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Quercetin is a substrate for the transmembrane oxidoreductase Dcytb
Free Radical Biology & Medicine 48 (2010) 1366–1369
Contents lists available at ScienceDirect
Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d
Original Contribution
Quercetin is a substrate for the transmembrane oxidoreductase Dcytb Evangelia Vlachodimitropoulou a, Richard J. Naftalin a,⁎, Paul A. Sharp b a b
Department of Physiology, King's College London, Waterloo Campus, London SE1 9HN, UK Division of Nutritional Sciences, King's College London, Waterloo Campus, London SE1 9HN, UK
a r t i c l e
i n f o
Article history: Received 15 October 2009 Revised 10 February 2010 Accepted 13 February 2010 Available online 22 February 2010 Keywords: Duodenal cytochrome b Flavonoids Ascorbate Iron absorption Redox balance Free radicals
a b s t r a c t Duodenal cytochrome b (Dcytb) is a transmembrane oxidoreductase protein found in apical membranes of duodenal enterocytes, as well as human erythrocytes, with the capacity to transport electrons donated by cytosolic ascorbate to extracellular electron receptors such as Fe(III), dehydroascorbate, or molecular O2. We have investigated the capacity of the flavonoid quercetin to act as an electron donor for Dcytb in a manner similar to that of ascorbate by observing the reduction of extracellular Fe(III) to Fe(II) in either Madin–Darby canine kidney (MDCK) cells overexpressing Dcytb (Dcytb+) or Dcytb-null MDCK cells. In Dcytb+ cells there is a saturable increase in extracellular Fe(III) reduction in response to increasing intracellular quercetin concentrations (Km = 6.53 ± 1.57 μM), in addition to a small linear response, whereas in Dcytb-null cells there is only a small linear increase in extracellular Fe(III) reduction. No extracellular Fe(III) reduction occurs in Dcytb-null cells when the cells are preloaded with ascorbate. Flavonoids such as quercetin at their physiological concentrations can therefore function as modulators of ferric reductases, enhancing the import of Fe(II) and also providing extracellular reducing potential. © 2010 Elsevier Inc. All rights reserved.
Flavonoids are polyphenolic compounds known to exert a number of biological effects, some of which may be attributed to their antioxidant properties. Quercetin is thought to be the most prevalent flavonoid in the Western diet. Its consumption has been estimated at 10–20 mg/day [1]. It has been shown to accumulate in rat adipocytes via the facilitated glucose transporters GLUTs 1 and 4 [2]. Quercetin also inhibits glucose and dehydroascorbate (DHA) transport via the GLUTs 1, 2, 3, and 4 in a reversible competitive manner [3,4]. In addition quercetin acts as a substrate for a plasma membrane oxidoreductase (PMOR) in erythrocytes, whose identity until now was unclear [5]. Recent work indicates that duodenal cytochrome b (Dcytb) fulfills this role [6]. Dcytb is a protein highly expressed on the brush border of duodenal enterocytes. It is localized alongside the divalent metal ion transporter (DMT1) on the apical membrane, and because of its ferric reductase activity Dcytb is believed to play an essential role in the absorption of nonheme Fe(II) iron from the human diet [7]. Its expression is iron regulated and it shares a structural homology
Abbreviations: AFR, ascorbyl free radical; Dcytb, duodenal cytochrome b; Fe(III), ferric iron; Fe(II), ferrous iron; DHA, dehydroascorbate; MDCK, Madin–Darby canine kidney cells; Dcytb+, MDCK cells overexpressing Dcytb; Dcytb−, MDCK cells lacking the Dcytb protein; Mops, 3-(N-morpholino)propanesulfonic acid; Mes, 2-(N-morpholino) ethanesulfonic acid; PMOR, plasma membrane oxidoreductase; DMT1, divalent metal ion transporter. ⁎ Corresponding author. Fax: +207 848 4600. E-mail address: [email protected] (R.J. Naftalin). 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.02.021
of approximately 40% with other members of the cytochrome b561 family. There are a total of six predicted α-helical transmembrane domains, four of which bind a histidine residue and form two heme-containing complexes. Evidence shows that these heme groups are reducible by 67% by the DHA/ascorbate redox couple [8]. There is general consensus that Dcytb is the primary ferric reductase responsible for uptake of dietary nonheme iron in the duodenum. However, recent studies involving Dcytb-knockout mice have given rise to some controversy as the rodents do not present with iron deficiency. Nonetheless, there is a clear decrease in iron uptake at the level of the enterocyte, and the lack of an irondeficient phenotype is most likely due to compensatory changes, possibly involving the protein ferroportin [9]. The rate of iron absorption is modulated by intracellular ascorbate concentrations, in that preloading cells with DHA leads to a highly significant extracellular ferric, as well as cupric, reduction [10]. DHA enters duodenal enterocytes via GLUTs 1 and 4 and increases intracellular ascorbate concentration [11,12]. Following evidence that flavonoids efficiently accumulate via GLUT transporters and act as intracellular substrates for a PMOR in erythrocytes [13], we here have investigated the potential modulatory role of quercetin in iron uptake via a mechanism of electron donation to the Dcytb protein, in a manner similar to that of ascorbate. Our findings demonstrate that the electron-donating capacity of Dcytb-expressing MDCK cells loaded with either DHA [10] or quercetin is greatly increased compared to wild-type MDCK cells (Fig. 1).
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Fig. 1. In our proposed model, GLUT transporters 1 and 4 aid DHA and quercetin accumulation in the intracellular space. In the cytosol DHA is reduced to ascorbate. Ascorbate and quercetin can donate electrons to the transmembrane oxidoreductase, Dcytb. This exports electrons across duodenal enterocyte apical membranes (and erythrocyte plasma membranes) and reduces nonbioavailable ferric to ferrous iron, thus aiding its assimilation from the gut via the transporter DMT1. Additionally, Dcytb is able to convert extracellular DHA to ascorbate via a two-electron-donating process, possibly by single-electron transference between Dcytb to AFR followed by dismutation of 2 AFR to ascorbate and DHA [23,25].
Materials and methods Transfection process and cell culture Cell transfection was carried out as previously described by Wyman et al. [10]. Briefly, PCR was used to amplify murine Dcytb cDNA from duodenal mRNA and create a pTRE2hyg-Dcytb-EGFP vector construct. The vector was transfected into Tetracycline-Off (TET-Off) MDCK cells, which were cultured for 2 weeks in Dulbecco's modified Eagle's medium, 10% TET-system-approved fetal bovine serum, 1 ng/ml puromycin, penicillin/streptomycin, and 200 μg/ml hygromycin. The expression of Dcytb-EGFP was controlled by the addition of 20 ng/ml doxycycline for a further 4 days. Ferric reductase assays The assay was performed on transfected Dcytb-EGFP and untransfected TET-Off MDCK cells that were grown on 24-well plates for at least 48 h to allow for cell attachment and confluency. Cells were treated with 0.5 ml of medium per well containing the reagents applicable to each experiment, for 30 min. The cells were then washed three times with phosphate-buffered saline (PBS) at pH 7.0 and incubated in physiological buffer (25 mM Mops, 25 mM Mes, 5.4 mM KCl, 5 mM glucose, 140 mM NaCl, 1.8 mM CaCl2, 800 μM MgCl2), 50 μM Fe(III)-nitrilotriacetic acid (NTA), and 200 μM 3-(2-pyridyl)-5,6diphenyl-1,2,4-triazine-4′,4″-disulfonic acid sodium salt (ferrozine) over a period of half an hour. Extracellular iron reduction was recorded via the formation of the colored Fe(II)–ferrozine complex monitored at 562 nm. All reactions were carried out in the dark, at 37°C, and absorbance values were converted into iron concentrations via the generation of standard curves [10]. It was noted, by recording the spectrophotometric change in ferrozine absorbance during the ferric reductase assay, that there is a direct concentration-dependent effect of quercetin on Fe(III) reduction to Fe(II) iron. Formation of the colored Fe(II)–ferrozine complex and the concentration of quercetin are positively correlated. There-
fore, to eliminate the possibility of flavonoid efflux leading to false assumptions that quercetin is responsible for the additional reducing effect seen in the presence of Dcytb and confirm our observations shown in Fig. 2, an alternative experimental approach was adopted. MDCK and Dcytb-transfected cells were treated with 100 μM quercetin solution, washed three times with PBS, and exposed to DMEM over a period of 1 h. The supernatant solution was then spectrophotometrically assayed, and with the aid of the reductase assay, only a minimal proportion of the original iron reduction could be accounted for by flavonoid efflux and hence a direct effect of quercetin on ferrozine. The percentage ranges between 4 and 6% and is consistent with the findings of Fiorani et al. [13] regarding flavonoid efflux from erythrocytes. They observed that the bulk of intracellular flavonones bind loosely to intracellular structures. This binding retards flavonone exit rates. These results reinforce the original hypothesis that the source of extracellular electrons stems from electrons donated to Dcytb at its intracellular site. Dcytb and MDCK cells were also preloaded with quercetin for variable time intervals between 0 and 90 min and then lysed to assess the intracellular flavonoid content with the aid of the ferrozine assay. Results indicated that the flavonoid readily accumulated in both cell types in less than 2 min. Results We have investigated whether quercetin has a similar modulatory role compared to ascorbate in iron uptake via a transmembrane electron-donating mechanism to the Dcytb protein. Cultured MDCK cells overexpressing the Dcytb gene (Dcytb+), as well as Dcytb-null MDCK cells, from which the protein is absent (Dcytb−), were loaded with variable concentrations of quercetin (0– 100 μM). In Dcytb+ cells, with increasing quercetin concentration, there is a saturable component, Km = 6.43 ± 1.57 μM (four experiments) and Vm = 0.92 ± 0.04 μmol μg cell protein−1 · h−1 (Figs. 2A and 2B). The Michaelis–Menten kinetics noted are similar to those obtained with preloading of cells with DHA; however, the Km and Vm
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for ascorbate is ≈ 100 times higher than observed here with quercetin, using the same cell culture system (see Discussion) [6]. This saturable response to quercetin is absent from Dcytb− cells. However, quercetin preloading also induced a quantitatively similar linear increase in extracellular iron reduction in both Dcytb+ and Dcytb− cells (Figs. 2A and 2B), which was absent with DHA (Fig. 3). In Fig. 3 it can be observed that the ratio of extracellular reduction in the Dcytb-expressing cells to that of the Dcytb-null cells when loaded with DHA is 18.00 ± 0.03, whereas when loaded with quercetin the ratio is 6.00 ± 0.153 (n = 6; P b 0.017). This indicates that the Dcytb-null cells allow approximately three times as much quercetin to escape from the cells as ascorbate. There are a number of potential pathways through which quercetin can pass, in particular, GLUTs and the monocarboxylate acid transporter pathways, both of which are expressed in MDCK cells and have been observed to bind and transport flavonones [4,14]. These findings indicate that quercetin acts as an electron donor to Dcytb and that the intracellular flavonoid reservoir [15], together with ascorbate, could act as a central modulator of inorganic iron assimilation in the duodenum and in erythrocytes, providing a source of electrons to maintain the reduced state of extracellular oxidizable substrates, e.g., ascorbate in plasma, where normally the concentration ratio of ascorbate:dehydroascorbate is maintained at ∼100 [16].
Discussion Our results further the understanding of the function of Dcytb in the maintenance of redox potentials and metal homeostasis. We have demonstrated that Dcytb has the capacity to reduce Fe(III), not only when the cells are loaded with DHA, but also in the presence of intracellular flavonoids, such as quercetin. Although Dcytb was discovered in the duodenal enterocytes [7], it is now known that it is expressed in a number of cell types, such as erythrocytes [6], Caco-2 cells [17], and lung epithelium [18]. Thus, electron donation to Dcytb by flavonoid compounds may play a crucial biological role in a variety of tissues.
Fig. 2. Dcytb-transfected and Dcytb-null cells were preloaded with variable quercetin concentrations (0–100 μΜ) over a period of 30 min, at 37°C. The cells were then washed three times with PBS and extracellular iron reduction was assessed with the aid of the ferrozine assay. Results have been adjusted for protein using the Bio-Rad DC protein assay kit. An increase in extracellular Fe3+ reduction is noted in both cells expressing Dcytb and Dcytb-null cells. There is a direct effect of quercetin on ferric iron reduction, as confirmed by the ferrozine assay. Increasing quercetin concentrations, 200 μM ferrozine, and 50 μM Fe (III)–NTA solution were incubated for 30 min, at 37°C, in the dark. (A) When cells are loaded over a wider concentration range the reductive capacity of Dcytb-expressing cells is observed to be saturable. At 50 μΜ extracellular quercetin, electron export is noted to be 0.65± 0.1 μM/μg protein/h in untransfected and 1.4 ± 0.1 μM/μg protein/h in Dcytbtransfected cells. No saturable effect is observed with Dcytb-null cells. (B) The difference between the two cell types is plotted. The best-fit Michaelis–Menten kinetic curve of the difference between the reducing capacities of Dcytb-expressing and Dcytb-null cells gives a Km ≈ 5 μM for the saturable component of quercetin-dependent extracellular reduction of ferrozine. (C) Effects of varying concentrations of quercetin from 0 to 10 μM on extracellular Fe3+ reduction in Dcytb+ and Dcytb− cells. There is a linear increase in extracellular reductive capacity in this concentration range (P b 0.001).
Fig. 3. Ferric reductase activity of 20 ng/ml Dcytb-transfected and untransfected MDCK cells treated with 100 μΜ DHA and 100 μΜ quercetin was recorded using the ferrozine assay. The data show that both ascorbate and quercetin loading at 100 μΜ led to similar rates of extracellular ferric reductase activity in Dcytb-expressing cells. There is a highly significant reduction in extracellular ferric reductase activity in Dcytb-null cells when preloaded with either ascorbate or quercetin. However, the data also show that a significantly higher proportion of quercetin than of ascorbate leaks from Dcytb-null cells, possibly because of the high affinity of quercetin for GLUT1 or lactate transporter expressed in these cells (see Discussion).
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There has been some debate about the precise nature of ascorbate– Dcytb interactions. It has been suggested that ascorbate is released directly into the extracellular fluid, perhaps via GLUTs or an alternative transport process, and directly interacts with ferricyanide (Fe(III)), at the external surface of the cell [19–21]. External ascorbate concentrations may be maintained by DHA reductase enzyme activity recycling either DHA or ascorbate free radical [22]. Wyman et al. [10] showed that the presence of ascorbate oxidase in extracellular fluid had no effect on extracellular copper or iron reduction in Dcytb-expressing cells preloaded with ascorbate. This indicates that the reducing capacity of ascorbate is entirely dependent on electron export via Dcytb and not on direct extracellular interaction between ascorbate and metal electron acceptors. The results in this paper support this view for two reasons. First, as Wyman et al. [10] observed with Dcytb-null MDCK cells, there is virtually no effect of intracellular ascorbate on ferrozine reduction, whereas a large reduction is observed when Dcytb is expressed (Fig. 3). Second, in contrast to ascorbate, which has a Km of 500– 1000 μM for Dcytb [6], quercetin has a much higher affinity (5–10 μM) and therefore the saturable response of the ferrozine reduction in Dcytb+ cells is more unambiguously indicative of a saturable process mediated by Dcytb than that seen with high intracellular ascorbate concentrations, observed previously [6,10]. The smaller linear component of quercetin-dependent extracellular ferrozine reduction could be due to a parallel release of quercetin, for example, via GLUT 1, which is present in MDCK cells [23] and to which quercetin binds with very high affinity, KD ≈ 500 nM [4]. In addition to its interaction with GLUT 1 [24], quercetin has also been reported to have the capacity to inhibit a number of other transporters, including the l-lactate transporter in hepatocytes [25] and lactate transport in human erythrocytes and Ehrlich ascites tumor cells [26]. The absence of this linear component with ascorbate as the substrate is probably due to very low intracellular concentrations of DHA that result from efficient intracellular conversion of membrane-permeative DHA to impermeative ascorbate [27]. The ascorbyl reductase system has been shown to maintain low extracellular concentrations of ascorbyl free radical [28], both in the gut lumen and in the blood plasma, and plays a protective role against a variety of extracellular oxidant stressors. It has been previously estimated that transmembrane oxidoreductase activity recycles human plasma ascorbate from DHA within 3 min and maintains it at a level of 50 μM during normal states of metabolism [27]. This intracellular ascorbate concentration is consistent with the KD of Dcytb [10]. Similarly, the concentration of unbound quercetin in plasma under normal physiological conditions is in the range 0.1–1 μM, which is of the same order as the Ki that we observed with quercetin in the preceding experiments. The antioxidant and iron-chelating properties of flavonoids have attracted attention for a number of years [29,30]. Their wide bioavailability in fruits and vegetables, their lack of toxicity in comparison with ascorbate, and their capacity to act as substrates for plasma oxidoreductases apparently provide an efficient supplemental role to ascorbate in maintaining redox balance [31]. Thus, both ascorbate and quercetin can be used within their physiological concentration ranges to reduce oxidized extracellular substrates. Acknowledgement EV is grateful to the Physiological Society and King's College London for a summer studentship and summer vacation award in 2008 and 2009 respectively. References [1] Hertog, M. G. L.; Hollman, P. C. H.; Katan, M. B.; Kromhout, D. Intake of potentially anticarcinogenic flavonoids and their determinants in adults in the Netherlands. Nutr. Cancer Int. J. 20:21–29; 1993.
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[2] Strobel, P.; Allard, C.; Perez-Acle, T.; Calderon, R.; Aldunate, R.; Leighton, F. Myricetin, quercetin and catechin-gallate inhibit glucose uptake in isolated rat adipocytes. Biochem. J. 386:471–478; 2005. [3] Park, J. B.; Levine, M. Intracellular accumulation of ascorbic acid is inhibited by flavonoids via blocking of dehydroascorbic acid and ascorbic acid uptakes in HL60, U937 and Jurkat cells. J. Nutr. 130:1297–1302; 2000. [4] Cunningham, P.; Afzal-Ahmed, I.; Naftalin, R. J. Docking studies show that dglucose and quercetin slide through the transporter GLUT1. J. Biol. Chem. 281: 5797–5803; 2006. [5] Fiorani, M.; Accorsi, A. Dietary flavonoids as intracellular substrates for an erythrocyte trans-plasma membrane oxidoreductase activity. Br. J. Nutr. 94:338–345; 2005. [6] Su, D.; May, J. M.; Koury, M. J.; Asard, H. Human erythrocyte membranes contain a cytochrome b(561) that may be involved in extracellular ascorbate recycling. J. Biol. Chem. 281:39852–39859; 2006. [7] McKie, A. T.; Barrow, D.; Latunde-Dada, G. O.; Rolfs, A.; Sager, G.; Mudaly, E.; Mudaly, M.; Richardson, C.; Barlow, D.; Bomford, A.; Peters, T. J.; Raja, K. B.; Shirali, S.; Hediger, M. A.; Farzaneh, F.; Simpson, R. J. An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 291:1755–1759; 2001. [8] Oakhill, J. S.; Marritt, S. J.; Gareta, E. G.; Cammack, R.; McKie, A. T. Functional characterization of human duodenal cytochrome b (Cybrd1): redox properties in relation to iron and ascorbate metabolism. Biochim. Biophys. Acta Bioenerg. 1777: 260–268; 2008. [9] McKie, A. T. The role of Dcytb in iron metabolism: an update. Biochem. Soc. Transact. 36:1239–1241; 2008. [10] Wyman, S.; Simpson, R. J.; McKie, A. T.; Sharp, P. A. Dcytb (Cybrd1) functions as both a ferric and a cupric reductase in vitro. FEBS Lett. 582:1901–1906; 2008. [11] Wang, Y. H.; Russo, T. A.; Kwon, O.; Chanock, S.; Rumsey, S. C.; Levine, M. Ascorbate recycling in human neutrophils: induction by bacteria. Proc. Natl. Acad. Sci. USA 94:13816–13819; 1997. [12] Song, J.; Kwon, O.; Chen, S. L.; Daruwala, R.; Eck, P.; Park, J. B.; Levine, M. Flavonoid inhibition of sodium-dependent vitamin C transporter 1 (SVCT1) and glucose transporter isoform 2 (GLUT2), intestinal transporters for vitamin C and glucose. J. Biol. Chem. 277:15252–15260; 2002. [13] Fiorani, M.; De Sanctis, R.; De Bellis, R.; Dacha, M. Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes. Free Radic. Biol. Med. 32:64–72; 2002. [14] Wang, Q.; Morris, M. Flavonoids modulate monocarboxylate transporter-1mediated transport of gamma-hydroxybutyrate in vitro and in vivo. Drug Metab. Dispos. 35:201–208; 2007. [15] Fiorani, M.; Accorsi, A.; Cantoni, O. Human red blood cells as a natural flavonoid reservoir. Free Radic. Res. 37:1331–1338; 2003. [16] Galleano, M.; Aimo, L.; Puntarulo, S. Ascorbyl radical/ascorbate ratio in plasma from iron overloaded rats as oxidative stress indicator. Toxicol. Lett. 133:193–201; 2002. [17] Balusikova, K.; Neubauerova, J.; Dostalikova-Cimburova, M.; Horak, J.; Kovar, J. Differing expression of genes involved in non-transferrin iron transport across plasma membrane in various cell types under iron deficiency and excess. Mol. Cell. Biochem. 321:123–133; 2009. [18] Ghio, A. J.; Turi, J. L.; Yang, F. M.; Garrick, L. M.; Garrick, M. D. Iron homeostasis in the lung. Biol. Res. 39:67–77; 2006. [19] Davies, N. P.; Rahmanto, Y. S.; Chitambar, C. R.; Richardson, D. R. Resistance to the antineoplastic agent gallium nitrate results in marked alterations in intracellular iron and gallium trafficking: identification of novel intermediates. J. Pharmacol. Exp. Ther. 317:153–162; 2006. [20] Upston, J. M.; Karjalainen, A.; Bygrave, F. L.; Stocker, R. Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C. Biochem. J. 342: 49–56; 1999. [21] Lane, D. J. R.; Lawen, A. Ascorbate and plasma membrane electron transport— enzymes vs efflux. Free Radic. Biol. Med. 47:485–495; 2009. [22] Schweinzer, E.; Goldenberg, H. Monodehydroascorbate reductase activity in the surface membrane of leukemic cells—characterization by a ferricyanide-driven redox cycle. Eur. J. Biochem. 218:1057–1062; 1993. [23] Pascoe, W. S.; Inukai, K.; Oka, Y.; Slot, J. W.; James, D. E. Differential targeting of facilitative glucose transporters in polarized epithelial cells. Am. J. Physiol. Cell Physiol 271:C547–C554; 1996. [24] Martin, H.; Kornmann, F.; Fuhrmann, G. The inhibitory effects of flavonoids and antiestrogens on the Glut1 glucose transporter in human erythrocytes. Chem. Biol. Interact 146:225–235; 2003. [25] Edlund, G.; Halestrap, A. The kinetics of transport of lactate and pyruvate into rat hepatocytes: evidence for the presence of a specific carrier similar to that in erythrocytes. Biochem. J. 249:117–126; 1988. 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Contents lists available at ScienceDirect
Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d
Original Contribution
Quercetin is a substrate for the transmembrane oxidoreductase Dcytb Evangelia Vlachodimitropoulou a, Richard J. Naftalin a,⁎, Paul A. Sharp b a b
Department of Physiology, King's College London, Waterloo Campus, London SE1 9HN, UK Division of Nutritional Sciences, King's College London, Waterloo Campus, London SE1 9HN, UK
a r t i c l e
i n f o
Article history: Received 15 October 2009 Revised 10 February 2010 Accepted 13 February 2010 Available online 22 February 2010 Keywords: Duodenal cytochrome b Flavonoids Ascorbate Iron absorption Redox balance Free radicals
a b s t r a c t Duodenal cytochrome b (Dcytb) is a transmembrane oxidoreductase protein found in apical membranes of duodenal enterocytes, as well as human erythrocytes, with the capacity to transport electrons donated by cytosolic ascorbate to extracellular electron receptors such as Fe(III), dehydroascorbate, or molecular O2. We have investigated the capacity of the flavonoid quercetin to act as an electron donor for Dcytb in a manner similar to that of ascorbate by observing the reduction of extracellular Fe(III) to Fe(II) in either Madin–Darby canine kidney (MDCK) cells overexpressing Dcytb (Dcytb+) or Dcytb-null MDCK cells. In Dcytb+ cells there is a saturable increase in extracellular Fe(III) reduction in response to increasing intracellular quercetin concentrations (Km = 6.53 ± 1.57 μM), in addition to a small linear response, whereas in Dcytb-null cells there is only a small linear increase in extracellular Fe(III) reduction. No extracellular Fe(III) reduction occurs in Dcytb-null cells when the cells are preloaded with ascorbate. Flavonoids such as quercetin at their physiological concentrations can therefore function as modulators of ferric reductases, enhancing the import of Fe(II) and also providing extracellular reducing potential. © 2010 Elsevier Inc. All rights reserved.
Flavonoids are polyphenolic compounds known to exert a number of biological effects, some of which may be attributed to their antioxidant properties. Quercetin is thought to be the most prevalent flavonoid in the Western diet. Its consumption has been estimated at 10–20 mg/day [1]. It has been shown to accumulate in rat adipocytes via the facilitated glucose transporters GLUTs 1 and 4 [2]. Quercetin also inhibits glucose and dehydroascorbate (DHA) transport via the GLUTs 1, 2, 3, and 4 in a reversible competitive manner [3,4]. In addition quercetin acts as a substrate for a plasma membrane oxidoreductase (PMOR) in erythrocytes, whose identity until now was unclear [5]. Recent work indicates that duodenal cytochrome b (Dcytb) fulfills this role [6]. Dcytb is a protein highly expressed on the brush border of duodenal enterocytes. It is localized alongside the divalent metal ion transporter (DMT1) on the apical membrane, and because of its ferric reductase activity Dcytb is believed to play an essential role in the absorption of nonheme Fe(II) iron from the human diet [7]. Its expression is iron regulated and it shares a structural homology
Abbreviations: AFR, ascorbyl free radical; Dcytb, duodenal cytochrome b; Fe(III), ferric iron; Fe(II), ferrous iron; DHA, dehydroascorbate; MDCK, Madin–Darby canine kidney cells; Dcytb+, MDCK cells overexpressing Dcytb; Dcytb−, MDCK cells lacking the Dcytb protein; Mops, 3-(N-morpholino)propanesulfonic acid; Mes, 2-(N-morpholino) ethanesulfonic acid; PMOR, plasma membrane oxidoreductase; DMT1, divalent metal ion transporter. ⁎ Corresponding author. Fax: +207 848 4600. E-mail address: [email protected] (R.J. Naftalin). 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.02.021
of approximately 40% with other members of the cytochrome b561 family. There are a total of six predicted α-helical transmembrane domains, four of which bind a histidine residue and form two heme-containing complexes. Evidence shows that these heme groups are reducible by 67% by the DHA/ascorbate redox couple [8]. There is general consensus that Dcytb is the primary ferric reductase responsible for uptake of dietary nonheme iron in the duodenum. However, recent studies involving Dcytb-knockout mice have given rise to some controversy as the rodents do not present with iron deficiency. Nonetheless, there is a clear decrease in iron uptake at the level of the enterocyte, and the lack of an irondeficient phenotype is most likely due to compensatory changes, possibly involving the protein ferroportin [9]. The rate of iron absorption is modulated by intracellular ascorbate concentrations, in that preloading cells with DHA leads to a highly significant extracellular ferric, as well as cupric, reduction [10]. DHA enters duodenal enterocytes via GLUTs 1 and 4 and increases intracellular ascorbate concentration [11,12]. Following evidence that flavonoids efficiently accumulate via GLUT transporters and act as intracellular substrates for a PMOR in erythrocytes [13], we here have investigated the potential modulatory role of quercetin in iron uptake via a mechanism of electron donation to the Dcytb protein, in a manner similar to that of ascorbate. Our findings demonstrate that the electron-donating capacity of Dcytb-expressing MDCK cells loaded with either DHA [10] or quercetin is greatly increased compared to wild-type MDCK cells (Fig. 1).
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Fig. 1. In our proposed model, GLUT transporters 1 and 4 aid DHA and quercetin accumulation in the intracellular space. In the cytosol DHA is reduced to ascorbate. Ascorbate and quercetin can donate electrons to the transmembrane oxidoreductase, Dcytb. This exports electrons across duodenal enterocyte apical membranes (and erythrocyte plasma membranes) and reduces nonbioavailable ferric to ferrous iron, thus aiding its assimilation from the gut via the transporter DMT1. Additionally, Dcytb is able to convert extracellular DHA to ascorbate via a two-electron-donating process, possibly by single-electron transference between Dcytb to AFR followed by dismutation of 2 AFR to ascorbate and DHA [23,25].
Materials and methods Transfection process and cell culture Cell transfection was carried out as previously described by Wyman et al. [10]. Briefly, PCR was used to amplify murine Dcytb cDNA from duodenal mRNA and create a pTRE2hyg-Dcytb-EGFP vector construct. The vector was transfected into Tetracycline-Off (TET-Off) MDCK cells, which were cultured for 2 weeks in Dulbecco's modified Eagle's medium, 10% TET-system-approved fetal bovine serum, 1 ng/ml puromycin, penicillin/streptomycin, and 200 μg/ml hygromycin. The expression of Dcytb-EGFP was controlled by the addition of 20 ng/ml doxycycline for a further 4 days. Ferric reductase assays The assay was performed on transfected Dcytb-EGFP and untransfected TET-Off MDCK cells that were grown on 24-well plates for at least 48 h to allow for cell attachment and confluency. Cells were treated with 0.5 ml of medium per well containing the reagents applicable to each experiment, for 30 min. The cells were then washed three times with phosphate-buffered saline (PBS) at pH 7.0 and incubated in physiological buffer (25 mM Mops, 25 mM Mes, 5.4 mM KCl, 5 mM glucose, 140 mM NaCl, 1.8 mM CaCl2, 800 μM MgCl2), 50 μM Fe(III)-nitrilotriacetic acid (NTA), and 200 μM 3-(2-pyridyl)-5,6diphenyl-1,2,4-triazine-4′,4″-disulfonic acid sodium salt (ferrozine) over a period of half an hour. Extracellular iron reduction was recorded via the formation of the colored Fe(II)–ferrozine complex monitored at 562 nm. All reactions were carried out in the dark, at 37°C, and absorbance values were converted into iron concentrations via the generation of standard curves [10]. It was noted, by recording the spectrophotometric change in ferrozine absorbance during the ferric reductase assay, that there is a direct concentration-dependent effect of quercetin on Fe(III) reduction to Fe(II) iron. Formation of the colored Fe(II)–ferrozine complex and the concentration of quercetin are positively correlated. There-
fore, to eliminate the possibility of flavonoid efflux leading to false assumptions that quercetin is responsible for the additional reducing effect seen in the presence of Dcytb and confirm our observations shown in Fig. 2, an alternative experimental approach was adopted. MDCK and Dcytb-transfected cells were treated with 100 μM quercetin solution, washed three times with PBS, and exposed to DMEM over a period of 1 h. The supernatant solution was then spectrophotometrically assayed, and with the aid of the reductase assay, only a minimal proportion of the original iron reduction could be accounted for by flavonoid efflux and hence a direct effect of quercetin on ferrozine. The percentage ranges between 4 and 6% and is consistent with the findings of Fiorani et al. [13] regarding flavonoid efflux from erythrocytes. They observed that the bulk of intracellular flavonones bind loosely to intracellular structures. This binding retards flavonone exit rates. These results reinforce the original hypothesis that the source of extracellular electrons stems from electrons donated to Dcytb at its intracellular site. Dcytb and MDCK cells were also preloaded with quercetin for variable time intervals between 0 and 90 min and then lysed to assess the intracellular flavonoid content with the aid of the ferrozine assay. Results indicated that the flavonoid readily accumulated in both cell types in less than 2 min. Results We have investigated whether quercetin has a similar modulatory role compared to ascorbate in iron uptake via a transmembrane electron-donating mechanism to the Dcytb protein. Cultured MDCK cells overexpressing the Dcytb gene (Dcytb+), as well as Dcytb-null MDCK cells, from which the protein is absent (Dcytb−), were loaded with variable concentrations of quercetin (0– 100 μM). In Dcytb+ cells, with increasing quercetin concentration, there is a saturable component, Km = 6.43 ± 1.57 μM (four experiments) and Vm = 0.92 ± 0.04 μmol μg cell protein−1 · h−1 (Figs. 2A and 2B). The Michaelis–Menten kinetics noted are similar to those obtained with preloading of cells with DHA; however, the Km and Vm
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for ascorbate is ≈ 100 times higher than observed here with quercetin, using the same cell culture system (see Discussion) [6]. This saturable response to quercetin is absent from Dcytb− cells. However, quercetin preloading also induced a quantitatively similar linear increase in extracellular iron reduction in both Dcytb+ and Dcytb− cells (Figs. 2A and 2B), which was absent with DHA (Fig. 3). In Fig. 3 it can be observed that the ratio of extracellular reduction in the Dcytb-expressing cells to that of the Dcytb-null cells when loaded with DHA is 18.00 ± 0.03, whereas when loaded with quercetin the ratio is 6.00 ± 0.153 (n = 6; P b 0.017). This indicates that the Dcytb-null cells allow approximately three times as much quercetin to escape from the cells as ascorbate. There are a number of potential pathways through which quercetin can pass, in particular, GLUTs and the monocarboxylate acid transporter pathways, both of which are expressed in MDCK cells and have been observed to bind and transport flavonones [4,14]. These findings indicate that quercetin acts as an electron donor to Dcytb and that the intracellular flavonoid reservoir [15], together with ascorbate, could act as a central modulator of inorganic iron assimilation in the duodenum and in erythrocytes, providing a source of electrons to maintain the reduced state of extracellular oxidizable substrates, e.g., ascorbate in plasma, where normally the concentration ratio of ascorbate:dehydroascorbate is maintained at ∼100 [16].
Discussion Our results further the understanding of the function of Dcytb in the maintenance of redox potentials and metal homeostasis. We have demonstrated that Dcytb has the capacity to reduce Fe(III), not only when the cells are loaded with DHA, but also in the presence of intracellular flavonoids, such as quercetin. Although Dcytb was discovered in the duodenal enterocytes [7], it is now known that it is expressed in a number of cell types, such as erythrocytes [6], Caco-2 cells [17], and lung epithelium [18]. Thus, electron donation to Dcytb by flavonoid compounds may play a crucial biological role in a variety of tissues.
Fig. 2. Dcytb-transfected and Dcytb-null cells were preloaded with variable quercetin concentrations (0–100 μΜ) over a period of 30 min, at 37°C. The cells were then washed three times with PBS and extracellular iron reduction was assessed with the aid of the ferrozine assay. Results have been adjusted for protein using the Bio-Rad DC protein assay kit. An increase in extracellular Fe3+ reduction is noted in both cells expressing Dcytb and Dcytb-null cells. There is a direct effect of quercetin on ferric iron reduction, as confirmed by the ferrozine assay. Increasing quercetin concentrations, 200 μM ferrozine, and 50 μM Fe (III)–NTA solution were incubated for 30 min, at 37°C, in the dark. (A) When cells are loaded over a wider concentration range the reductive capacity of Dcytb-expressing cells is observed to be saturable. At 50 μΜ extracellular quercetin, electron export is noted to be 0.65± 0.1 μM/μg protein/h in untransfected and 1.4 ± 0.1 μM/μg protein/h in Dcytbtransfected cells. No saturable effect is observed with Dcytb-null cells. (B) The difference between the two cell types is plotted. The best-fit Michaelis–Menten kinetic curve of the difference between the reducing capacities of Dcytb-expressing and Dcytb-null cells gives a Km ≈ 5 μM for the saturable component of quercetin-dependent extracellular reduction of ferrozine. (C) Effects of varying concentrations of quercetin from 0 to 10 μM on extracellular Fe3+ reduction in Dcytb+ and Dcytb− cells. There is a linear increase in extracellular reductive capacity in this concentration range (P b 0.001).
Fig. 3. Ferric reductase activity of 20 ng/ml Dcytb-transfected and untransfected MDCK cells treated with 100 μΜ DHA and 100 μΜ quercetin was recorded using the ferrozine assay. The data show that both ascorbate and quercetin loading at 100 μΜ led to similar rates of extracellular ferric reductase activity in Dcytb-expressing cells. There is a highly significant reduction in extracellular ferric reductase activity in Dcytb-null cells when preloaded with either ascorbate or quercetin. However, the data also show that a significantly higher proportion of quercetin than of ascorbate leaks from Dcytb-null cells, possibly because of the high affinity of quercetin for GLUT1 or lactate transporter expressed in these cells (see Discussion).
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There has been some debate about the precise nature of ascorbate– Dcytb interactions. It has been suggested that ascorbate is released directly into the extracellular fluid, perhaps via GLUTs or an alternative transport process, and directly interacts with ferricyanide (Fe(III)), at the external surface of the cell [19–21]. External ascorbate concentrations may be maintained by DHA reductase enzyme activity recycling either DHA or ascorbate free radical [22]. Wyman et al. [10] showed that the presence of ascorbate oxidase in extracellular fluid had no effect on extracellular copper or iron reduction in Dcytb-expressing cells preloaded with ascorbate. This indicates that the reducing capacity of ascorbate is entirely dependent on electron export via Dcytb and not on direct extracellular interaction between ascorbate and metal electron acceptors. The results in this paper support this view for two reasons. First, as Wyman et al. [10] observed with Dcytb-null MDCK cells, there is virtually no effect of intracellular ascorbate on ferrozine reduction, whereas a large reduction is observed when Dcytb is expressed (Fig. 3). Second, in contrast to ascorbate, which has a Km of 500– 1000 μM for Dcytb [6], quercetin has a much higher affinity (5–10 μM) and therefore the saturable response of the ferrozine reduction in Dcytb+ cells is more unambiguously indicative of a saturable process mediated by Dcytb than that seen with high intracellular ascorbate concentrations, observed previously [6,10]. The smaller linear component of quercetin-dependent extracellular ferrozine reduction could be due to a parallel release of quercetin, for example, via GLUT 1, which is present in MDCK cells [23] and to which quercetin binds with very high affinity, KD ≈ 500 nM [4]. In addition to its interaction with GLUT 1 [24], quercetin has also been reported to have the capacity to inhibit a number of other transporters, including the l-lactate transporter in hepatocytes [25] and lactate transport in human erythrocytes and Ehrlich ascites tumor cells [26]. The absence of this linear component with ascorbate as the substrate is probably due to very low intracellular concentrations of DHA that result from efficient intracellular conversion of membrane-permeative DHA to impermeative ascorbate [27]. The ascorbyl reductase system has been shown to maintain low extracellular concentrations of ascorbyl free radical [28], both in the gut lumen and in the blood plasma, and plays a protective role against a variety of extracellular oxidant stressors. It has been previously estimated that transmembrane oxidoreductase activity recycles human plasma ascorbate from DHA within 3 min and maintains it at a level of 50 μM during normal states of metabolism [27]. This intracellular ascorbate concentration is consistent with the KD of Dcytb [10]. Similarly, the concentration of unbound quercetin in plasma under normal physiological conditions is in the range 0.1–1 μM, which is of the same order as the Ki that we observed with quercetin in the preceding experiments. The antioxidant and iron-chelating properties of flavonoids have attracted attention for a number of years [29,30]. Their wide bioavailability in fruits and vegetables, their lack of toxicity in comparison with ascorbate, and their capacity to act as substrates for plasma oxidoreductases apparently provide an efficient supplemental role to ascorbate in maintaining redox balance [31]. Thus, both ascorbate and quercetin can be used within their physiological concentration ranges to reduce oxidized extracellular substrates. Acknowledgement EV is grateful to the Physiological Society and King's College London for a summer studentship and summer vacation award in 2008 and 2009 respectively. References [1] Hertog, M. G. L.; Hollman, P. C. H.; Katan, M. B.; Kromhout, D. Intake of potentially anticarcinogenic flavonoids and their determinants in adults in the Netherlands. Nutr. Cancer Int. J. 20:21–29; 1993.
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