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Differential effects of dietary flavonoids on reactive oxygen and nitrogen species generation and changes in antioxidant enzyme expression induced by proinflammatory cytokines in Chang Liver cells
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Food and Chemical Toxicology 46 (2008) 1555–1569 www.elsevier.com/locate/foodchemtox
Differential effects of dietary flavonoids on reactive oxygen and nitrogen species generation and changes in antioxidant enzyme expression induced by proinflammatory cytokines in Chang Liver cells Irene Crespo, Marı´a V. Garcı´a-Mediavilla, Mar Almar, Paquita Gonza´lez, Marı´a J. Tun˜o´n, Sonia Sa´nchez-Campos, Javier Gonza´lez-Gallego * Ciberehd and Institute of Biomedicine, University of Leo´n, 24071 Leo´n, Spain Received 16 January 2007; accepted 10 December 2007
Abstract This study was aimed to investigate the differential protective effect of dietary flavonoids against oxidative stress induced by proinflammatory stimuli in parenchymal liver cells. Chang Liver cells were incubated with a cytokine mixture (CM) supplemented with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin (5–50 lM). Concentrations of oxidised and reduced glutathione, generation of different ROS/RNS, and expression of antioxidant enzymes were measured. Oxidised glutathione concentration and the oxidised/reduced glutathione ratio were increased by the CM. These effects were significantly prevented by quercetin, kaempferol and taxifolin at all tested concentrations. Effects of apigenin reached a lesser extent and were not significant at 25 lM. Treatment with quercetin and kaempferol prevented the production of peroxides, superoxide anion and nitric oxide induced by CM. Taxifolin 50 lM and apigenin 25–50 lM caused a significant increase in peroxides and nitric oxide generation. Protein concentration of the different antioxidant enzymes was generally reduced by kaempferol and quercetin in comparison to CM, although quercetin 25 and 50 lM increased Mn SOD protein concentration. GPx protein level was significantly increased by apigenin 25 and 50 lM. Changes in mRNA tended to be parallel to those in protein concentration. Our study reveals that important differences exist between flavonoids with different structural features in their capacity to abrogate the generation of different ROS/RNS, and suggests that the modulation of antioxidant enzymes by flavonoids may be also important in their antioxidant effects in liver cells. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Flavonoids; Quercetin; Kaempferol; Taxifolin; Apigenin; Liver
1. Introduction Flavonoids are a group of plant polyphenols that are abundant in fruits and vegetables, and represent substantial constituents of the non-energetic part of the human diet. Apart from their physiological role in plants, flavonoids are known to possess a therapeutic potential in some diseases, including cancer, ischemic heart disease, atherosclerosis and liver diseases (Moreira et al., 2004; Kwon *
Corresponding author. Tel.: +34 987 291258; fax: +34 987 291267. E-mail address: [email protected] (J. Gonza´lez-Gallego).
0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.12.014
et al., 2005). Their action can be explained by the antioxidant activity exhibited by many flavonoids, including the inhibition of enzymes involved in the formation of reactive oxygen and nitrogen species (ROS/RNS), the direct scavenging of ROS/RNS, the chelation of trace elements or the upregulation of antioxidant genes (Gong and Chen, 2003). As the oxidative stress plays a central role in liver diseases pathogenesis and progression, the use of antioxidants has been proposed as therapeutic agents, as well as drug coadjuvants, to counteract liver damage (Vitaglione et al., 2004). Protective effects of flavonoids have been reported
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in animal models of fibrosis (Pavanato et al., 2003), biliary cirrhosis (Peres et al., 2000), alcoholic liver disease (Nanji et al., 2003), steatohepatitis (Leclercq et al., 2004), diabetes (Simo˜es et al., 2005), portal hypertension-associated gastropathy (Moreira et al., 2004), or drug toxicity (Galisteo et al., 2004). Although evidence that flavonoids per se have a beneficial effect in human disease, as opposed to animal models, is still lacking, placebo-controlled studies are currently being carried out to investigate the usefulness of flavonoids administration as a therapeutic strategy in different liver pathologies (Emerit et al., 2005). Kupffer cells appear to be centrally involved in the protective effect exerted by flavonoids (Uesugi et al., 2001). However, there are different reports indicating a contribution by parenchymal cells. Thus, curcumin reduces iNOS and nitrotyrosine levels in hepatocytes (Nanji et al., 2003), and alleviates the severity of hepatic inflammation in mice with experimental steatohepatitis (Leclercq et al., 2004). It is known that quercetin protects human hepatoma cells against oxidative stress induced by tert-butyl hydroperoxide (Alia et al., 2006), and we have recently reported that quercetin inhibits ROS/RNS generation, inducible nitric oxide synthase expression and NF-jB activation in IL-1b-activated rat hepatocytes (Martı´nez-Flo´rez et al., 2005). Flavonoids may vary significantly in their antioxidant activities depending on slight variations in their structural features. In particular it has been shown that only flavonoids with a hydroxyl group at the C3 and C4 and the 2–3 double bond show significant antioxidant activity (Sartor et al., 2002). In addition, other characteristics such as the spatial conformation or the lipophilicity of the molecule have an important influence on their pharmacological activity (Heijnen et al., 2002). Evaluation of differences in the molecular mechanisms of flavonoid-induced antioxidant effects may thus help to choose selected flavonoids
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or combinations of flavonoids for clinical development, and may uncover new strategies for the treatment of liver diseases. The objective of the present study was to investigate the differential protective effect of dietary flavonoids against oxidative stress induced by a mixture of proinflammatory cytokines in parenchymal liver cells. The flavonols quercetin (3,30 ,40 ,5,7-pentahydroxyflavone) and kaempferol (3,40 ,5,7-tetrahydroxyflavone), the flavanone taxifolin (3,30 ,40 ,5,7-pentahydroxyflavanone) and the flavone apigenin (40 ,5,7-trihydroxyflavone) were investigated. Although cell lines derived from human tumors, such as HepG2 cells, are easy to culture and well characterized, and have been extensively used as in vitro models of various liver diseases, it is known that marked differences exist in protein expression between transformed cell lines and normal liver. In spite of still differing in many aspects from hepatocytes, non transformed cells show the greatest resemblance in their two-dimensional electrophoresis map of hepatic proteins to those found in normal human liver (Seow et al., 2001). For this reason, concentrations of oxidised and reduced glutathione, generation of different ROS/RNS, and expression of antioxidant enzymes were tested in the present study using the human hepatocyte-derived cell line Chang Liver (Libra et al., 2006; Garcı´a-Mediavilla et al., 2007). 2. Materials and methods 2.1. Cells, cell culture and cytokine activation protocol The human hepatocyte-derived cell line termed Chang Liver (CHL) obtained from American Type Culture Collection (ATCC) (Manassas, VA) has been used. Cells were suspended in culture medium at 5.5– 6.0 105 cell/mL, seeded onto plastic dishes (2 mL/dish, 35 mm 10 mm:9 cm2, Falcon Plastic) and then cultured at monolayers
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Fig. 1. Effect of flavonoids on cell viability in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Cell viability was determined by the MTT assay. Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control or CM-treated group.
I. Crespo et al. / Food and Chemical Toxicology 46 (2008) 1555–1569 in a 5% CO2 humidified incubator at 37 °C. The cultured medium used was Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and 50 lg/mL gentamycin. After 48 h, the medium was changed to include a cytokine mixture (CM) containing human recombinant interleukin 1b (IL-1b), tumor necrosis factor a (TNF-a) and interferon c (IFN-c) (250 UI each) (Genzyme Corp., Boston, MA), as previously described (Garcı´a-Mediavilla et al., 2007) with or without quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A) (5 lM, 25 lM or 50 lM) dissolved in DMSO (0.05%). This vehicle was also added to control and CM-treated groups. After 24 h, cells were trypsinized, pelleted and washed with cold phosphate-buffered saline (PBS) and stored at 70 °C until assayed.
2.2. Cell viability in cell culture The cell viability was assessed by the mitochondrial function, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
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reduction activity as previously reported (Mosmann, 1983). Briefly, cells were seeded in a 24-well plate and incubated with cytokine mixture with or without quercetin, kaempferol, taxifolin or apigenin (5 lM, 25 lM or 50 lM). After 24 h, the cells were incubated with 0.5 mg/mL MTT (Sigma–Aldrich) for 2 h at 37 °C. Subsequently, the media were aspirated and the cells were lysed DMSO, where after the absorbance was read at 560 nm, with background susbstraction at 650 nm, using a microplate reader (Bio-Rad Laboratories, Veenendaal, The Netherlands).
2.3. Reduced and oxidised glutathione concentrations Oxidised and reduced glutathione analysis was performed fluorimetrically by the method of Hissin and Hill (1976). Briefly, cells were homogenised in 0.1 M sodium phosphate, 5 mM EDTA buffer (pH 8.0) with 25% phosphoric acid at a proportion of 1:20. The mixture was centrifuged at 100,000g for 30 min at 4 °C, the supernatant was collected and 500 lL were diluted with 4.5 mL of buffer. Two spectrophotometry
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Fig. 2. Effect of flavonoids on GSSG concentration (A) and on the GSSH/GSH ratio (B) in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
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Fig. 3. Effect of flavonoids on intracellular ROS/RNS generation in CHL cells measured by flow cytometry with DCF-DHA. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). (A) Representative histograms of DCF fluorescence in CM cells and flavonoid 50 lM-treated cells. The fluorescence (FL1-H) is plotted against the number of cells. (B) Fluorescence intensity as percentage of control values. Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CMtreated group.
cuvettes per sample were prepared with 1.8 mL phosphate-EDTA buffer, 100 lL supernatant and 100 lL O-phthalaldehyde (7.5 lM). After incubating for 15 min at 4 °C, a spectrofluorometric reading was obtained at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. To find the percentage of glutathione corresponding to oxidised and reduced forms, 500 lL of the sample supernatant was incubated with 20 lL 4-vinylpyridine for 30 min; to this mixture 4.5 mL of 0.1 M NaOH was added. A 100 lL portion of this mixture was then processed as described above to determine GSSG. GSH was obtained by subtracting GSSG from total glutathione.
2.4. Generation of reactive oxygen and nitrogen species The ROS and RNS production was assessed by flow cytometry as the fluorescence of 20 ,70 -dichlorofluorescein (DCF), diaminofluorescein triazol (DAF-2T) and ethidium (ETH), which are the oxidation products of 20 ,70 dichlorofluorescein diacetate (DCFH-DA; Sigma–Aldrich, Madrid, Spain), 4,5-diaminofluorescein diacetate (DAF-2 DA, Sigma–Aldrich) and dihydroethidium (DHE; Molecular Probes, Leiden, The Netherlands) with a sensitivity for H2O2/NO-based radicals, NO and O 2 , respectively (Garcı´a-Mediavilla et al., 2005). At the end of the incubation period cells
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Fig. 4. Effect of flavonoids on intracellular superoxide anion generation in CHL cells measured by flow cytometry with DHE. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Representative histograms of ETH fluorescence in CM cells and flavonoid 50 lM-treated cells. The fluorescence (FL2-H) is plotted against the number of cells. (B) Fluorescence intensity as percentage of control values. Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group. were incubated with 5 lM DCFH-DA or DHE for 45 min or 90 min for DAF-2 DA at 37 °C then washed twice, resuspended in PBS, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA). ETH fluorescence (FL-2 channel) and DCF and DAF-2T fluorescences (FL-1 channel) of 10,000 cells were analyzed using Cell Quest software (Becton Dickinson Biosciences), and average fluorescence intensity of all trials was calculated.
2.5. Western blot Protein extraction and Western blotting were performed as described (Tun˜o´n et al., 2003). Cell lysates were prepared in 0.25 mmol/L sucrose, 1 mmol/L EDTA, 10 mmol/L Tris and 1% protease inhibitor cocktail. The
mixture was incubated for 30 min at 4 °C and centrifuged 30 min at 13,000g and 4 °C. The supernatant was kept as CHL cell extracts. Samples containing 75 lg of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (9% acrylamide) and transferred to nitrocellulose. Non-specific binding was blocked by preincubation of the nitrocellulose in phosphate-buffered saline containing 5% bovine serum albumin for 1 h. The nitrocellulose was then incubated overnight at 4 °C with rabbit polyclonal anti-glutathione reductase (Abcam, Cambridge, UK), anti-glutathione peroxidase (Abcam) anti-catalase (Calbiochem, San Diego, CA), anti-MnSOD (Stressgen, Ann Arbor, Michigan), and antiCuZnSOD (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies. Bound primary antibody was detected with HRP-conjugated anti-rabbit antibody (DAKO, Glostrup, Denmark) and blots were developed using an
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Fig. 5. Effect of flavonoids on intracellular nitric oxide generation in CHL cells measured by flow cytometry with DAF-2DA. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). (A) Representative histograms of DAF-2T fluorescence in CM cells and flavonoid 50 lM-treated cells. The fluorescence (FL1-H) is plotted against the number of cells. (B) Fluorescence intensity as percentage of control values. Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CMtreated group.
enhanced chemiluminescence detection system (ECL kit, Amersham Pharmacia, Uppsala, Sweden). The density of the specific bands was quantified with an imaging densitometer (Scion Image, Maryland, MA).
2.6. Real-Time RT-PCR Total RNA was obtained by using a Trizol reagent (Life Technologies, Carlsbad, CA) and quantified by the fluorescent method Ribogreen RNA Quantitation Kit (Molecular Probes). Residual genomic DNA was removed by incubating RNA with RQ1 RNase-free DNase (Promega, Madison, WI). First-standard cDNA was synthesized using HighCapacity cDNA Archive Kit (Applied Biosystems, Weiterstadt, Ger-
many). The negative control (no transcriptase control) was performed in parallel. cDNA was amplified using TaqMan Universal PCR Master Mix (Applied Biosystems) on an ABI 7000 (Applied Biosystems). TaqMan primers and probes for glutathione reductase (GenBank accession No. X15722 and Hs00167317_m1), glutathione peroxidase (GenBank accession No. Y00433 and Hs00829989_gH), catalase (GenBank accession No. X04076 and Hs00156308_m1), superoxide dismutase 2 (GenBank accession No. M36693 and Hs00167309_ml), superoxide dismutase 1 (GenBank accession No. X02317 and Hs00533490_m1), and 18S rRNA (GenBank accession No. X03205.1 and Hs99999901_s1) genes were derived from the commercially available TaqManÒ Gene Expression Assays (Applied Biosystems). Relative changes in gene expression levels were determined using the 2DDCT method as described previously (Livak and Schmittgen,
I. Crespo et al. / Food and Chemical Toxicology 46 (2008) 1555–1569 2001). The cycle number at which the transcripts were detectable (CT) was normalized to the cycle number of 18S detection, referred to as DCT.
2.7. Statistical analysis Means and SEMs were calculated. Data were analyzed using ANOVA. Post-hoc comparisons were carried out by the Newman Keuls test. Statistical significance was set at the p < 0.05. SPSS+ vers. 13.0 statistical software (Chicago, IL) was used.
3. Results 3.1. Cell viability The MTT assay showed quercetin and taxifolin to be slightly cytotoxic, with 11% and 13% cell death, respectively, at 50 lM. Apigenin at 25 lM and 50 lM reduced significantly cell viability, with 21% and 35% cell death, respectively (Fig. 1). 3.2. Glutathione concentration Oxidised glutathione concentration was significantly increased by the cytokine mixture. This was significantly prevented by quercetin, kaempferol and taxifolin at all tested concentrations. The oxidised to reduced glutathione
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ratio was used as a marker of oxidative stress. The treatment with the cytokine mixture evoked a marked increase of this ratio that was significantly prevented by quercetin, kaempferol and taxifolin. Effects of apigenin reached a lesser extent and were not significant at 25 lM (Fig. 2). 3.3. Generation of ROS/RNS We investigated generation of ROS/RNS by flow cytometry using DCF-DA. Analysis of histograms in which the fluorescence, detected with the FL1-H channel, was plotted against the relative number of cells (Fig. 3A) and quantification of the corresponding fluorescence intensity (Fig. 3B), indicated that the cytokine mixture induced a significant increase in ROS/RNS production as compared to unstressed controls. Treatment of cells with 50 lM quercetin or kaempferol significantly decreased ROS/RNS production. When cells were treated with taxifolin 50 lM or apigenin 25 lM and 50 lM, ROS/RNS production increased significantly as compared to the CM-treated group (Fig. 3B). Flow cytometry using ETH was used to detect superoxide anion generation. The increase induced by the cytokine mixture was significantly prevented by quercetin and kaempferol 25 lM and 50 lM and by 5
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Fig. 6. Effect of flavonoids on Mn SOD protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-Mn SOD antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
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Fig. 7. Effect of flavonoids on Cu/Zn SOD protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-Cu/Zn SOD antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
taxifolin 5–50 lM, whereas apigenin had no preventive effect (Fig. 4B). Generation of nitric oxide, measured by DAF-2T fluorescence, was significantly decreased by quercetin 50 lM, kaempferol 5–50 lM, and taxifolin 5 lM. Taxifolin 50 lM or apigenin 25 lM and 50 lM caused a significant increase in nitric oxide production (Fig. 5B). 3.4. Gene expression of antioxidant enzymes Data presented in Fig. 6 show the effects of the different flavonoids on Mn SOD protein level. The significant increase induced by the cytokine mixture was partially prevented by kaempferol and taxifolin 25 lM or 50 lM. Quercetin 25 lM and 50 lM caused a further increase, while apigenin had no significant effect. Cu/Zn SOD protein level (Fig. 7) was significantly decreased by quercetin 25 lM and 50 lM, kaempferol 5–50 lM, and taxifolin 50 lM. GPx protein level was significantly decreased by kaempferol 5– 50 lM, quercetin 25 lM and 50 lM, and taxifolin 50 lM. When cells were treated with apigenin, all concentrations increased significantly GPx protein level as compared to the CM group (Fig. 8). Quercetin 5–50 lM and kaempferol 25–50 lM partially prevented the increase in CAT protein
level induced in the CM-group, with no effect of the different taxifolin or apigenin concentrations (Fig. 9). GR protein level was significantly reduced as compared to both control and CM groups, by quercetin 5–50 lM and kaempferol 25–50 lM. Values were significantly increased by taxifolin 50 lM (Fig. 10). Since protein levels of the different antioxidant enzymes were down- or up-regulated by the different flavonoids, RT-PCR analysis was performed to assess the effects on the mRNA level. The fold change in expression of the Mn SOD, Cu/Zn SOD, GPx, CAT, and GR genes was expressed as 2DDCT, in which DDCT equals the DCT of the flavonoid-treated group minus the DCT of the control group, that was normalized to 1. No significant changes in Mn SOD RNA levels were observed as compared to the CM-treated group, except for the increase induced by quercetin 25 lM or 50 lM and the decrease by kaempferol 50 lM. Kaempferol 25 lM and 50 lM or quercetin 50 lM induced a decrease in mRNA level for Cu/Zn SOD, GPx, CAT and GR. Quercetin 25 lM also decreased mRNA level for CAT and GR. GR mRNA levels was significantly increased by taxifolin 50 lM and reduced by apigenin 50 lM (Fig. 11).
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Fig. 8. Effect of flavonoids on GPx protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-GPx antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
4. Discussion This study demonstrates that flavonoids may have the ability to protect CHL cells against oxidative stress by modulating ROS/RNS generation, the reduced to oxidised glutathione ratio and the main antioxidant enzymes expression. However, important differential effects were detected between the various molecules tested. Reduced glutathione (GSH) is the main nonenzymatic antioxidant defense within the cell, reducing different peroxides and hydroperoxides by oxidation to GSSG. Thus, the GSSG to GSH ratio has been widely employed as a marker of oxidative stress (Gutie´rrez et al., 2006). By using this method, we found that the GSSG/GSH ratio increase induced by the cytokine mixture was prevented, although to a different extent, by the different flavonoids. Quercetin data coincide with previous reports that this molecule protects against liver-induced oxidative stress, with changes in the GSSG/GSH ratio, both in rats with biliary obstruction (Peres et al., 2000) or following chronic ethanol administration in mice (Molina et al., 2003). Although it has been recently indicated that quercetin protects HepG2 cells
against a decrease of GSH levels mainly by preventing the formation of GSH conjugates rather than oxidation to GSSG (Lima et al., 2006), our data demonstrate that in CHL cells treated with a cytokine mixture quercetin decreased GSSG concentration, and that this effect appeared not to be due to an increase conversion back to GSH, because both GR protein concentration and mRNA levels were significantly reduced by treatment with the flavonoid. Similar changes for GR expression were detected in the case of kaempferol. As for the effects of apigenin, in vitro studies indicate that this molecule forms upon oxidation phenoxyl radicals with high redox potentials which cooxidise GSH, resulting in GSSG formation. In contrast, GSH is not oxidised by the low redox potential of quercetin or kaempferol-derived radicals (Galati et al., 2001). This could contribute to explain the lesser ability of apigenin to prevent the increase in GSSG concentration and in the GSSG/GSH ratio. The lowered generation of GSSG by quercetin and kaempferol was probably related to the effects of both flavonoids on the formation/elimination of hydrogen peroxide and organic peroxides, which are the main substrates
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Fig. 9. Effect of flavonoids on CAT protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-CAT antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
of GPx. Expression of this antioxidant enzyme was reduced by both flavonoids, and this effect was accompanied at high concentrations by a lowered DCF fluorescence intensity, which is proportional to the amount of peroxides produced by the cells (Tun˜o´n et al., 2003). Our data coincide with previous reports that both flavonols suppress lipid peroxidation in cultured hepatocytes (Sugihara et al., 1999). It has been shown that flavonoids with a hydroxyl group at the C3 and C4 and the 2–3 double bond show significant antioxidant activity (Sartor et al., 2002; Kachadourian and Day, 2006), and both quercetin and kaempferol share these characteristics (Ueda et al., 2004). When flow cytometry was used to compare generation of specific ROS/RNS, the two flavonols, which are known to exhibit an inhibitory effect on xanthine oxidase and superoxide scavenging activity (Lin et al., 2002; Masuoka et al., 2006), were observed to inhibit superoxide generation to a similar extent at the different tested concentrations. However, kaempferol suppressed more effectively than quercetin nitric oxide generation, in spite of lacking a radical scavenging catechol moiety in the B ring, which has been suggested to be essential for the inhibitory action of quercetin on iNOS gene expression (Banerjee et al., 2002). This does not necessarily indicate a better anti-inflammatory capacity by kaempfer-
ol, because nitric oxide cytoxicity is for the major part mediated by peroxynitrite, formed in the reaction of nitric oxide with superoxide (Garcı´a-Mediavilla et al., 2005), and quercetin has shown a much higher peroxynitrite scavenging capacity, apparently related to the lack of free aromatic hydroxyl groups (Haenen et al., 1997; Sadeghipour et al., 2005). The behavior of the flavone apigenin and the flavanone taxifolin is more complex. Apigenin 25 lM and 50 lM, reduced cell viability, increased significantly the generation of peroxides, and also caused a specific increase in nitric oxide generation. Although apigenin has been reported to block COX-2 expression in macrophages and keratinocytes (Van Dross et al., 2005), different findings have shown that this molecule has minimal antioxidant activity in comparison to structurally related flavonoids such as quercetin (Safari and Sheikh, 2003). In human leukemic cells it has demonstrated a high capacity to act as proteosome inhibitor and apoptosis inducer, an effect which appears to be related to the absence of a C3 hydroxyl group (Chen et al., 2005). However, although apigenin has been previously indicated to reduce cell viability in macrophages in the presence of LPS (Raso et al., 2001) and to result cytotoxic to human normal cells, such as lung fibroblasts (Mat-
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suo et al., 2005), induction of cell death appears to be absent in non-transformed cells (Chen et al., 2005; Vargo et al., 2006). The exact mechanism for the toxic effects of apigenin in CHL cells is not clear, but oxidative stress due to the formation of ROS/RNS may play a crucial role, and it is known that apigenin radicals are very reactive, being able to act as radical initiators (Galati and O’Brien, 2004; Sadeghipour et al., 2005). Taxifolin 5 lM was able to reduce the generation of superoxide and nitric oxide, but had no effect on DCF fluorescence, which could be indicating a reduced capacity to scavenge hydrogen peroxide and organic peroxides. A low antioxidant capacity of taxifolin in comparison to quercetin has been previously reported in retinal cells after ascorbate/Fe2+-induced oxidative stress, and it has been suggested that differences may arise from the less planar structure of taxifolin which results in strong hydrogen bonds (Areias et al., 2001). At high concentrations (50 lM), however, taxifolin caused a marked elevation in nitric oxide generation and in DCF fluorescence. It is known that certain flavonoids may switch from antioxidant to oxidant action under some conditions, such as changes in the molar ratio to metal ions
CON
A
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and decreases in the redox potential of metal–flavonoid complexes (Sugihara et al., 1999; Texeira et al., 2005). Thus, these flavonoids with various effects on oxidation may have dual biochemical and pharmacological actions. The role of dietary flavonoids as free radical scavengers is well know, but their regulatory roles on antioxidant proteins remains to be fully elucidated. Organisms have developed a variety of antioxidant defense systems as protection from ROS/RNS. The major endogenous antioxidant systems include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). SOD catalyzes the dismutation of the superoxide radical anion, and there is a Mn SOD localized in the mitochondria and a Cu/Zn SOD mainly localized in the cytosol. CAT and GPX convert H2O2 to H2O, and GR recycles oxidised glutathione back to reduced glutathione (Alia et al., 2006). In our study, treatment of CHL cells with a cytokine cocktail caused a transcriptional induction of antioxidant enzymes, with significant increases in both the RNA level and protein concentration. Because quercetin, kaempferol and taxifolin decreased superoxide anion generation to a similar extent, and given the existence of
CM
5
25
50
(μM) GR
Quercetin Kaempferol Taxifolin Apigenin
Q
B
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T ab
Relative GR protein level
200 180
a
a
160 140
A
a
a
120
ab
100
ab
80 60
ab ab ab
40 20 0
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+
5 25 50 + + +
5 25 50 + + +
5 25 50 + + +
5 25 50 (μmol/L) + + +
Fig. 10. Effect of flavonoids on GR protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-GR antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
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Mn SOD mRNA fold increase
Q
K
T
A
ab
6 5
a ab 4
a
a
a a
3
a a a
a a
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CM -
+
3 2
5 25 50 + + +
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a a
a a ab
a b
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+
3
GPx mRNA fold increase
5 25 50 + + +
2.5
a
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a
a a
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CM 2
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+
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CM 3
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Fig. 11. Effect of flavonoids on mRNA level of Mn SOD, Cu/Zn SOD, GPx, CAT and GR as quantitated by real time polymerase chain reaction analysis in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Relative changes in gene expression levels were determined using the 2DDCT method. Data, normalized against 18S rRNA, are presented as fold change from the control group and represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
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a feedback mechanism on the antioxidant enzymes triggered by the flavonoids (Breinholt et al., 1999), the decrease in substrate concentration could contribute to explain a reduction in the expression of SOD isoforms. However, quercetin caused a post-transcriptional induction of Mn SOD, which is difficult to conciliate with a report indicating that this flavonol induces in hepatoma cells a decrease in Mn SOD mRNA levels, probably resulting from inhibition of NF-jB activation (Rohrdanz et al., 2003; Martı´nezFlo´rez et al., 2005). It has been demonstrated that nitric oxide causes an induction of Mn SOD protein (Keller et al., 2003) in glomerular mesangial cells. Because in our experiments, differently from kaempferol, quercetin was not able to significantly reduce nitric oxide generation (Keller et al., 2003), Mn SOD induction could represent a mechanism by which nitric oxide reduces the concentration of superoxide anion and the formation of peroxynitrite to ensure a self-limited inflammatory response (Pfeilschifter et al., 2002). Effects of flavonoids on antioxidant enzyme expression could be also mediated by modification of signal transduction pathways. Sequence analysis of GPx and CAT has revealed in different species binding motifs for NF-jB and AP-1 (Zhou et al., 2001), transcriptional regulators that are activated in response to oxidative stress in various tissues (Surth et al., 2005). Although there are conflicting reports concerning the relationship between flavonoids and the NF-jB/AP-1 pathways, and it has been found that dietary quercetin does not reduce NF-jB activation in the renal cortex of rats with chronic glomerular disease (Rangan et al., 2002), different studies indicate that flavonols inhibit AP-1 activation in lipopolysaccharide-activated RAW264.7 cells (Kim et al., 2005), and that quercetin prevents LPS-IjB phosphorylation in bone marrow derived macrophages (Comalada et al., 2005), and inhibits NF-jB activation induced by interleukin-1 in murine fibroblast l-TK cells (Muraoka et al., 2002), interleukin-1b activated rat hepatocytes (Martı´nez-Flo´rez et al., 2005) or H2O2 in HepG2 cells (Musonda and Chipman, 1998). We have recently reported that quercetin and kaempferol inhibit the activation of NFjB induced by a cytokine mixture in Chang Liver cells (Garcı´a-Mediavilla et al., 2007). In our study, the generation of peroxides was reduced by quercetin and kaempferol, and the lower exposure to levels of oxidizing species could, through changes in the activation of the redox-sensitive transcription factors, contribute to a decrease in the expression of antioxidant enzymes. The NF-E2 related factor 2 (Nrf2), a member of the cap0 n0 collar family of basic leucine zipper transcription factors, is another redox-sensitive factor whose nuclear translocation and binding to the antioxidant response elements (ARE) in their promoter regions may result in induction of antioxidant enzymes (BrigeliusFlohe and Banning, 2006). Up-regulation by apigenin of GPx could, thus, be also a consequence of the increased generation of ROS/RNS and their effects on nuclear factors and other regulatory elements. Increased expression of GPx could, in turn, contribute to the formation, upon oxidation
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by H2O2 of the apigenin phenoxyl radical which catalyzes GSH or NADH co-oxidation and generate ROS/RNS (Galati et al., 2001; Galati and O’Brien, 2004). In conclusion, our study reveals that important differences exist between flavonoids with different structural features in their capacity to abrogate the generation of different ROS/RNS, and suggests that the modulation of antioxidant enzymes by flavonoids may be also important in their antioxidant effects in liver cells. Although validation in animal models or human systems is necessary, and extrapolation to the in vivo environment requires consideration of flavonoid bioavailability and metabolism, data here reported showed clear protective effects by some flavonoids against oxidative damage induced in CHL cells. This could be of use against liver diseases where it is known that oxidative stress plays a relevant role. Acknowledgment This work was supported by the Plan Nacional de I+D, Spain (Grant No. BFI2003-03114). References Alia, M., Ramos, S., Mateos, R., Granado-Serrano, A.B., Bravo, L., Goya, L., 2006. Quercetin protects human hepatoma HepG2 against oxidative stress induced by tert-butyl hydroperoxide. Toxicol. Appl. Pharmacol. 21, 110–118. Areias, F.M., Rego, A.C., Oliveira, C.R., Seabra, R.M., 2001. Antioxidant effects of flavonoids after ascorbate/Fe2+-induced oxidative stress in cultured retinal cells. Biochem. Pharmacol. 62, 111–118. Banerjee, T., Van der Vliet, A., Ziboh, V.A., 2002. Downregulation of COX-2 and iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line. Prostag. Leukotr. Essent. Fatty Acid 66, 485–492. Breinholt, V., Lauridsen, S.T., Dragsted, L.O., 1999. Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rats. Xenobiotica 29, 1227–1240. Brigelius-Flohe, R., Banning, A., 2006. Part of the series: from dietary antioxidants to regulators in cellular signalling and gene regulation. Free Radic. Biol. Med. 40, 775–787. Chen, D., Daniel, K.G., Chen, M.S., Kuhn, D.J., Landis-Piwowar, K.R., Dou, Q.P., 2005. Dietary flavonoids as proteosome inhibitors and apoptosis inducers in human leukemic cells. Biochem. Pharmacol. 69, 1421–1432. Comalada, M., Camuesco, D., Sierra, S., Ballester, I., Xaus, J., Galvez, J., Zarzuelo, A., 2005. In vivo quercitrin anti-inflammatory effect involves releases of quercetin, which inhibits inflammation through down-regulation of NF-jB pathway. Eur. J. Immunol. 35, 584–592. Emerit, I., Huang, C.Y., Serejo, F., Filipe, P., Fernandes, A., Costa, A., Freitas, J., Baptista, A., Carneiro de Moura, M., 2005. Oxidative stress in chronic hepatitis C: a preliminary study on the protective effects of antioxidant flavonoids. Hepatogastroenterology 52, 530–536. Galati, G., Moridani, M.Y., Chan, T.S., O’Brien, P.J., 2001. Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation. Free Radic. Biol. Med. 34, 370–382. Galati, G., O’Brien, P.J., 2004. Potential toxicity of flavonoids and other dietary phenolics: significance for their chemoprotective and anticancer properties. Free Radic. Biol. Med. 37, 287–303. Galisteo, M., Garcı´a Saura, M.F., Jime´nez, R., Villar, I.C., Zarzuelo, A., Vargas, F., Duarte, J., 2004. Effects of chronic quercetin treatment on
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Matsuo, M., Sasaki, N., Saga, K., Kaneko, T., 2005. Citotoxicity of flavonoids towards cultured normal human cells. Biol. Pharm. Bull. 28, 253–259. Molina, M.F., Sanchez Reus, I., Iglesias, I., Benedi, J., 2003. Quercetin, a flavonoid antioxidant, prevents and protects against ethanol-induced oxidative stress in mouse liver. Biol. Pharm. Bull. 26, 1398–1402. Moreira, A.J., Fraga, C., Alonso, M., Collado, P.S., Zetller, C., Marroni, C., Gonza´lez-Gallego, J., 2004. Quercetin prevents oxidative stress and NF-jB activation in gastric mucosa of portal hypertensive rats. Biochem. Pharmacol. 268, 1939–1946. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Muraoka, K., Shimizu, K., Sun, X., Tani, T., Izumumi, R., Miwa, K., Yamamoto, K., 2002. Flavonoids exert diverse inhibitory effects on the activation of NF-jB. Transplant. Proc. 34, 1335–1340. Musonda, C.A., Chipman, J.K., 1998. Quercetin inhibits hydrogen peroxide (H2O2)-induced NF-jB DNA binding activity and DNA damage in HepG2 cells. Carcinogenesis 19, 1583–1589. Nanji, A.A., Jokelainen, K., Tipoe, G.L., Rahemtulla, A., Thomas, P., Dannenberg, A.J., 2003. Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-jB-dependent genes. Am. J. Physiol. 284, G321–G327. Pavanato, A., Tun˜o´n, M.J., Sa´nchez-Campos, S., Llesuy, C.S., Gonza´lezGallego, J., Marroni, N., 2003. Effects of quercetin on liver damage in rats with carbon tetrachloride-induced cirrhosis. Dig. Dis. Sci. 48, 824– 829. Peres, W., Tun˜o´n, M.J., Mato, S., Collado, P.S., Gonza´lez-Gallego, J., 2000. Hepatoprotective effects of the flavonoid quercetin in rats with biliary obstruction. J. Hepatol. 33, 742–750. Pfeilschifter, J., Beck, K.F., Eberhardt, W., Huwiler, A., 2002. Changing gears in the course of glomerulonephritis by shifting superoxide to nitric oxide-dominated chemistry. Kidney Int. 61, 809–815. Rangan, G.K., Wang, Y., Harris, D.C., 2002. Dietary quercetin augments activator protein-1 and does not reduce nuclear factor-kappa B in the renal cortex of rats with established chronic glomerular disease. Nephron 90, 313–319. Raso, G.M., Meli, R., Di Carlo, G., Pacilio, M., Di Carlo, R., 2001. Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1. Life Sci. 68, 921– 931. Rohrdanz, E., Bittner, A., Tran-Thi, Q.H., Kahl, R., 2003. The effect of quercetin on the mRNA expression of antioxidant enzymes in hepatoma cells. Arch. Toxicol. 77, 506–510. Sadeghipour, M., Terreux, R., Phipps, J., 2005. Flavonoids and tyrosine nitration: structure activity relationship correlation with enthalpy of formation. Toxicol. In Vitro 19, 155–165. Safari, M.R., Sheikh, H., 2003. Effects of flavonoids on the susceptibility of low density lipoprotein to oxidative modification. Prostag. Leukot. Essents. Fatty Acid 69, 73–77. Sartor, L., Pezzato, E., Dell’Aica, I., Caniato, R., Biggin, S., Garbisa, S., 2002. Inhibition of matrix-proteases by polyphenols: chemical insights for anti-inflammatory and anti-invasion drug design. Biochem. Pharmacol. 64, 229–237. Seow, T.K., Liang, R.C.M., Leow, C.K., Chung, M.C.M., 2001. Hepatocellular carcinoma: from bedside to proteomics. Proteomics 1, 1249– 1263. Simo˜es, A., Porawski, M., Alonso, M., Collado, P.S., Marroni, N., Gonza´lez-Gallego, J., 2005. Quercetin prevents oxidative stress and NF-jB activation in liver of type 1 diabetic rats. J. Nutr. 135, 2299– 2304. Sugihara, N., Arakawa, T., Ohnishi, M., Furuno, K., 1999. Anti-and prooxidative effects of flavonoids on metal-induced lipid hydroperoxidedependent lipid peroxidation in cultured hepatocytes loaded with alpha-linoleic acid. Free Radic. Biol. Med. 27, 1313–1323. Surth, Y.J., Kundu, J.K., Na, H.K., Lee, J.S., 2005. Redox-sensitive transcription factors as prime targets for chemoprevention with anti-
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Food and Chemical Toxicology 46 (2008) 1555–1569 www.elsevier.com/locate/foodchemtox
Differential effects of dietary flavonoids on reactive oxygen and nitrogen species generation and changes in antioxidant enzyme expression induced by proinflammatory cytokines in Chang Liver cells Irene Crespo, Marı´a V. Garcı´a-Mediavilla, Mar Almar, Paquita Gonza´lez, Marı´a J. Tun˜o´n, Sonia Sa´nchez-Campos, Javier Gonza´lez-Gallego * Ciberehd and Institute of Biomedicine, University of Leo´n, 24071 Leo´n, Spain Received 16 January 2007; accepted 10 December 2007
Abstract This study was aimed to investigate the differential protective effect of dietary flavonoids against oxidative stress induced by proinflammatory stimuli in parenchymal liver cells. Chang Liver cells were incubated with a cytokine mixture (CM) supplemented with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin (5–50 lM). Concentrations of oxidised and reduced glutathione, generation of different ROS/RNS, and expression of antioxidant enzymes were measured. Oxidised glutathione concentration and the oxidised/reduced glutathione ratio were increased by the CM. These effects were significantly prevented by quercetin, kaempferol and taxifolin at all tested concentrations. Effects of apigenin reached a lesser extent and were not significant at 25 lM. Treatment with quercetin and kaempferol prevented the production of peroxides, superoxide anion and nitric oxide induced by CM. Taxifolin 50 lM and apigenin 25–50 lM caused a significant increase in peroxides and nitric oxide generation. Protein concentration of the different antioxidant enzymes was generally reduced by kaempferol and quercetin in comparison to CM, although quercetin 25 and 50 lM increased Mn SOD protein concentration. GPx protein level was significantly increased by apigenin 25 and 50 lM. Changes in mRNA tended to be parallel to those in protein concentration. Our study reveals that important differences exist between flavonoids with different structural features in their capacity to abrogate the generation of different ROS/RNS, and suggests that the modulation of antioxidant enzymes by flavonoids may be also important in their antioxidant effects in liver cells. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Flavonoids; Quercetin; Kaempferol; Taxifolin; Apigenin; Liver
1. Introduction Flavonoids are a group of plant polyphenols that are abundant in fruits and vegetables, and represent substantial constituents of the non-energetic part of the human diet. Apart from their physiological role in plants, flavonoids are known to possess a therapeutic potential in some diseases, including cancer, ischemic heart disease, atherosclerosis and liver diseases (Moreira et al., 2004; Kwon *
Corresponding author. Tel.: +34 987 291258; fax: +34 987 291267. E-mail address: [email protected] (J. Gonza´lez-Gallego).
0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.12.014
et al., 2005). Their action can be explained by the antioxidant activity exhibited by many flavonoids, including the inhibition of enzymes involved in the formation of reactive oxygen and nitrogen species (ROS/RNS), the direct scavenging of ROS/RNS, the chelation of trace elements or the upregulation of antioxidant genes (Gong and Chen, 2003). As the oxidative stress plays a central role in liver diseases pathogenesis and progression, the use of antioxidants has been proposed as therapeutic agents, as well as drug coadjuvants, to counteract liver damage (Vitaglione et al., 2004). Protective effects of flavonoids have been reported
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in animal models of fibrosis (Pavanato et al., 2003), biliary cirrhosis (Peres et al., 2000), alcoholic liver disease (Nanji et al., 2003), steatohepatitis (Leclercq et al., 2004), diabetes (Simo˜es et al., 2005), portal hypertension-associated gastropathy (Moreira et al., 2004), or drug toxicity (Galisteo et al., 2004). Although evidence that flavonoids per se have a beneficial effect in human disease, as opposed to animal models, is still lacking, placebo-controlled studies are currently being carried out to investigate the usefulness of flavonoids administration as a therapeutic strategy in different liver pathologies (Emerit et al., 2005). Kupffer cells appear to be centrally involved in the protective effect exerted by flavonoids (Uesugi et al., 2001). However, there are different reports indicating a contribution by parenchymal cells. Thus, curcumin reduces iNOS and nitrotyrosine levels in hepatocytes (Nanji et al., 2003), and alleviates the severity of hepatic inflammation in mice with experimental steatohepatitis (Leclercq et al., 2004). It is known that quercetin protects human hepatoma cells against oxidative stress induced by tert-butyl hydroperoxide (Alia et al., 2006), and we have recently reported that quercetin inhibits ROS/RNS generation, inducible nitric oxide synthase expression and NF-jB activation in IL-1b-activated rat hepatocytes (Martı´nez-Flo´rez et al., 2005). Flavonoids may vary significantly in their antioxidant activities depending on slight variations in their structural features. In particular it has been shown that only flavonoids with a hydroxyl group at the C3 and C4 and the 2–3 double bond show significant antioxidant activity (Sartor et al., 2002). In addition, other characteristics such as the spatial conformation or the lipophilicity of the molecule have an important influence on their pharmacological activity (Heijnen et al., 2002). Evaluation of differences in the molecular mechanisms of flavonoid-induced antioxidant effects may thus help to choose selected flavonoids
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Cell viability (% of control)
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or combinations of flavonoids for clinical development, and may uncover new strategies for the treatment of liver diseases. The objective of the present study was to investigate the differential protective effect of dietary flavonoids against oxidative stress induced by a mixture of proinflammatory cytokines in parenchymal liver cells. The flavonols quercetin (3,30 ,40 ,5,7-pentahydroxyflavone) and kaempferol (3,40 ,5,7-tetrahydroxyflavone), the flavanone taxifolin (3,30 ,40 ,5,7-pentahydroxyflavanone) and the flavone apigenin (40 ,5,7-trihydroxyflavone) were investigated. Although cell lines derived from human tumors, such as HepG2 cells, are easy to culture and well characterized, and have been extensively used as in vitro models of various liver diseases, it is known that marked differences exist in protein expression between transformed cell lines and normal liver. In spite of still differing in many aspects from hepatocytes, non transformed cells show the greatest resemblance in their two-dimensional electrophoresis map of hepatic proteins to those found in normal human liver (Seow et al., 2001). For this reason, concentrations of oxidised and reduced glutathione, generation of different ROS/RNS, and expression of antioxidant enzymes were tested in the present study using the human hepatocyte-derived cell line Chang Liver (Libra et al., 2006; Garcı´a-Mediavilla et al., 2007). 2. Materials and methods 2.1. Cells, cell culture and cytokine activation protocol The human hepatocyte-derived cell line termed Chang Liver (CHL) obtained from American Type Culture Collection (ATCC) (Manassas, VA) has been used. Cells were suspended in culture medium at 5.5– 6.0 105 cell/mL, seeded onto plastic dishes (2 mL/dish, 35 mm 10 mm:9 cm2, Falcon Plastic) and then cultured at monolayers
K 5 μM
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Fig. 1. Effect of flavonoids on cell viability in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Cell viability was determined by the MTT assay. Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control or CM-treated group.
I. Crespo et al. / Food and Chemical Toxicology 46 (2008) 1555–1569 in a 5% CO2 humidified incubator at 37 °C. The cultured medium used was Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and 50 lg/mL gentamycin. After 48 h, the medium was changed to include a cytokine mixture (CM) containing human recombinant interleukin 1b (IL-1b), tumor necrosis factor a (TNF-a) and interferon c (IFN-c) (250 UI each) (Genzyme Corp., Boston, MA), as previously described (Garcı´a-Mediavilla et al., 2007) with or without quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A) (5 lM, 25 lM or 50 lM) dissolved in DMSO (0.05%). This vehicle was also added to control and CM-treated groups. After 24 h, cells were trypsinized, pelleted and washed with cold phosphate-buffered saline (PBS) and stored at 70 °C until assayed.
2.2. Cell viability in cell culture The cell viability was assessed by the mitochondrial function, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
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reduction activity as previously reported (Mosmann, 1983). Briefly, cells were seeded in a 24-well plate and incubated with cytokine mixture with or without quercetin, kaempferol, taxifolin or apigenin (5 lM, 25 lM or 50 lM). After 24 h, the cells were incubated with 0.5 mg/mL MTT (Sigma–Aldrich) for 2 h at 37 °C. Subsequently, the media were aspirated and the cells were lysed DMSO, where after the absorbance was read at 560 nm, with background susbstraction at 650 nm, using a microplate reader (Bio-Rad Laboratories, Veenendaal, The Netherlands).
2.3. Reduced and oxidised glutathione concentrations Oxidised and reduced glutathione analysis was performed fluorimetrically by the method of Hissin and Hill (1976). Briefly, cells were homogenised in 0.1 M sodium phosphate, 5 mM EDTA buffer (pH 8.0) with 25% phosphoric acid at a proportion of 1:20. The mixture was centrifuged at 100,000g for 30 min at 4 °C, the supernatant was collected and 500 lL were diluted with 4.5 mL of buffer. Two spectrophotometry
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Fig. 2. Effect of flavonoids on GSSG concentration (A) and on the GSSH/GSH ratio (B) in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
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Fig. 3. Effect of flavonoids on intracellular ROS/RNS generation in CHL cells measured by flow cytometry with DCF-DHA. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). (A) Representative histograms of DCF fluorescence in CM cells and flavonoid 50 lM-treated cells. The fluorescence (FL1-H) is plotted against the number of cells. (B) Fluorescence intensity as percentage of control values. Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CMtreated group.
cuvettes per sample were prepared with 1.8 mL phosphate-EDTA buffer, 100 lL supernatant and 100 lL O-phthalaldehyde (7.5 lM). After incubating for 15 min at 4 °C, a spectrofluorometric reading was obtained at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. To find the percentage of glutathione corresponding to oxidised and reduced forms, 500 lL of the sample supernatant was incubated with 20 lL 4-vinylpyridine for 30 min; to this mixture 4.5 mL of 0.1 M NaOH was added. A 100 lL portion of this mixture was then processed as described above to determine GSSG. GSH was obtained by subtracting GSSG from total glutathione.
2.4. Generation of reactive oxygen and nitrogen species The ROS and RNS production was assessed by flow cytometry as the fluorescence of 20 ,70 -dichlorofluorescein (DCF), diaminofluorescein triazol (DAF-2T) and ethidium (ETH), which are the oxidation products of 20 ,70 dichlorofluorescein diacetate (DCFH-DA; Sigma–Aldrich, Madrid, Spain), 4,5-diaminofluorescein diacetate (DAF-2 DA, Sigma–Aldrich) and dihydroethidium (DHE; Molecular Probes, Leiden, The Netherlands) with a sensitivity for H2O2/NO-based radicals, NO and O 2 , respectively (Garcı´a-Mediavilla et al., 2005). At the end of the incubation period cells
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Fig. 4. Effect of flavonoids on intracellular superoxide anion generation in CHL cells measured by flow cytometry with DHE. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Representative histograms of ETH fluorescence in CM cells and flavonoid 50 lM-treated cells. The fluorescence (FL2-H) is plotted against the number of cells. (B) Fluorescence intensity as percentage of control values. Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group. were incubated with 5 lM DCFH-DA or DHE for 45 min or 90 min for DAF-2 DA at 37 °C then washed twice, resuspended in PBS, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA). ETH fluorescence (FL-2 channel) and DCF and DAF-2T fluorescences (FL-1 channel) of 10,000 cells were analyzed using Cell Quest software (Becton Dickinson Biosciences), and average fluorescence intensity of all trials was calculated.
2.5. Western blot Protein extraction and Western blotting were performed as described (Tun˜o´n et al., 2003). Cell lysates were prepared in 0.25 mmol/L sucrose, 1 mmol/L EDTA, 10 mmol/L Tris and 1% protease inhibitor cocktail. The
mixture was incubated for 30 min at 4 °C and centrifuged 30 min at 13,000g and 4 °C. The supernatant was kept as CHL cell extracts. Samples containing 75 lg of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (9% acrylamide) and transferred to nitrocellulose. Non-specific binding was blocked by preincubation of the nitrocellulose in phosphate-buffered saline containing 5% bovine serum albumin for 1 h. The nitrocellulose was then incubated overnight at 4 °C with rabbit polyclonal anti-glutathione reductase (Abcam, Cambridge, UK), anti-glutathione peroxidase (Abcam) anti-catalase (Calbiochem, San Diego, CA), anti-MnSOD (Stressgen, Ann Arbor, Michigan), and antiCuZnSOD (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies. Bound primary antibody was detected with HRP-conjugated anti-rabbit antibody (DAKO, Glostrup, Denmark) and blots were developed using an
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Fig. 5. Effect of flavonoids on intracellular nitric oxide generation in CHL cells measured by flow cytometry with DAF-2DA. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). (A) Representative histograms of DAF-2T fluorescence in CM cells and flavonoid 50 lM-treated cells. The fluorescence (FL1-H) is plotted against the number of cells. (B) Fluorescence intensity as percentage of control values. Data represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CMtreated group.
enhanced chemiluminescence detection system (ECL kit, Amersham Pharmacia, Uppsala, Sweden). The density of the specific bands was quantified with an imaging densitometer (Scion Image, Maryland, MA).
2.6. Real-Time RT-PCR Total RNA was obtained by using a Trizol reagent (Life Technologies, Carlsbad, CA) and quantified by the fluorescent method Ribogreen RNA Quantitation Kit (Molecular Probes). Residual genomic DNA was removed by incubating RNA with RQ1 RNase-free DNase (Promega, Madison, WI). First-standard cDNA was synthesized using HighCapacity cDNA Archive Kit (Applied Biosystems, Weiterstadt, Ger-
many). The negative control (no transcriptase control) was performed in parallel. cDNA was amplified using TaqMan Universal PCR Master Mix (Applied Biosystems) on an ABI 7000 (Applied Biosystems). TaqMan primers and probes for glutathione reductase (GenBank accession No. X15722 and Hs00167317_m1), glutathione peroxidase (GenBank accession No. Y00433 and Hs00829989_gH), catalase (GenBank accession No. X04076 and Hs00156308_m1), superoxide dismutase 2 (GenBank accession No. M36693 and Hs00167309_ml), superoxide dismutase 1 (GenBank accession No. X02317 and Hs00533490_m1), and 18S rRNA (GenBank accession No. X03205.1 and Hs99999901_s1) genes were derived from the commercially available TaqManÒ Gene Expression Assays (Applied Biosystems). Relative changes in gene expression levels were determined using the 2DDCT method as described previously (Livak and Schmittgen,
I. Crespo et al. / Food and Chemical Toxicology 46 (2008) 1555–1569 2001). The cycle number at which the transcripts were detectable (CT) was normalized to the cycle number of 18S detection, referred to as DCT.
2.7. Statistical analysis Means and SEMs were calculated. Data were analyzed using ANOVA. Post-hoc comparisons were carried out by the Newman Keuls test. Statistical significance was set at the p < 0.05. SPSS+ vers. 13.0 statistical software (Chicago, IL) was used.
3. Results 3.1. Cell viability The MTT assay showed quercetin and taxifolin to be slightly cytotoxic, with 11% and 13% cell death, respectively, at 50 lM. Apigenin at 25 lM and 50 lM reduced significantly cell viability, with 21% and 35% cell death, respectively (Fig. 1). 3.2. Glutathione concentration Oxidised glutathione concentration was significantly increased by the cytokine mixture. This was significantly prevented by quercetin, kaempferol and taxifolin at all tested concentrations. The oxidised to reduced glutathione
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ratio was used as a marker of oxidative stress. The treatment with the cytokine mixture evoked a marked increase of this ratio that was significantly prevented by quercetin, kaempferol and taxifolin. Effects of apigenin reached a lesser extent and were not significant at 25 lM (Fig. 2). 3.3. Generation of ROS/RNS We investigated generation of ROS/RNS by flow cytometry using DCF-DA. Analysis of histograms in which the fluorescence, detected with the FL1-H channel, was plotted against the relative number of cells (Fig. 3A) and quantification of the corresponding fluorescence intensity (Fig. 3B), indicated that the cytokine mixture induced a significant increase in ROS/RNS production as compared to unstressed controls. Treatment of cells with 50 lM quercetin or kaempferol significantly decreased ROS/RNS production. When cells were treated with taxifolin 50 lM or apigenin 25 lM and 50 lM, ROS/RNS production increased significantly as compared to the CM-treated group (Fig. 3B). Flow cytometry using ETH was used to detect superoxide anion generation. The increase induced by the cytokine mixture was significantly prevented by quercetin and kaempferol 25 lM and 50 lM and by 5
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Mn SOD
Kaempferol Taxifolin Apigenin
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Relative Mn SOD protein level
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Fig. 6. Effect of flavonoids on Mn SOD protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-Mn SOD antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
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Fig. 7. Effect of flavonoids on Cu/Zn SOD protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-Cu/Zn SOD antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
taxifolin 5–50 lM, whereas apigenin had no preventive effect (Fig. 4B). Generation of nitric oxide, measured by DAF-2T fluorescence, was significantly decreased by quercetin 50 lM, kaempferol 5–50 lM, and taxifolin 5 lM. Taxifolin 50 lM or apigenin 25 lM and 50 lM caused a significant increase in nitric oxide production (Fig. 5B). 3.4. Gene expression of antioxidant enzymes Data presented in Fig. 6 show the effects of the different flavonoids on Mn SOD protein level. The significant increase induced by the cytokine mixture was partially prevented by kaempferol and taxifolin 25 lM or 50 lM. Quercetin 25 lM and 50 lM caused a further increase, while apigenin had no significant effect. Cu/Zn SOD protein level (Fig. 7) was significantly decreased by quercetin 25 lM and 50 lM, kaempferol 5–50 lM, and taxifolin 50 lM. GPx protein level was significantly decreased by kaempferol 5– 50 lM, quercetin 25 lM and 50 lM, and taxifolin 50 lM. When cells were treated with apigenin, all concentrations increased significantly GPx protein level as compared to the CM group (Fig. 8). Quercetin 5–50 lM and kaempferol 25–50 lM partially prevented the increase in CAT protein
level induced in the CM-group, with no effect of the different taxifolin or apigenin concentrations (Fig. 9). GR protein level was significantly reduced as compared to both control and CM groups, by quercetin 5–50 lM and kaempferol 25–50 lM. Values were significantly increased by taxifolin 50 lM (Fig. 10). Since protein levels of the different antioxidant enzymes were down- or up-regulated by the different flavonoids, RT-PCR analysis was performed to assess the effects on the mRNA level. The fold change in expression of the Mn SOD, Cu/Zn SOD, GPx, CAT, and GR genes was expressed as 2DDCT, in which DDCT equals the DCT of the flavonoid-treated group minus the DCT of the control group, that was normalized to 1. No significant changes in Mn SOD RNA levels were observed as compared to the CM-treated group, except for the increase induced by quercetin 25 lM or 50 lM and the decrease by kaempferol 50 lM. Kaempferol 25 lM and 50 lM or quercetin 50 lM induced a decrease in mRNA level for Cu/Zn SOD, GPx, CAT and GR. Quercetin 25 lM also decreased mRNA level for CAT and GR. GR mRNA levels was significantly increased by taxifolin 50 lM and reduced by apigenin 50 lM (Fig. 11).
I. Crespo et al. / Food and Chemical Toxicology 46 (2008) 1555–1569
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Fig. 8. Effect of flavonoids on GPx protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-GPx antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
4. Discussion This study demonstrates that flavonoids may have the ability to protect CHL cells against oxidative stress by modulating ROS/RNS generation, the reduced to oxidised glutathione ratio and the main antioxidant enzymes expression. However, important differential effects were detected between the various molecules tested. Reduced glutathione (GSH) is the main nonenzymatic antioxidant defense within the cell, reducing different peroxides and hydroperoxides by oxidation to GSSG. Thus, the GSSG to GSH ratio has been widely employed as a marker of oxidative stress (Gutie´rrez et al., 2006). By using this method, we found that the GSSG/GSH ratio increase induced by the cytokine mixture was prevented, although to a different extent, by the different flavonoids. Quercetin data coincide with previous reports that this molecule protects against liver-induced oxidative stress, with changes in the GSSG/GSH ratio, both in rats with biliary obstruction (Peres et al., 2000) or following chronic ethanol administration in mice (Molina et al., 2003). Although it has been recently indicated that quercetin protects HepG2 cells
against a decrease of GSH levels mainly by preventing the formation of GSH conjugates rather than oxidation to GSSG (Lima et al., 2006), our data demonstrate that in CHL cells treated with a cytokine mixture quercetin decreased GSSG concentration, and that this effect appeared not to be due to an increase conversion back to GSH, because both GR protein concentration and mRNA levels were significantly reduced by treatment with the flavonoid. Similar changes for GR expression were detected in the case of kaempferol. As for the effects of apigenin, in vitro studies indicate that this molecule forms upon oxidation phenoxyl radicals with high redox potentials which cooxidise GSH, resulting in GSSG formation. In contrast, GSH is not oxidised by the low redox potential of quercetin or kaempferol-derived radicals (Galati et al., 2001). This could contribute to explain the lesser ability of apigenin to prevent the increase in GSSG concentration and in the GSSG/GSH ratio. The lowered generation of GSSG by quercetin and kaempferol was probably related to the effects of both flavonoids on the formation/elimination of hydrogen peroxide and organic peroxides, which are the main substrates
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Fig. 9. Effect of flavonoids on CAT protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-CAT antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
of GPx. Expression of this antioxidant enzyme was reduced by both flavonoids, and this effect was accompanied at high concentrations by a lowered DCF fluorescence intensity, which is proportional to the amount of peroxides produced by the cells (Tun˜o´n et al., 2003). Our data coincide with previous reports that both flavonols suppress lipid peroxidation in cultured hepatocytes (Sugihara et al., 1999). It has been shown that flavonoids with a hydroxyl group at the C3 and C4 and the 2–3 double bond show significant antioxidant activity (Sartor et al., 2002; Kachadourian and Day, 2006), and both quercetin and kaempferol share these characteristics (Ueda et al., 2004). When flow cytometry was used to compare generation of specific ROS/RNS, the two flavonols, which are known to exhibit an inhibitory effect on xanthine oxidase and superoxide scavenging activity (Lin et al., 2002; Masuoka et al., 2006), were observed to inhibit superoxide generation to a similar extent at the different tested concentrations. However, kaempferol suppressed more effectively than quercetin nitric oxide generation, in spite of lacking a radical scavenging catechol moiety in the B ring, which has been suggested to be essential for the inhibitory action of quercetin on iNOS gene expression (Banerjee et al., 2002). This does not necessarily indicate a better anti-inflammatory capacity by kaempfer-
ol, because nitric oxide cytoxicity is for the major part mediated by peroxynitrite, formed in the reaction of nitric oxide with superoxide (Garcı´a-Mediavilla et al., 2005), and quercetin has shown a much higher peroxynitrite scavenging capacity, apparently related to the lack of free aromatic hydroxyl groups (Haenen et al., 1997; Sadeghipour et al., 2005). The behavior of the flavone apigenin and the flavanone taxifolin is more complex. Apigenin 25 lM and 50 lM, reduced cell viability, increased significantly the generation of peroxides, and also caused a specific increase in nitric oxide generation. Although apigenin has been reported to block COX-2 expression in macrophages and keratinocytes (Van Dross et al., 2005), different findings have shown that this molecule has minimal antioxidant activity in comparison to structurally related flavonoids such as quercetin (Safari and Sheikh, 2003). In human leukemic cells it has demonstrated a high capacity to act as proteosome inhibitor and apoptosis inducer, an effect which appears to be related to the absence of a C3 hydroxyl group (Chen et al., 2005). However, although apigenin has been previously indicated to reduce cell viability in macrophages in the presence of LPS (Raso et al., 2001) and to result cytotoxic to human normal cells, such as lung fibroblasts (Mat-
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suo et al., 2005), induction of cell death appears to be absent in non-transformed cells (Chen et al., 2005; Vargo et al., 2006). The exact mechanism for the toxic effects of apigenin in CHL cells is not clear, but oxidative stress due to the formation of ROS/RNS may play a crucial role, and it is known that apigenin radicals are very reactive, being able to act as radical initiators (Galati and O’Brien, 2004; Sadeghipour et al., 2005). Taxifolin 5 lM was able to reduce the generation of superoxide and nitric oxide, but had no effect on DCF fluorescence, which could be indicating a reduced capacity to scavenge hydrogen peroxide and organic peroxides. A low antioxidant capacity of taxifolin in comparison to quercetin has been previously reported in retinal cells after ascorbate/Fe2+-induced oxidative stress, and it has been suggested that differences may arise from the less planar structure of taxifolin which results in strong hydrogen bonds (Areias et al., 2001). At high concentrations (50 lM), however, taxifolin caused a marked elevation in nitric oxide generation and in DCF fluorescence. It is known that certain flavonoids may switch from antioxidant to oxidant action under some conditions, such as changes in the molar ratio to metal ions
CON
A
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and decreases in the redox potential of metal–flavonoid complexes (Sugihara et al., 1999; Texeira et al., 2005). Thus, these flavonoids with various effects on oxidation may have dual biochemical and pharmacological actions. The role of dietary flavonoids as free radical scavengers is well know, but their regulatory roles on antioxidant proteins remains to be fully elucidated. Organisms have developed a variety of antioxidant defense systems as protection from ROS/RNS. The major endogenous antioxidant systems include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). SOD catalyzes the dismutation of the superoxide radical anion, and there is a Mn SOD localized in the mitochondria and a Cu/Zn SOD mainly localized in the cytosol. CAT and GPX convert H2O2 to H2O, and GR recycles oxidised glutathione back to reduced glutathione (Alia et al., 2006). In our study, treatment of CHL cells with a cytokine cocktail caused a transcriptional induction of antioxidant enzymes, with significant increases in both the RNA level and protein concentration. Because quercetin, kaempferol and taxifolin decreased superoxide anion generation to a similar extent, and given the existence of
CM
5
25
50
(μM) GR
Quercetin Kaempferol Taxifolin Apigenin
Q
B
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T ab
Relative GR protein level
200 180
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a
160 140
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100
ab
80 60
ab ab ab
40 20 0
CM -
+
5 25 50 + + +
5 25 50 + + +
5 25 50 + + +
5 25 50 (μmol/L) + + +
Fig. 10. Effect of flavonoids on GR protein concentration in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Total cellular protein was separated on 9% SDS-polyacrylamide gels and blotted with anti-GR antibody. (A) Representative Western blots. (B) Densitometric analysis of Western blots. Data represent means ± SEM from four separate experiments and are normalized to levels of b-actin. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
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Mn SOD mRNA fold increase
Q
K
T
A
ab
6 5
a ab 4
a
a
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a a a
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Cu/Zn SOD mRNA fold increase
CM -
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3 2
5 25 50 + + +
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a a ab
a b
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GPx mRNA fold increase
5 25 50 + + +
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a
a a
2
(μmol/L)
a ab
a ab
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ab ab
a
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CAT mRNA fold increase
CM 2
1.5
+
5 25 50 + + +
5 25 50 + + +
5 25 50 + + +
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(μmol/L)
a a
a ab
1
ab
ab ab
0.5 0
GR mRNA fold increase
CM 3
+
5 25 50 + + +
5 25 50 + + +
5 25 50 + + +
5 25 50 + + +
(μmol/L)
ab
2.5 2
a
1.5
a b
ab
1
ab
ab
a ab
0.5 0 CM -
+
5 25 50 + + +
5 25 50 + + +
5 25 50 + + +
5 25 50 + + +
(μmol/L)
Fig. 11. Effect of flavonoids on mRNA level of Mn SOD, Cu/Zn SOD, GPx, CAT and GR as quantitated by real time polymerase chain reaction analysis in CHL cells. Cells were incubated with a cytokine mixture (CM) and 5–50 lM quercetin (Q), kaempferol (K), taxifolin (T) or apigenin (A). Relative changes in gene expression levels were determined using the 2DDCT method. Data, normalized against 18S rRNA, are presented as fold change from the control group and represent means ± SEM from four separate experiments. ap < 0.05 compared with control group. bp < 0.05 compared with CM-treated group.
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a feedback mechanism on the antioxidant enzymes triggered by the flavonoids (Breinholt et al., 1999), the decrease in substrate concentration could contribute to explain a reduction in the expression of SOD isoforms. However, quercetin caused a post-transcriptional induction of Mn SOD, which is difficult to conciliate with a report indicating that this flavonol induces in hepatoma cells a decrease in Mn SOD mRNA levels, probably resulting from inhibition of NF-jB activation (Rohrdanz et al., 2003; Martı´nezFlo´rez et al., 2005). It has been demonstrated that nitric oxide causes an induction of Mn SOD protein (Keller et al., 2003) in glomerular mesangial cells. Because in our experiments, differently from kaempferol, quercetin was not able to significantly reduce nitric oxide generation (Keller et al., 2003), Mn SOD induction could represent a mechanism by which nitric oxide reduces the concentration of superoxide anion and the formation of peroxynitrite to ensure a self-limited inflammatory response (Pfeilschifter et al., 2002). Effects of flavonoids on antioxidant enzyme expression could be also mediated by modification of signal transduction pathways. Sequence analysis of GPx and CAT has revealed in different species binding motifs for NF-jB and AP-1 (Zhou et al., 2001), transcriptional regulators that are activated in response to oxidative stress in various tissues (Surth et al., 2005). Although there are conflicting reports concerning the relationship between flavonoids and the NF-jB/AP-1 pathways, and it has been found that dietary quercetin does not reduce NF-jB activation in the renal cortex of rats with chronic glomerular disease (Rangan et al., 2002), different studies indicate that flavonols inhibit AP-1 activation in lipopolysaccharide-activated RAW264.7 cells (Kim et al., 2005), and that quercetin prevents LPS-IjB phosphorylation in bone marrow derived macrophages (Comalada et al., 2005), and inhibits NF-jB activation induced by interleukin-1 in murine fibroblast l-TK cells (Muraoka et al., 2002), interleukin-1b activated rat hepatocytes (Martı´nez-Flo´rez et al., 2005) or H2O2 in HepG2 cells (Musonda and Chipman, 1998). We have recently reported that quercetin and kaempferol inhibit the activation of NFjB induced by a cytokine mixture in Chang Liver cells (Garcı´a-Mediavilla et al., 2007). In our study, the generation of peroxides was reduced by quercetin and kaempferol, and the lower exposure to levels of oxidizing species could, through changes in the activation of the redox-sensitive transcription factors, contribute to a decrease in the expression of antioxidant enzymes. The NF-E2 related factor 2 (Nrf2), a member of the cap0 n0 collar family of basic leucine zipper transcription factors, is another redox-sensitive factor whose nuclear translocation and binding to the antioxidant response elements (ARE) in their promoter regions may result in induction of antioxidant enzymes (BrigeliusFlohe and Banning, 2006). Up-regulation by apigenin of GPx could, thus, be also a consequence of the increased generation of ROS/RNS and their effects on nuclear factors and other regulatory elements. Increased expression of GPx could, in turn, contribute to the formation, upon oxidation
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by H2O2 of the apigenin phenoxyl radical which catalyzes GSH or NADH co-oxidation and generate ROS/RNS (Galati et al., 2001; Galati and O’Brien, 2004). In conclusion, our study reveals that important differences exist between flavonoids with different structural features in their capacity to abrogate the generation of different ROS/RNS, and suggests that the modulation of antioxidant enzymes by flavonoids may be also important in their antioxidant effects in liver cells. Although validation in animal models or human systems is necessary, and extrapolation to the in vivo environment requires consideration of flavonoid bioavailability and metabolism, data here reported showed clear protective effects by some flavonoids against oxidative damage induced in CHL cells. This could be of use against liver diseases where it is known that oxidative stress plays a relevant role. Acknowledgment This work was supported by the Plan Nacional de I+D, Spain (Grant No. BFI2003-03114). References Alia, M., Ramos, S., Mateos, R., Granado-Serrano, A.B., Bravo, L., Goya, L., 2006. Quercetin protects human hepatoma HepG2 against oxidative stress induced by tert-butyl hydroperoxide. Toxicol. Appl. Pharmacol. 21, 110–118. Areias, F.M., Rego, A.C., Oliveira, C.R., Seabra, R.M., 2001. Antioxidant effects of flavonoids after ascorbate/Fe2+-induced oxidative stress in cultured retinal cells. Biochem. Pharmacol. 62, 111–118. Banerjee, T., Van der Vliet, A., Ziboh, V.A., 2002. Downregulation of COX-2 and iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line. Prostag. Leukotr. Essent. Fatty Acid 66, 485–492. Breinholt, V., Lauridsen, S.T., Dragsted, L.O., 1999. Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rats. Xenobiotica 29, 1227–1240. Brigelius-Flohe, R., Banning, A., 2006. Part of the series: from dietary antioxidants to regulators in cellular signalling and gene regulation. Free Radic. Biol. Med. 40, 775–787. Chen, D., Daniel, K.G., Chen, M.S., Kuhn, D.J., Landis-Piwowar, K.R., Dou, Q.P., 2005. Dietary flavonoids as proteosome inhibitors and apoptosis inducers in human leukemic cells. Biochem. Pharmacol. 69, 1421–1432. Comalada, M., Camuesco, D., Sierra, S., Ballester, I., Xaus, J., Galvez, J., Zarzuelo, A., 2005. In vivo quercitrin anti-inflammatory effect involves releases of quercetin, which inhibits inflammation through down-regulation of NF-jB pathway. Eur. J. Immunol. 35, 584–592. Emerit, I., Huang, C.Y., Serejo, F., Filipe, P., Fernandes, A., Costa, A., Freitas, J., Baptista, A., Carneiro de Moura, M., 2005. Oxidative stress in chronic hepatitis C: a preliminary study on the protective effects of antioxidant flavonoids. Hepatogastroenterology 52, 530–536. Galati, G., Moridani, M.Y., Chan, T.S., O’Brien, P.J., 2001. Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation. Free Radic. Biol. Med. 34, 370–382. Galati, G., O’Brien, P.J., 2004. Potential toxicity of flavonoids and other dietary phenolics: significance for their chemoprotective and anticancer properties. Free Radic. Biol. Med. 37, 287–303. Galisteo, M., Garcı´a Saura, M.F., Jime´nez, R., Villar, I.C., Zarzuelo, A., Vargas, F., Duarte, J., 2004. Effects of chronic quercetin treatment on
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