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Institute of Toxicology, Heinrich-Heine-University, 40001 Düsseldorf, Germany; and * Institute of Pharmaceutical Biology, Heinrich-Heine-University, 40225 Düsseldorf, Germany
3To whom correspondence should be addressed. E-mail: wim.waetjen{at}uni-duesseldorf.de.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • apoptosis • comet assay • fisetin • quercetin • uptake
Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin; >6000 flavonoids, low-molecular-weight phenylbenzopyrones, have been identified in plant sources. This class of compounds has become increasingly popular in terms of health protection because they possess a remarkable spectrum of biochemical and pharmacologic activities (1). Flavonoids affect basic cell functions such as growth, differentiation, and apoptosis. Epidemiologic studies have suggested that flavonoids may protect against various stages of the cancer process and are associated with a reduced incidence of coronary heart disease (2,3). Flavonoids were shown to be potent antioxidants because of their radical-scavenging activity. It was also shown that flavonoids are able to complex heavy metal ions, e.g., iron and copper, which are involved in Fenton-like reactions (4). The biological actions of flavonoids have long been thought to be due to their antioxidant potential but at present, it is by no means clear whether other mechanisms of action contribute to their overall effect or are even more important than their radical-scavenging properties.
Although some flavonoids act as powerful antioxidants, it was also shown that in high concentrations, they can generate reactive oxygen species by autoxidation and redox-cycling (5–9). Consequently, many reports described adverse actions of flavonoids on a cellular level. For a number of the flavonoids used in this study, cytoprotective as well as cytotoxic and proapoptotic effects were shown in various cell culture models. Thus, protection against apoptotic stimuli was found with quercetin (10), epicatechin (11), and rutin (12) but on the other hand, induction of apoptosis was described for quercetin (13,14), fisetin (15), morin (16), myricetin (17), and rutin (16). In the comet assay, a protection against DNA strand breaks was demonstrated for quercetin (18,19), myricetin (19,20), and rutin (20,21); however, strand breaks were also induced by quercetin (18,22) and myricetin (22) in that assay. Given this wide spectrum of biological actions, it is quite understandable that numerous health claims that are in part mutually exclusive have been linked with flavonoids. Notably, in cancer, but also in infections or autoimmune disease, a deficiency in apoptosis is one of the key events. On the other hand, overefficient apoptosis, as observed in fulminant liver failure, or the long-term accumulation of apoptotic events in neurodegenerative disorders may be equally harmful to the organism.
Given that both beneficial and adverse effects can in principle be caused by flavonoids, it must be assumed that in addition to the cell type or tissue involved and to the presence or absence of a stressor, dose determines which action prevails. Dose dependency, however, has not been studied systematically. The importance of the chemical structure of the flavonoids in relation to their biological activity is also far from obvious. Dietary flavonoids are predominantly present in a glycosidic form, e.g., in fruits (23), although free flavonoids also occur, e.g., myricetin in red wine. The basic structure of flavonoids consists of an O-heterocyclic ring (C) fused to an aromatic ring (A) with a third ring system (B) attached at C2 of the heterocyclic ring. Out of the great variety of structures, we selected 7 representative flavonoids to investigate their pro- and antioxidative effects and their pro- and antiapoptotic properties (Fig. 1). Quercetin, a major flavonoid in the diet, can be regarded as the lead structure. The structures of the other flavonoids differ from quercetin in the number and position of hydroxy substituents (fisetin, morin, myricetin), the C2-C3 double bond (taxifolin) or the existence of a glycoside moiety (rutin). In the case of (±)-catechin, the only flavanol analyzed, no 4-keto oxygen or C2-C3 double bond is present.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell culture. The metabolically active and differentiated rat H4IIE hepatoma cell line was used as a model system for further experiments in vivo on the influence of flavonoids on enzyme activation in rat liver. Cells were grown in DMEM (4.5 g/L glucose, 2 mmol/L L-glutamine, 100 kU/L penicillin and 100 mg/L streptomycin, and 10% fetal calf serum) in a humidified atmosphere at 37°C with 5% CO2.
Cellular uptake of flavonoids. We used HPLC (Eurosphere C-18, photodiode array detector) to investigate intracellular concentrations of flavonoids as described earlier (24). HPLC-MS-MS was performed to analyze the formation of flavonoid metabolites. The intracellular distribution of quercetin was analyzed by fluorescence microscopy (excitation: 450–490 nm, emission: 515 nm). Nuclei were stained with Hoechst 33342 (100 µmol/L).
Cytotoxicity. Cell viability was determined using the MTT-assay (25) and the neutral red assay (26), with slight modifications. Briefly, for measurement of flavonoid toxicity, 10,000 cells/well (96-multiwell dish) were plated; for protective effects 25,000 cells/well (24-multiwell dish) were plated. Color development (20 mg/L MTT, 16 mg/L neutral red) was performed for 3 h.
Lipid peroxidation. Malondialdehyde (MDA)5 was quantified by the thiobarbituric acid assay and subsequent HPLC (27) as described earlier (24).
Apoptotic DNA-fragmentation. DNA was isolated using phenol:chloroform extraction and oligonucleosomal fragmentation of DNA was analyzed electrophoretically as described earlier (24).
Caspase activity. Colorimetrically labeled substrates for VDVADase activity (caspase-2), DEVDase activity (caspase-3/7), and LEHDase activity (caspase-9) were used according to the manufacturer’s protocol (Calbiochem).
DNA strand breaks ("comet assay"). Cells (3 x 106) were seeded out in a 6-well dish and incubated 24 h later with either flavonoid alone for 3 h or with 500 µmol/L H2O2 (2 h) in the presence or absence of a preincubation (1 h) with flavonoids. For determination of DNA strand breaks, the comet assay (28) was used.
Microscopic analysis of nuclear fragmentation. To investigate nuclear fragmentation as a further feature of apoptotic cell death, fluorescent staining with Hoechst 33342 (100 µmol/L) was used as described earlier (24).
TEAC assay. Trolox equivalent antioxidative capacity (TEAC) was determined as described earlier (29), and absorption was measured after 4 min of mixing the substances with the ABTS solution.
Protein. Protein concentration was determined spectrophotometrically according to Bradford (30) using bovine serum albumin as the standard.
Statistics. Data are given as means ± SEM of at least 3 independent experiments. The significance of differences was assessed using 1-way ANOVA followed by the LSD test (Analyze-it). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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50% (Fig. 2A). Preincubation with other flavonoids at a concentration of 50 µmol/L caused only minor (taxifolin, myricetin) or no reduction (morin, rutin, catechin) in H2O2-mediated DNA strand breakage (Table 1).
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50% (Fig. 2B). Quercetin and fisetin at 10 µmol/L provided 50% protection against the loss of cell viability induced by 150–250 µmol/L H2O2. Other flavonoids showed no protective effect against H2O2-mediated caspase activation (Table 1). Uptake of flavonoids. To investigate whether the difference of in vitro and in vivo potency of the flavonoids was due to differences in cellular uptake, we analyzed their intracellular concentration by HPLC at different incubation times (0.5–24 h). Quercetin, fisetin, morin, taxifolin, and myricetin were detected in the cells after 1 h of incubation although to a very different extent (quercetin > fisetin >> morin >>taxifolin > myricetin, Table 1) which might explain why only quercetin and fisetin are protective in the cells. Rutin and catechin were not taken up by H4IIE cells even after an incubation of 24 h. Incubation with 50 µmol/L quercetin led to an intracellular concentration of 0.82 ± 0.14 nmol/106 cells. Quercetin uptake was not saturated at high concentrations [3.5 ± 0.7, 33.4 ± 10.7, and 109 ± 21 nmol quercetin/106 cells after incubation (1 h) with 250, 500, and 750 µmol/L quercetin, respectively].
The intracellular concentration of quercetin decreased exponentially with time (15.9 ± 4.7, 9.7 ± 3.5, and 1.7 ± 0.6 mmol quercetin/106 cells after 2, 4, and 8 h, respectively) due to its metabolism; 3 peaks at 13.53, 13.78, and 15.61 min were identified as glucuronidated metabolites of quercetin due to spectral characteristics and MS fragmentation spectra. The m/z of [M-H]-ions was 477, which were fragmented to [M-H]-ions with an m/z of 301. We further analyzed the intracellular distribution of quercetin in the cells by fluorescence microscopy. After 1 h of incubation with 50 µmol/L quercetin, a bright nuclear fluorescence was observed, suggesting that the flavonoid accumulates in the nucleus (Fig. 3).
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-tocopherol, 500 µmol/L ascorbic acid, 500 µmol/L N-acetylcysteine, 1000 µmol/L glutathione) or the metal chelator desferoxamine (25 µmol/L) did not protect against quercetin-induced cytotoxicity. In the case of ascorbic acid, a further increase in flavonoid-mediated cytotoxicity occurred (data not shown). Incubation of H4IIE cells with high concentrations of flavonoids (up to 500 µmol/L) for 24 h did not increase the formation of MDA, a marker of oxidative stress (Table 2). Induction of DNA strand breaks by flavonoids. Incubation of H4IIE cells with quercetin increased comet formation in a time- and concentration-dependent manner. A dose-response curve was found with saturation at 250 µmol/L (500 µmol/L quercetin: image length, 36.0 ± 5.7 µm). Fisetin also induced DNA breakage (Fig. 4). There was a slight increase in DNA strand breaks after incubation with morin but no increase in DNA "comet" formation after incubation with taxifolin, rutin, catechin, or myricetin (Table 2).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All flavonoids tested proved to be good antioxidants in a cell-free assay; their potency equaled that of the standard antioxidant, Trolox. However, the pharmacologic activity of the flavonoids tested did not correlate with their antioxidant potential measured in this in vitro assay. The data suggest that this might be due to differences in cellular uptake. There was a convincing relation between cellular uptake and both cytoprotective and proapoptotic actions indicating that these events require the presence of the compounds in the cell. Intrinsic cytotoxicity of the flavonoids, however, does not fit into this scheme. Morin and myricetin in particular had only low intracellular concentrations, but their cytotoxicity was comparable to that of quercetin and fisetin. Various explanations for the cytotoxicity of flavonoids have been proposed, including the inhibition of enzymes such as protein kinase C, cGMP-dependent protein kinase, adenylate cyclase, DNA topoisomerases, or glutathione S-transferase (1,23), leading to disturbances in cell cycle, for example, followed by apoptotic or necrotic events. There are several ways in which a flavonoid could exert pharmacologic activity in the absence of cellular uptake, e.g., extracellular H2O2 formation may occur (31,32). Alternatively, the attack may take place at a membrane structure, e.g., a growth hormone receptor. For the green tea polyphenol, epigallocatechin gallate, the existence of a cell surface receptor was suggested (33). Myricetin and morin caused growth inhibition in HT29 cells at concentrations very similar to those that led to cytotoxicity in our study (47 or 117 µmol/L, respectively) and myricetin activated caspase-3 in these cells but not in MCF-7 cells and LLC-PK1 cells (17). In our study the apoptosis-inducing potential of myricetin in H4IIE cells was negligible.
Controversy exists concerning whether the cytotoxic and DNA-damaging effects of flavonoids involve the formation of reactive oxygen species. In our study, we did not find evidence for increased lipid peroxidation even at high concentrations of flavonoids. Also, no protection against quercetin- and fisetin-induced cytotoxicity was achieved by a number of antioxidants, indicating that ROS production may not be involved. Incubation of human lymphocytes with high concentrations of quercetin increased DNA strand breakage but did not induce oxidative damage to DNA bases (22). Yamashita et al. (34) reported that the quercetin-induced DNA damage was dependent on the presence of copper(II)-ions because it was inhibited by bathocuproine, a copper-specific chelator. We investigated the effects of desferoxamine on quercetin-induced cytotoxicity but again found no protective effects.
Only limited conclusions can be derived from our results with respect to the structure-activity relation of the compounds tested. We demonstrated that the antioxidative potential in vitro of the flavonoids increased with the number of their phenolic hydroxyl groups because myricetin, which has the highest number of hydroxyl groups, exhibited the strongest antioxidant action in the TEAC assay. In view of the correlation between intracellular concentration and the range of pharmacologic activity that includes protective, DNA-damaging, and apoptotic effects, structural hindrance is likely to occur at the level of cellular uptake. It was shown previously that the rutinoside moiety of flavonoids inhibits membrane crossing (35); therefore the inactivity of rutin was not unexpected. It cannot be excluded that in addition to uptake, the intracellular actions of the flavonoids are also influenced by structural differences. Current results do not allow us to distinguish these 2 levels of effect. In any case, it appears that the double bond in the ring C causing the planarity of the ring system is supportive but not sufficient for uptake and/or biological activity.
Quercetin is one of the most abundant flavonoids in the diet; it is also considered to be one of the most promising members of this class for both general chemoprevention and cancer chemotherapy. For this reason, the pharmacokinetics of quercetin were studied repeatedly in rats as well as in humans. Manach et al. (36) reported that rats fed quercetin (single meal, 0.2%) exhibited constant plasma concentrations of quercetin metabolites of 50 µmol/L for at least 16 h. Rats adapted to quercetin (0.2%) maintained plasma concentrations of 100 µmol/L (quercetin + metabolites). The authors suggested that the elimination of quercetin metabolites is low and that high plasma concentrations are easily maintained with a regular supply of quercetin in the diet. More data on the pharmacokinetics of quercetin in humans are also available. Both the aglycone and the glucosides can enter the body via the small intestine, whereas other conjugates can be taken up only after hydrolysis by colonic bacteria (37,38). One study with 4 doses (250 mg)/day of quercetin administered as capsules resulted in plasma concentrations of 1.5 µmol/L (39); however, later studies using quercetin glucosides yielded relatively higher plasma concentrations, i.e.,7 µmol/L after quercetin-4'-O-glucoside equivalent to 100 mg quercetin (40), 4.5 µmol/L after 150 mg quercetin 4'-glucoside, and 5 µmol/L after 150 mg quercetin 3-glucoside (41). When administered in onions (a plant source that contains quercetin as glucosides) the following plasma concentrations of total quercetin as related to dose were reported: 0.05 µmol/L after 15 mg (42), 0.63 µmol/L after 68–94 mg (43), 7.65 µmol/L after 100 mg (40), and 4 µmol/L after 300 mg (44). Half-lives between 10 and 30 h were reported (40,41,45), suggesting that continuous daily intake will result in a steady-state concentration. All kinetic variables measured in humans were measured in plasma, and no information on flavonoid concentrations in different tissues is available. In some studies, no free quercetin was found in blood samples [e.g., (43)], whereas in others, free quercetin was detected (46). Isorhamnetin is the only phase I metabolite described (40). It is unclear at present whether the metabolites still possess pharmacologic activity. Data suggest that flavonoids can be deglucuronidated at the site of action, e.g., in endothelial cells (47,48). In contrast to quercetin, data on fisetin plasma concentrations in humans are lacking, probably because it is minimally present in the diet. Fisetin, like quercetin, is methylated in human liver (49). Taken together, the data on quercetin pharmacokinetics in humans suggest that a dietary supplement of 1–2 g of quercetin, an amount proposed by supplement makers, may result in plasma concentrations exceeding 10 µmol/L but probably not exceeding 50 µmol/L if taken properly.
In summary, we found that quercetin and fisetin were readily taken up into H4IIE cells and protected against H2O2-induced cytotoxicity, DNA strand breaks, and apoptosis at concentrations of 10–25 µmol/L; however, these compounds themselves induced cytotoxicity, DNA strand breaks, oligonucleosomal DNA fragmentation and caspase activation at concentrations between 50 and 250 µmol/L. The other flavonoids tested were good antioxidants in a cell-free assay; their pharmacologic activity did not correlate with in vitro antioxidant potential but rather with cellular uptake. Our data suggest that cytoprotective concentrations of some flavonoids are lower by a factor of 5–10 than their DNA-damaging and proapoptotic concentrations. These results have implications also for humans in terms of risk assessment and in the modulation of isolated food constituents; they should be carefully studied because flavonoids are used increasingly in dietary supplements.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by DFG International Graduate College 738: Molecular Mechanisms of Food Toxicology.
4 This work represents part of a doctoral thesis.
5 Abbreviations used: EC50, 50% effective concentration; LSD, least significant difference; MDA, malondialdehyde; TEAC, Trolox equivalent antioxidative capacity.
Manuscript received 10 September 2004. Initial review completed 28 October 2004. Revision accepted 27 December 2004.
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LITERATURE CITED |
|---|
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Middleton, E., Jr, Kandaswami, C. & Theoharides, T. C. (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52:673-751.
2. Hollman, P.C.H., Hertog, M.G.L. & Katan, M. B. (1996) Role of dietary flavonoids in protection against cancer and coronary heart disease. Biochem. Soc. Trans. 24:785-789.[Medline]
3. Knekt, P., Kumpulainen, J., Jarvinen, R., Rissanen, H., Heliovaara, M., Reunanen, A., Hakulinen, T. & Aromaa, A. (2002) Flavonoid intake and risk of chronic diseases. Am. J. Clin. Nutr. 76:560-568.
4. Mira, L., Fernandez, M. T., Santos, M., Rocha, R., Florencio, M. H. & Jennings, K. R. (2002) Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity. Free Radic. Res. 36:1199-1208.[Medline]
5. Gaspar, J., Rodrigues, A., Laires, A., Silva, F., Costa, S., Monteiro, M. J., Monteiro, C. & Rueff, J. (1994) On the mechanisms of genotoxicity and metabolism of quercetin. Mutagenesis 9:445-449.
6. Ochiai, M., Nagao, M., Wakabayashi, K. & Sugimura, T. (1984) Superoxide dismutase acts as an enhancing factor for quercetin mutagenesis in rat-liver cytosol by preventing its decomposition. Mutat. Res. 129:19-24.[Medline]
7. Hodnick, W. F., Kung, F. S., Roettger, W. J., Bohmont, C. W. & Pardini, R. S. (1986) Inhibition of mitochondrial respiration and production of toxic oxygen radicals by flavonoids. A structure-activity study. Biochem. Pharmacol. 35:2345-2357.[Medline]
8. Metodiewa, D., Jaiswal, A. K., Cenas, N., Dickancaite, E. & Segura-Aguilar, J. (1999) Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radic. Biol. Med. 26:107-116.[Medline]
9. Yoshino, M., Haneda, M., Naruse, M. & Murakami, K. (1999) Prooxidant activity of flavonoids: copper-dependent strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine in DNA. Mol. Genet. Metab. 68:468-472.[Medline]
10. Oyama, Y., Noguchi, S., Nakata, M., Okada, Y., Yamazaki, Y., Funai, M., Chikahisa, L. & Kanemaru, K. (1999) Exposure of rat thymocytes to hydrogen peroxide increases annexin V binding to membranes: inhibitory actions of deferoxamine and quercetin. Eur. J. Pharmacol. 384:47-52.[Medline]
11. Schroeter, H., Spencer, J. P., Rice-Evans, C. & Williams, R. J. (2001) Flavonoids protect neurons from oxidized low-density-lipoprotein-induced apoptosis involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3. Biochem. J. 358:547-557.[Medline]
12. Chen, C., Wei, T., Gao, Z., Zhao, B., Hou, J., Xu, H., Xin, W. & Packer, L. (1999) Different effects of the constituents of EGb761 on apoptosis in rat cerebellar granule cells induced by hydroxyl radicals. Biochem. Mol. Biol. Int. 47:397-405.[Medline]
13. Richter, M., Ebermann, R. & Marian, B. (1999) Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling. Nutr. Cancer 34:88-99.[Medline]
14. Choi, J. A., Kim, J. Y., Lee, J. Y., Kang, C. M., Kwon, H. J., Yoo, Y. D., Kim, T. W., Lee, Y. S. & Lee, S. J. (2001) Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin. Int. J. Oncol. 19:837-844.[Medline]
15. Chen, Y. C., Shen, S. C., Lee, W. R., Lin, H. Y., Ko, C. H., Shih, C. M. & Yang, L. L. (2002) Wogonin and fisetin induction of apoptosis through activation of caspase 3 cascade and alternative expression of p21 protein in hepatocellular carcinoma cells SK-HEP-1. Arch. Toxicol. 76:351-359.[Medline]
16. Romero, I., Paez, A., Ferruelo, A., Lujan, M. & Berenguer, A. (2002) Polyphenols in red wine inhibit the proliferation and induce apoptosis of LNCaP cells. BJU Int. 89:950-954.[Medline]
17. Kuntz, S., Wendel, U. & Daniel, H. (1999) Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines. Eur. J. Nutr. 38:133-142.[Medline]
18. Musonda, C. A. & Chipman, J. K. (1998) Quercetin inhibits hydrogen peroxide-induced NFkB DNA binding activity and DNA damage in HepG2 cells. Carcinogenesis 19:1583-1589.
19. Duthie, S. J. & Dobson, V. L. (1999) Dietary flavonoids protect human colonocyte DNA from oxidative attack in vitro. Eur. J. Nutr. 38:28-34.[Medline]
20. O’Brien, N. M., Woods, J. A., Aherne, S. A. & O’Callaghan, Y. C. (2000) Cytotoxicity, genotoxicity and oxidative reactions in cell-culture models: modulatory effects of phytochemicals. Biochem. Soc. Trans. 28:22-26.[Medline]
21. Undeger, U., Aydin, S., Basaran, A. A. & Basaran, N. (2004) The modulating effects of quercetin and rutin on the mitomycin C induced DNA damage. Toxicol. Lett. 151:143-149.[Medline]
22. Duthie, S. J., Johnson, W. & Dobson, V. L. (1997) The effect of dietary flavonoids on DNA damage (strand breaks and oxidised pyrimdines) and growth in human cells. Mutat. Res. 390:141-151.[Medline]
23. Formica, J. V. & Regelson, W. (1995) Review of the biology of quercetin and related bioflavonoids. Food Chem. Toxicol. 33:1061-1080.[Medline]
24. Michels, G., Wätjen, W., Niering, P., Steffan, B., Tran-Thi, Q.-H., Chovolou, Y., Kampkötter, A., Bast, A., Proksch, P. & Kahl, R. (2005) Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells. Toxicology 206:337-348.[Medline]
25. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63.[Medline]
26. Babich, H., Shopsis, C. & Borenfreund, E. (1986) In vitro cytotoxicity testing of aquatic pollutants using established fish cell lines. Ecotoxicol. Environ. Saf. 11:91-99.[Medline]
27. Draper, H. H., Squires, E. J., Mahmoodi, H., Wu, J., Agarwal, S. & Hadley, M. (1993) A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic. Biol. Med. 15:353-363.[Medline]
28. Singh, N. P., McCoy, M. T., Tice, R. R. & Schneider, E. L. (1988) A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell. Res. 175:184-191.[Medline]
29. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. & Rice-Evans, C. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26:1231-1237.[Medline]
30. Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 175:7931-7937.
31. Sakagami, H., Arakawa, H., Maeda, M., Satoh, K., Kadofuku, T., Fukuchi, K. & Gomi, K. (2001) Production of hydrogen peroxide and methionine sulfoxide by epigallocatechin gallate and antioxidants. Anticancer Res. 21:2633-2641.[Medline]
32. Morita, K., Arimochi, H. & Ohnishi, Y. (2003) In vitro cytotoxicity of 4-methylcatechol in murine tumor cells: induction of apoptotic cell death by extracellular pro-oxidant action. J. Pharmacol. Exp. Ther. 306:317-323.
33. Tachibana, H., Koga, K., Fujimura, Y. & Yamada, K. (2004) A receptor for green tea polyphenol EGCG. Nat. Struct. Mol. Biol. 11:380-381.[Medline]
34. Yamashita, N., Tanemura, H. & Kawanishi, S. (1999) Mechanism of oxidative DNA damage induced by quercetin in the presence of Cu(II). Mutat. Res. 425:107-115.[Medline]
35. Scalbert, A. & Williamson, G. (2000) Dietary intake and bioavailability of polyphenols. J. Nutr. 130:2073S-2085S.
36. Manach, C., Morand, C., Demigné, C., Texier, O., Regerat, F. & Rémésy, C. (1997) Bioavailability of rutin and quercetin in rats. FEBS Lett. 409:12-16.[Medline]
37. Hollman, P. C., Bijsman, M. N., van Gameren, Y., Cnossen, E. P., de Vries, J. H. & Katan, M. B. (1999) The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man. Free Radic. Res. 31:569-573.[Medline]
38. Manach, C. & Donovan, J. L. (2004) Pharmacokinetics and metabolism of dietary flavonoids in humans. Free Radic. Res. 38:771-785.[Medline]
39. Conquer, J. A., Maiani, G., Azzini, E., Raguzzini, A. & Holub, B. J. (1998) Supplementation with quercetin markedly increases plasma quercetin concentration without effect on selected risk factors for heart disease in healthy subjects. J. Nutr. 128:593-597.
40. Graefe, E. U., Wittig, J., Mueller, S., Riethling, A. K., Uehleke, B., Drewelow, B., Pforte, H., Jacobasch, G., Derendorf, H. & Veit, M. (2001) Pharmacokinetics and bioavailability of quercetin glycosides in humans. J. Clin. Pharmacol. 41:492-499.[Abstract]
41. Olthof, M. R., Hollman, P. C., Vree, T. B. & Katan, M. B. (2000) Bioavailabilities of quercetin-3-glucoside and quercetin-4'-glucoside do not differ in humans. J. Nutr. 130:1200-1203.
42. de Vries, J. H., Hollman, P. C., van Amersfoort, I., Olthof, M. R. & Katan, M. B. (2001) Red wine is a poor source of bioavailable flavonols in men. J. Nutr. 131:745-748.
43. Moon, J. H., Nakata, R., Oshima, S., Inakuma, T. & Terao, J. (2000) Accumulation of quercetin conjugates in blood plasma after the short-term ingestion of onion by women. Am. J. Physiol. 279:R461-R467.
44. de Pascual-Teresa, S., Johnston, K. L., DuPont, M. S., O’Leary, K. A., Needs, P. W., Morgan, L. M., Clifford, M. N., Bao, Y. & Williamson, G. (2004) Quercetin metabolites downregulate cyclooxygenase-2 transcription in human lymphocytes ex vivo but not in vivo. J. Nutr. 134:552-557.
45. Hollman, P. C., van Trijp, J. M., Buysman, M. N., van der Gaag, M. S., Mengelers, M. J., de Vries, J. H. & Katan, M. B. (1997) Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man. FEBS Lett. 418:152-156.[Medline]
46. Erlund, I., Kosonen, T., Alfthan, G., Maenpaa, J., Perttunen, K., Kenraali, J., Parantainen, J. & Aro, A. (2000) Pharmacokinetics of quercetin from quercetin aglycone and rutin in healthy volunteers. Eur. J. Clin. Pharmacol. 56:545-553.[Medline]
47. Shimoi, K., Saka, N., Kaji, K., Nozawa, R. & Kinae, N. (2000) Metabolic fate of luteolin and its functional activity at focal site. Biofactors 12:181-186.[Medline]
48. Wittig, J., Smolenski, A., Thalheimer, P. & Veit, M. (2000) Beta-glucuronidase activity in human endothelial tissues in an in-vitro model using primary monolayer cultures of human umbilical vein endothelial cells (HUVEC). Polyphenols Commun. 2:459-460.
49. De Santi, C., Pietrabissa, A., Mosca, F. & Pacifici, G. M. (2002) Methylation of quercetin and fisetin, flavonoids widely distributed in edible vegetables, fruits and wine, by human liver. Int. J. Clin. Pharmacol. Ther. 40:207-212.[Medline]
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