Flavonoid conjugates interact with organic anion transporters (OATs) and attenuate cytotoxicity of adefovir mediated by organic anion transporter 1 (OAT1/SLC22A6)
Graphical abstract
Introduction
Quercetin is a chemopreventive and anti-inflammatory flavonoid that interacts with nuclear factor E2-related factor 2 (Nrf2) and activates antioxidant response elements (ARE) [1], [2], contributing to enhanced cellular protection against carcinogens and oxidative stress [3]. Isoflavones are phytoestrogens that bind to the estrogen receptor [4] and exert preventive effects on hormone-dependent cancer [5], cardiovascular diseases [6], and post-menopausal osteoporosis [7]. Any biological activities of flavonoids, however, are influenced by their metabolism. Dietary flavonoids are metabolized extensively in the intestine and liver, and major forms found in vivo are glucuronide, sulfate and/or methyl-conjugates of the parent aglycone [8]. Quercetin is metabolized into quercetin-3-O-glucuronide, 3′-methylquercetin-3-O-glucuronide and quercetin-3′-O-sulfate following consumption of onions [9]. After soy ingestion, soy isoflavones such as daidzein and genistein are present in the circulation predominantly as their 7-O-glucuronide and 4′-O-glucuronide, and sulfates were minor metabolites [10]. Concentrations of these metabolites in human plasma are typically low after intake of 50 mg aglycone equivalent and rarely exceed 10 μM even at much higher ingested doses [11].
Interaction with uptake and efflux transporters is considered to be one of the factors that limit the bioavailability of flavonoids, by facilitating the elimination of hydrophilic conjugates into bile and/or urine [12]. Urinary excretion of flavonoid conjugates has been well documented and varies according to the individual flavonoid aglycones. For the isoflavones daidzein and glycitein, about half of the intake could be recovered in the urine as conjugated metabolites [13]. A high urinary recovery of conjugates was also observed with catechins (∼25%) [14]; while for quercetin the urinary excretion is much less than 10% of the intake [15]. Many drugs and their conjugated metabolites are eliminated in the urine via tubular secretion [16]. Vectorial transport of hydrophilic substances across the renal proximal tubules involves the interplay of uptake transporters on the basolateral membrane and the efflux transporters on the apical membrane. Active efflux is carried out by multidrug resistance proteins (MRP), which transport glucuronide and sulfate conjugates of flavonoids such as quercetin, baicalein and genistein [17], [18], [19]. The molecular mechanism underlying the possible uptake of flavonoid conjugates into renal tubules via basolateral transporters is not known.
OAT1 (SLC22A6) and OAT3 (SLC22A8) are highly expressed on the basolateral membrane of proximal tubules [20]. They play an important role in the sodium-dependent renal uptake of structurally diverse organic anions from the blood into proximal tubule cells. OAT1 and OAT3 are capable of mediating the uptake of these organic anions against their electrochemical gradients by coupling to efflux of intracellular α-ketoglutarate. OAT1 interacts with endogenous metabolites, drugs including antivirals, antibiotics, nonsteroidal anti-inflammatory agents and statins, as well as toxicants such as mercury and ochratoxin [21]. OAT3 recognizes many OAT1 substrates; in addition, it has the ability to transport corticosterone, estradiol-17β-glucuronide and taurocholate, that are not substrates of OAT1 [22]. As toxins and cytotoxic drugs are taken up by OAT1 and OAT3, these toxic substances may exert toxic effects on the proximal tubule cells and cause renal injury. In vitro studies with OAT1-transfected cells showed that OAT1 over-expression sensitizes the cytotoxicity caused by antiviral drugs, adefovir and cidofovir [23]. Co-administration of the inhibitors of OATs may reduce the extent of OAT-induced nephrotoxicity [24]. On the other hand, inhibition of OAT1 and OAT3 may also alter the pharmacokinetics of a variety of drugs that are OAT substrates. OAT1 inhibitors, such as probenecid, could be administered with other drugs to prolong plasma half lives and improve therapeutic effects [25].
To date, only limited information is available concerning the interaction between OATs and flavonoids. Ellagic acid is a substrate and a potent inhibitor of OAT1 [26], while several flavonoids (naringenin, morin, silybin and quercetin) were also found to inhibit OAT1 and OAT3 [27]. However, the flavonoid aglycones are mostly absent in human plasma. The aim of this work is to investigate the transport of glucuronidated and sulfated metabolites of quercetin and isoflavones, major metabolites in vivo, in OAT1- and OAT3-transfected human embryonic kidney 293H cells. We also examined the inhibition of OATs by flavonoid conjugates and the potential of quercetin-3′-O-sulfate to attenuate OAT1-induced cytotoxicity of adefovir.
Section snippets
Chemicals
Genistein and quercetin were obtained from Extrasynthese (Genay, France). Quercetin-7-O-glucuronide, quercetin-3-O-glucuronide and quercetin-3′-O-glucuronide were synthesized from quercetin using human liver S9 (Sigma–Aldrich, St. Louis, MO) and purified by HPLC [28]. Quercetin-3′-O-sulfate was chemically synthesized as described and purified by gel filtration [9]. The identity of quercetin-3-O-glucuronide and quercetin-3′-O-sulfate was further confirmed by comparing the retention time and the
Uptake of flavonoid and isoflavone conjugates by OAT1
To test whether flavonoid and isoflavone conjugates are transported by OAT1, 293H cells transfected with empty or OAT1 plasmid were incubated with 25 μM of the test compounds for 30 min. The results are shown in Fig. 1, Fig. 2. Sulfated metabolites quercetin-3′-O-sulfate and genistein-4′-O-sulfate appeared to be the best substrates of OAT1 (>10-fold over control), especially the former (Fig. 1, Fig. 2). However, daidzein-7,4′-O-disulfate was not significantly transported. The glucuronide
Discussion
Flavonoids are extensively metabolized in humans by the phase II enzyme families of sulfotransferases and UDP-glucuronosyltransferases following oral administration. Conjugates of glucuronide and/or sulfate are the predominant forms in human plasma, while the parent aglycones are usually absent or present in minute amounts [11]. In the present study, we examined transport of nine quercetin and isoflavone metabolites found in vivo in OAT1- or OAT3-overexpressing human embryonic kidney 293H
Acknowledgement
We thank the Nestle Research Center for funding.
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