5 ± 1.7 μM for MRP1 and 24.6 ± 1.3 μM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC50 value of 1.1 ± 0.1 μM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC50 values of 33.1 ± 1.9 and 22.2 ± 1.4 μM, respectively. In both the MRP1 and MRP2 cells, exposure to 25 μM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC50 values of 7.6 ± 0.5 and 5.8 ± 0.5 μM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance. © 2005 Elsevier Inc. All rights reserved.

The present study characterises the effect of phase II metabolism, especially methylation and glucuronidation, of the model flavonoid quercetin on its capacity to inhibit human MRP1 and MRP2 activity in Sf9 inside-out vesicles. The results obtained reveal that 3′-O-methylation does not affect the MRP inhibitory potential of quercetin. However, 4′-O-methylation appeared to reduce the potential to inhibit both MRP1 and MRP2. In contrast, glucuronidation in general, and especially glucuronidation at the 7-hydroxylmoiety, resulting in 7-O-glucuronosyl quercetin, significantly increased the potential of quercetin to inhibit MRP1 and MRP2 mediated calcein transport with inhibition of MRP1 being generally more effective than that of MRP2. Overall, the results of this study reveal that the major phase II metabolites of quercetin are equally potent or even better inhibitors of human MRP1 and MRP2 than quercetin itself. This finding indicates that phase II metabolism of quercetin could enhance the potential use of quercetin- or flavonoids in general-as an inhibitor to overcome MRP-mediated multidrug resistance. © 2007 Elsevier Inc. All rights reserved.

The present study describes the effect of different flavonoids on the absorption of the pro-carcinogen PhIP through Caco-2 monolayers and the development of an in silico model describing this process taking into account passive diffusion and active transport of PhIP. Various flavonoids stimulated the apical to basolateral PhIP transport. Using the in silico model for flavone, kaempferol and chrysoeriol, the apparent Ki value for inhibition of the active transport to the apical side was estimated to be below 53 μM and for morin, robinetin and taxifolin between 164 and 268 μM. For myricetin, luteolin, naringenin and quercetin, the apparent Ki values were determined more accurately and amounted to 37.3, 12.2, 11.7 and 5.6 μM respectively. Additional experiments revealed that the apical to basolateral PhIP transport was also increased in the presence of a typical BCRP or MRP inhibitor with apparent Ki values in the same range as those of the flavonoids. This observation together with the fact that flavonoids are known to be inhibitors of MRPs and BCRP, corroborates that inhibition of these apical membrane transporters is involved in the flavonoid-mediated increased apical to basolateral PhIP transport. Based on the apparent Ki values obtained, it is concluded that the flavonols, at the levels present in the regular Western diet, are capable of stimulating the transport of PhIP through Caco-2 monolayers from the apical to the basolateral compartment. This points to flavonoid-mediated stimulation of the bioavailability of PhIP and, thus, a possible adverse effect of these supposed beneficial food ingredients. © 2006 Elsevier Inc. All rights reserved.

To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of α,β-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. α,β-Unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition. © 2005 Elsevier Inc. All rights reserved.

In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated calcein transport in a cellular model. Several of the methoxylated flavonoids are among the best MRP1 inhibitors (IC50 values, ranging between 2.7 and 14.3 μM) followed by robinetin, myricetin and quercetin (IC50 values ranging between 13.6 and 21.8 μM). Regarding inhibition of MRP2 activity especially robinetin and myricetin appeared to be good inhibitors (IC 50 values of 15.0 and 22.2 μM, respectively). Kinetic characterization revealed that the two transporters differ marginally in the apparent Km for the substrate calcein. For one flavonoid, robinetin, the kinetics of inhibition were studied in more detail and revealed competitive inhibition with respect to calcein, with apparent inhibition constants of 5.0 μM for MRP1 and 8.5 μM for MRP2. For inhibition of MRP1, a quantitative structure activity relationship (QSAR) was obtained that indicates three structural characteristics to be of major importance for MRP1 inhibition by flavonoids: the total number of methoxylated moieties, the total number of hydroxyl groups and the dihedral angle between the B- and C-ring. Regarding MRP2 mediated calcein efflux inhibition, only the presence of a flavonol B-ring pyrogallol group seems to be an important structural characteristic. Overall, this study provides insight in the structural characteristics involved in MRP inhibition and explores the differences between inhibitors of these two transporters, MRP1 and MRP2. Ultimately, MRP2 displays higher selectivity for flavonoid type inhibition than MRP1. © 2004 Elsevier Inc. All rights reserved.

The objective of this study was to investigate the structural requirements necessary for inhibition of glutathione S-transferase P1-1 (GSTP1-1) and GS-X pump (MRP1 and MRP2) activity by structurally related flavonoids, in GSTP1-1 transfected MCF7 cells (pMTG5). The results reveal that GSTP1-1 activity in MCF7 pMTG5 cells can be inhibited by some flavonoids. Especially galangin was able to inhibit almost all cellular GSTP1-1 activity upon exposure of the cells to a concentration of 25μM. Other flavonoids like kaempferol, eriodictyol and quercetin showed a moderate GSTP1-1 inhibitory potential. For GSTP1-1 inhibition, no specific structural requirements necessary for potent inhibition could be defined. Most flavonoids appeared to be potent GS-X transport inhibitors with IC50 values ranging between 0.8 and 8μM. Luteolin and quercetin were the strongest inhibitors with IC50 values of 0.8 and 1.3μM, respectively. Flavonoids without a C2-C3 double bond like eriodictyol, taxifolin and catechin did not inhibit GS-X pump activity. The results of this study demonstrate that the structural features necessary for high potency GS-X pump inhibition by flavonoids are (1) the presence of hydroxyl groups, especially two of them generating the 3′,4′- catechol moiety; and (2) a planar molecule due to the presence of a C2-C3 double bond. Other factors, like lipophilicity and the total number of hydroxyl groups do not seem to be dominating the flavonoid-mediated GS-X pump inhibition. To identify the GS-X pump responsible for the DNP-SG efflux in MCF7 cells, the effects of three characteristic flavonoids quercetin, flavone and taxifolin on MRP1 and MRP2 activity were studied using transfected MDCKII cells. All three flavonoids as well as the typical MRP inhibitor (MK571) affected MRP1-mediated transport activity in a similar way as observed in the MCF7 cells. In addition, the most potent GS-X pump inhibitor in the MCF7 cells, quercetin, did not affect MRP2-mediated transport activity. These observations clearly indicate that the GS-X pump activity in the MCF7 cells is likely to be the result of flavonoid-mediated inhibition of MRP1 and not MRP2. Altogether, the present study reveals that a major site for flavonoid interaction with GSH-dependent toxicokinetics is the GS-X pump MRP1 rather than the conjugating GSTP1-1 activity itself. Of the flavonoids shown to be most active especially quercetin is frequently marketed in functional food supplements. Given the physiological levels expected to be reached upon supplement intake, the IC50 values of the present study point at possible flavonoid-drug and/or flavonoid-xenobiotic interactions especially regarding transport processes involved in toxicokinetics. © 2004 Elsevier Inc. All rights reserved.

1-{7-[(1-(3,5-Diethoxyphenyl)-3-{[(3,5-difluorophenyl)(ethyl)amino]carbo nyl}-4-oxo-1,4-dihydroquinolin-7-yl)oxy]heptyl}-1-methylpiperidinium bromide, R-146224, is a potent, specific ileum apical sodium-dependent bile acid transporter (ASBT) inhibitor; concentrations required for 50% inhibition of [3H]taurocholate uptake in human ASBT-expressing HEK-293 cells and hamster ileum tissues were 0.023 and 0.73 μM, respectively. In bile-fistula rats, biliary and urinary excretion 48 h after 10 mg/kg [14C]R-146224, were 1.49 ± 1.75% and 0.14 ± 0.05%, respectively, demonstrating extremely low absorption. In hamsters, R-146224 dose-dependently reduced gallbladder bile [3H]taurocholate uptake (ED50: 2.8 mg/kg). In basal diet-fed hamsters, 14-day 30-100 mg/kg R-146224 dose-dependently reduced serum total cholesterol (∼ 40%), high density lipoprotein (HDL) cholesterol (∼ 37%), non-HDL cholesterols (∼ 20%), and phospholipids (∼ 20%), without affecting serum triglycerides, associated with reduced free and esterified liver cholesterol contents. In normocholesterolemic cynomolgus monkeys, R-146224 specifically reduced non-HDL cholesterol. In human ileum specimens, R-146224 dose-dependently inhibited [3H]taurocholate uptake. Potent non-systemic ASBT inhibitor R-146224 decreases bile acid reabsorption by inhibiting the ileal bile acid active transport system, resulting in hypolipidemic activity. © 2006 Elsevier B.V. All rights reserved. Chemicals / CAS: carbon 14, 14762-75-5; cholesterol, 57-88-5; taurocholic acid, 145-42-6, 59005-70-8, 81-24-3; tritium, 10028-17-8; carrier protein, 80700-39-6; sodium, 7440-23-5; 1-(7-((1-(3,5-diethoxyphenyl)-3-(((3,5-difluorophenyl)(ethyl)amino)carbonyl)-4-oxo-1,4-dihydroquinolin-7-yl)oxy)heptyl)-1-methylpiperidinium bromide; Anticholesteremic Agents; Bile Acids and Salts; Cholesterol, 57-88-5; Membrane Transport Proteins; Organic Anion Transporters, Sodium-Dependent; Piperidines; Quinolines; Sodium, 7440-23-5; sodium-bile acid cotransporter, 145420-23-1; Symporters; Taurocholic Acid, 81-24-3