- Email: [email protected]
Quercetin Prevents DNA Single Strand Breakage and Cytotoxicity Caused By tert-Butylhydroperoxide: Free Radical Scavenging Versus Iron Chelating Mechanism
Free Radical Biology & Medicine, Vol. 25, No. 2, pp. 196 –200, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(98)00040-9
Original Contribution QUERCETIN PREVENTS DNA SINGLE STRAND BREAKAGE AND CYTOTOXICITY CAUSED BY tert-BUTYLHYDROPEROXIDE: FREE RADICAL SCAVENGING VERSUS IRON CHELATING MECHANISM PIERO SESTILI,* ANDREA GUIDARELLI,* MARINA DACHA` ,†
and
ORAZIO CANTONI*
*Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale; and †Istituto di Chimica Biologica “Giorgio Fornaini,” Universita` di Urbino, Urbino, Italy (Received 18 December 1997; Accepted 27 January 1998)
Abstract—Although the antioxidant properties of flavonoids are well documented, it is still unclear whether these effects are dependent on radical scavenging or iron chelating activities. By using an experimental approach based on the notion that iron chelators suppress DNA strand scission and cytotoxicity caused by tert-butylhydroperoxide, whereas radical scavenging antioxidants prevent only the latter response, we provide experimental evidence indicating that the most prominent activity of the flavonoid quercetin resides in its ability to chelate iron. This experimental approach can be utilized for the assessment of iron chelation in the biological activity of flavonoids or other antioxidants. © 1998 Elsevier Science Inc. Keywords—Quercetin, Flavonoids, tert-Butylhydroperoxide, Hydrogen peroxide, DNA damage, Cytotoxicity, Free radical
duced V79 cell death,15 lipid peroxidation initiated by ferric iron in the retina, purified rod segments and retinal pigment epithelium,16 buthionine sulfoximine-induced oxidative stress in cutaneous tissue-associated cell types,17 and H2O2induced histamine release in human basophils.18 In the present study we have investigated the effect of quercetin on DNA cleavage and cytotoxicity induced by tert-butylhydroperoxide (tB-OOH). The purpose of this study was to find out whether the effects of quercetin were better explained by a radical scavenging or iron chelating mechanism. Our approach was based on the notion that DNA cleavage evoked by tB-OOH is abolished by iron chelators19 –22 and insensitive to antioxidants,19 –22 whereas cell death induced by the hydroperoxide is abolished by both iron chelators and antioxidants.19,22 The results obtained indicate that the effects of quercetin are best explained by an iron chelating mechanism.
INTRODUCTION
A large and steadily growing family of naturally occurring flavonoids has received intensive study owing to their antioxidant activity. The early observation that these agents prolong the shelf-life of fat-containing foodstuff1 was later on extended by showing that flavonoids are efficient inhibitors of lipid peroxidation.2–5 Few studies, however, have investigated the molecular basis for these effects. Indeed, the polyphenol structure of flavonoids allows both the scavenging of free radicals, with concomitant formation of fairly stable aroxyl radicals,6 – 8 and the chelation of transition metals, including iron.6,9 –12 The question therefore arises as to whether the biological activities of flavonoids are primarily due to their radical scavenging or iron chelating properties. Quercetin is an important member of the flavonoid family and can be found in fairly large amounts in fruits, vegetables, olive oil, red wine, tea, and in the propolis of bee hives.13,14 Quercetin was shown to inhibit H2O2-in-
MATERIALS AND METHODS
Cell culture and treatments
Address correspondence to: Prof. Orazio Cantoni, Istituto di Farmacologia e Farmacognosia, Universita` di Urbino, Via S. Chiara 27, 61029 Urbino (PS), Italy; Tel: 39-722-2671; Fax: 39-722-327670; E-mail: [email protected].
U937 cells were grown in RPMI 1640 culture medium (HyClone Laboratories, Logan, UT) supplemented with 196
Quercetin and tB-OOH-induced cyto- and genotoxicity
10% Fetal clone III (HyClone), penicillin (50 units/ml), and streptomycin (50 mg/ml) at 37°C in T-75 tissue culture flasks (Corning, Corning, NY) gassed with an atmosphere of 95% air-5% CO2. Reagent-grade chemicals, tB-OOH, H2O2 and quercetin were obtained from Sigma-Aldrich (Milan, Italy). Desferroxamine was from Ciba-Geigy (Origgio, Italy). Stock solutions of tB-OOH, H2O2 and desferroxamine were freshly prepared in saline A (8.182 g/l NaCl, 0.372 g/l KCl, 0.336 g/l NaHCO3 and 0.9 g/l glucose). Trolox was dissolved in 1 M NaHCO3. Quercetin and o-phenanthroline were dissolved in dimethyl sulfoxide. Butylated hydroxytoluene (BHT), N,N9-diphenyl-1,4-phenylenediamine (DPPD) and a-tocopherol were dissolved in 95% ethanol. At the treatment stage the final dimethyl sulfoxide/ethanol concentration was never higher than 0.05%. Under these conditions ethanol and dimethyl sulfoxide were neither toxic nor DNA-damaging, nor did they affect the cytogenotoxic properties of tB-OOH. Cells (2.5 3 105/ml) were treated for 30 min in saline A (2 ml), washed and either analyzed immediately for DNA damage or postincubated for 6 h in complete medium and then analyzed for cell viability. Iron chelators, antioxidants, and quercetin were added to the cultures 5 min prior to addition of tB-OOH. Cytotoxicity assay After the treatments, the cells were washed with saline A and resuspended in prewarmed RPMI 1640 medium before being plated into 35-mm tissue culture dishes and incubated at 37°C for 6 h. Cytotoxicity was determined using the trypan blue exclusion assay. Briefly, an aliquot of the cell suspension was diluted 1:1 (v/v) with 0.4% trypan blue and the cells were counted using a haemocytometer. Results are expressed as the percentage of dead cells (ratio of stained cells versus the total number of cells). Comet assay DNA single strand breakage in individual cells was detected using the comet assay,23 with minor modifications. After treatments, U937 cells were resuspended at 2.0 3 104 cells/100 ml in 1.0% low-melting agarose in phosphate-buffered saline (8 g/l NaCl, 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4, 0.2 g/l KCl) containing 5 mM ethylenediaminetetraacetic acid (EDTA) and immediately pipetted onto agarose-coated slides. The slides were immersed in ice-cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% sarkosyl, 5% dimethyl sulfoxide, and 1% Triton X100 [pH 10.0]) for 60 min. The slides were then placed on an electrophoretic tray
197
with an alkaline buffer (300 mM NaOH and 1 mM EDTA) for 20 min to allow the DNA to unwind; electrophoresis was then performed at 300 mA for 20 min in the same alkaline buffer maintained at 14°C. The slides were then washed and stained with ethidium bromide. The DNA was visualized using a Bio Rad DVC 250 confocal laser microscope (Bio Rad, Richmond, CA, USA) and the resulting images were taken and processed with a Hamamatsu chilled CCD 5985 camera (Hamamatsu Italy S.p.a., Milan, Italy) coupled with a NIH Image 1.61 software. Quantification of the DNA damage per cell was calculated as the ratio between comet tail area and nucleus area (tail nucleus ratio) according to the method of Mu¨ller et al.24 The measurements from 50 –75 randomly selected cells per treatment condition were averaged.
Measurement of DNA single strand breaks in post-lysed DNA samples by alkaline elution Post-lysed DNA samples from [methyl-14C]-thymidine-labelled cells were prepared and treated as previously described.22 After treatments, the post-lysed samples were analyzed for DNA damage by the alkaline elution assay, using a procedure virtually identical to that described by Kohn et al.25 with minor modifications.26 Strand scission factor values were calculated from the resulting elution profiles by determining the absolute log of the ratio of the percentage of DNA retained in the filters of the drug-treated sample to that retained from the untreated control sample (both after 8 h of elution). RESULTS AND DISCUSSION
The effect of increasing concentrations of quercetin on U937 cell death caused by tB-OOH was investigated. As illustrated in Fig. 1A, treatment with 3 mM tB-OOH for 30 min, followed by posttreatment incubation for 6 h in fresh culture medium, was highly toxic in U937 cells. Addition of quercetin at the time of peroxide exposure mitigated the cytotoxic response and this effect was concentration dependent (IC50 5 12.67 mM 6 0.86). Importantly, quercetin alone was not cytotoxic at any of the concentrations tested (not shown). Under similar experimental conditions, the antioxidants BHT (200 mM), DPPD (10 mM), a-tocopherol (100 mM), and Trolox (1 mM), and the iron chelator o-phenanthroline (25 mM), also promoted cytoprotection (Fig. 1B). These results are consistent with previously published work from our20 as well as from other19 laboratories and indicate that the lethal response evoked by tB-OOH requires a source of iron and the formation of antioxidant-sensitive lipid peroxidation products. Thus, the cy-
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Fig. 1. The effect of quercetin, antioxidants and iron chelators on tB-OOH-induced cytotoxicity. U937 cells were plated at a density of 2.5 3 105 cells/ml in saline A and exposed for 30 min to 3 mM tB-OOH in the absence or presence of increasing concentrations of quercetin (A) or various antioxidant and iron chelators (B). The relative number of dead cells was measured after 6 h of post-challenge growth in fresh culture medium using the trypan blue exclusion assay. Results represent the mean 6 SEM of at least three separate experiments, each performed in duplicate.
toprotective effects of quercetin may be the consequence of its radical scavenging and iron chelation capacity, or both of these activities. The effect of quercetin on DNA cleavage produced by tB-OOH was also investigated. In these experiments the cells were exposed for 30 min to 200 mM tB-OOH, in the absence or presence of increasing concentrations of quercetin, and the level of DNA single strand breakage was measured immediately after the treatments using the microgel comet assay. As illustrated in Fig. 2A, the flavonoid effectively reduced DNA strand scission caused by the hydroperoxide and this effect was concentration dependent (IC50 5 2.73 mM 6 0.29). Figure 2B shows representative photomicrographs of ethidium bromide-stained nuclei and indicates that 30 mM quercetin virtually abolished the extensive DNA cleavage caused by the hydroperoxide. Quercetin alone was not DNA damaging at any of the concentrations tested (not shown). The bar graph shown in Fig. 2C demonstrates that o-phenanthroline (25 mM), unlike BHT (200 mM), DPPD (10 mM), a-tocopherol (100 mM), and Trolox (1
Fig. 2. The effect of quercetin, antioxidants and iron chelators on tB-OOH-induced DNA single strand breakage. The cells were treated for 30 min with 200 mM tB-OOH in saline A in the absence or presence of increasing concentrations of quercetin (A) or various antioxidant and iron chelators (C) and immediately assayed for DNA damage with the microgel comet assay. Results are expressed as the ratio of the comet tail versus the comet head area and represent the mean 6 SEM of at least three separate experiments, each performed in duplicate. (B) shows representative photomicrographs of microgel electrophoresed U937 cells treated under experimental conditions similar to those detailed in (A).
mM), prevented the DNA strand scission caused by tB-OOH. These results are also consistent with previously published work from our20 –22 as well as from other19 –21 laboratories and indicate that the DNA cleavage generated by tB-OOH requires a source of iron and is insensitive to radical scavenging antioxidants. Thus, the inhibitory effects of quercetin on DNA strand scission caused by the hydroperoxide can only be explained by an iron chelating mechanism. The final clue indicating that quercetin is an effective iron chelator is provided by experiments in which the effect of this flavonoid, as well as that of selected iron
Quercetin and tB-OOH-induced cyto- and genotoxicity
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support to the notion that the flavonoid is an effective iron chelator. The experimental approach utilized in this study may represent a valid system to discriminate radical scavenging versus iron chelating mechanisms in the antioxidant effects of different drugs and flavonoids. Acknowledgement—This work was supported by a grant from Specchiasol S.r.l. (Verona).
Fig. 3. The effect of quercetin, antioxidants and iron chelators on H2O2-induced DNA single strand breakage in post-lysed DNA. The cells (4 3 105) were lysed onto polycarbonate filters and, after extensive washing, were treated for 20 min in saline at room temperature. After the treatments, the extent of DNA single strand breakage was determined by the alkaline alution technique. The strand scission factor values were calculated from the resulting elution profiles, as detailed in the Methods section. Each point is the mean 6 SEM of three separate experiments.
chelators and antioxidants, was investigated in a preparation of partially purified DNA. In these experiments the cells were lysed onto polycarbonate filters and, after accurate washing, the post-lysed DNA samples were treated for 20 min with 100 mM H2O2. The extent of DNA strand scission was estimated using the alkaline elution technique. H2O2 was used as a DNA-damaging agent because tB-OOH does not cause strand scission in partially purified DNA.22 Indeed, our previous work demonstrated that the mechanism whereby the organic hydroperoxide generates DNA lesions involves calciumdependent mitochondrial formation of tB-OOH-derived DNA-damaging species mainly represented by H2O2.27 Furthermore, desferroxamine was used in place of ophenanthroline because the latter is known to form a complex with cupric ions that can directly generate lesions in preparations of purified DNA.28 The results illustrated in Fig. 3 demonstrate that the DNA cleavage generated by H2O2, although insensitive to 1 mM Trolox or 10 mM DPPD, was prevented by 100 mM quercetin or 10 mM desferroxamine. In conclusion, the results presented in this study demonstrate that iron chelation is a prominent effect of quercetin in U937 cells exposed to tB-OOH. Indeed, whereas the cytoprotective effects may be explained by radical chain-breaking or metal-binding activities, prevention of DNA strand scission in intact cells was entirely based on the iron chelating capacity of the flavonoid. The fact that cleavage of partially purified DNA caused by H2O2 was insensitive to antioxidants Trolox (hydrophilic) and DPPD (lipophilic), but abolished by the iron chelator desferroxamine and by quercetin, provides additional
REFERENCES 1. Ku¨hnau, J. Dietary flavonoids. World Rev. Nutr. Diet. 24:117–123; 1976. 2. Torel, J.; Cillard, J.; Cillard, P. Antioxidant activity of flavonoids and reactivity with peroxy radical. Phytochemistry 25:383–386; 1986. 3. Fraga, C. G.; Martino, V. S.; Ferraro, G. E.; Coussio, J. D.; Boveris, A. Flavonoids as antioxidants evaluated by in vitro and in situ liver chemiluminescence. Biochem. Pharmacol. 36:717–720; 1987. 4. Sgaragli, G. P.; Valoti, M.; Gorelli, B.; Fusi, F.; Palmi, M.; Mantovani, P. Calcium antagonist and antiperoxidant properties of some hindered phenols. Br. J. Pharmacol. 110:369 –377; 1993. 5. Terao, J.; Piskulli, M.; Yao, Q. Protective effect of epicatechin, epicatechin gallate and quercetin on lipid peroxidation in phospholipid bilayers. Arch. Biochem. Biophys. 308:278 –284; 1994. 6. Afanas’ev, I. B.; Dorohzhko, A. L.; Brodskii, A. V.; Korstyuk, V. A.; Potapovitch, A. I. Chelating and free radical scavenging mechanism of inhibitory action of rutin and quercetin in lipid peroxidation. Biochem. Pharmacol. 38:1763–1768; 1989. 7. Cotelle, N.; Bernier, J. L.; Henichart, J. P.; Catteau, J. P.; Gaydou, E.; Wallet, J. C. Scavenger and antioxidant properties of ten synthetic flavones. Free Radic. Biol. Med. 13:211–219; 1992. 8. Bors, W.; Michel, C.; Saran, M. Flavonoids antioxidants: Rate constants for reactions with oxygen radicals. Methods Enzymol. 234:420 – 429; 1994. 9. Laughton, M. J.; Evans, P. J.; Moroney, M. A.; Hoult, J. R.; Halliwell, B. Inhibition of mammalian 5-lipoxygenase and cyclooxygenase by flavonoids and phenolic dietary additives. Biochem. Pharmacol. 42:1673–1681; 1991. 10. Morel, I.; Lescoat, G.; Cogrel, P.; Sergent, O.; Pasdeloup, N.; Brissot, P.; Cillard, P.; Cillard, J. Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures. Biochem. Pharmacol. 7:13– 19; 1993. 11. Morel, I.; Lescoat, G.; Cillard, P.; Cillard, J. Role of flavonoids and iron chelation in antioxidant action. Methods Enzymol. 234:437– 443; 1994. 12. Rice-Evans, C. A.; Miller, N. J.; Bolwell, P. G.; Bramley, P. M.; Pridham, J. B. The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic. Res. 22:375–383; 1995. 13. Herman, K. Flavonols and flavones in food plants: A review. J. Food Technol. 11:443– 448; 1976. 14. Havsteen, B. Flavonoids, a class of natural products of high pharmacological potency. Biochem. Pharmacol. 32:1141–1148; 1983. 15. Nakayama, T.; Yamada, M.; Osawa, T.; Kawakishi, S. Suppression of active-oxygen-induced cytotoxicity by flavonoids. Biochem. Pharmacol. 45:265–267; 1993. 16. Ueda, T.; Armstrong, D. Preventive effect of natural and synthetic antioxidants on lipid peroxidation in the mammalian eye. Ophthalmic Res. 28:184 –192; 1996. 17. Skaper, S. D.; Fabris, M.; Ferrari, V.; Dalle Carbonare, M.; Leon, A. Quercetin protects cutaneous tissue-associated cell types including sensory neurons from oxidative stress induced by glutathione depletion: Cooperative effects of ascorbic acid. Free Radic. Biol. Med. 22:669 – 678; 1997. 18. Ogasawara, H.; Fujitani, T.; Drzewiecki, G.; Middleton, E. The
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P. SESTILI et al. role of hydrogen peroxide in basophil histamine release and the effect of selected flavonoids. Y. Allergy Clin. Immunol. 78:321– 328; 1986. Coleman, J. B.; Gilfor, D.; Farber, J. L. Dissociation of the accumulation of single-strand breaks in DNA from killing of cultured hepatocytes by an oxidative stress. Mol. Pharmacol. 36:193–200; 1989. Guidarelli, A.; Sestili, P.; Cossarizza, A.; Franceschi, C.; Cattabeni, F.; Cantoni, O. Evidence for dissimilar mechanisms of enhancement of inorganic and organic hydroperoxide cytotoxicity by L-histidine. J. Pharmacol. Exp. Ther. 275:1575–1582; 1995. Latour, I.; Demoulin, J. B.; Buc-Calderon, P. Oxidative DNA damage by t-butyl hydroperoxide causes DNA single strand breaks which is not linked to cell lysis. A mechanistic study in freshly isolated rat hepatocytes. FEBS Lett. 373:299 –302; 1995. Guidarelli, A.; Cattabeni, F.; Cantoni, O. Alternative mechanisms for hydroperoxide-induced DNA single strand breakage. Free Radic. Res. 26:537–547; 1997. Singh, N. P.; McCoy, M. T.; Tice, R. R.; Schneider, E. L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175:184 –191; 1988. Mu¨ller, W. U.; Bauch, T.; Streffer, C.; Niedereichholz, F.; Bo¨cker, W. Comet assay studies of radiation-induced DNA damage and repair in various tumor cell lines. Int. J. Radiat. Biol. 65:315–319; 1994. Kohn, K. W.; Ewig, R. A. G.; Erickson, L. C.; Zwelling, L. A.
Measurement of strand breaks and crosslinks by alkaline elution. In: Friedberg, E.; Hanawalt, P., eds. DNA Repair: A laboratory Manual of Research Procedures. Vol. 1. New York: Marcel Dekker; 1981:397– 401. 26. Cantoni, O.; Murray, D.; Meyn, R. E. Effect of 3-aminobenzamide on DNA strand-break rejoining and cytotoxicity in CHO cells treated with hydrogen peroxide. Biochim. Biophys. Acta 867:135– 143; 1986. 27. Guidarelli, A.; Clementi, F.; Sciorati, C.; Cattabeni, F.; Cantoni, O. Calcium-dependent mitochondrial formation of species mediating DNA single strand breakage in U937 cells exposed to sublethal concentrations of tert-butylhdroperoxide. J. Pharmacol. Exp. Ther. 283:66 –74; 1997. 28. Dizdaroglu, M.; Aruoma, O. I.; Halliwell, B. Modification of bases in DNA by copper ion-1,10-phenanthroline complexes. Biochemistry 29:8447– 8451; 1990.
ABBREVIATIONS
BHT— butylated hyroxytoluene DPPD—N,N9-diphenyl-1,4-phenylene-diamine EDTA— ethylenediaminetetraacetic acid tB-OOH—tert-butylhydroperoxide
PII S0891-5849(98)00040-9
Original Contribution QUERCETIN PREVENTS DNA SINGLE STRAND BREAKAGE AND CYTOTOXICITY CAUSED BY tert-BUTYLHYDROPEROXIDE: FREE RADICAL SCAVENGING VERSUS IRON CHELATING MECHANISM PIERO SESTILI,* ANDREA GUIDARELLI,* MARINA DACHA` ,†
and
ORAZIO CANTONI*
*Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale; and †Istituto di Chimica Biologica “Giorgio Fornaini,” Universita` di Urbino, Urbino, Italy (Received 18 December 1997; Accepted 27 January 1998)
Abstract—Although the antioxidant properties of flavonoids are well documented, it is still unclear whether these effects are dependent on radical scavenging or iron chelating activities. By using an experimental approach based on the notion that iron chelators suppress DNA strand scission and cytotoxicity caused by tert-butylhydroperoxide, whereas radical scavenging antioxidants prevent only the latter response, we provide experimental evidence indicating that the most prominent activity of the flavonoid quercetin resides in its ability to chelate iron. This experimental approach can be utilized for the assessment of iron chelation in the biological activity of flavonoids or other antioxidants. © 1998 Elsevier Science Inc. Keywords—Quercetin, Flavonoids, tert-Butylhydroperoxide, Hydrogen peroxide, DNA damage, Cytotoxicity, Free radical
duced V79 cell death,15 lipid peroxidation initiated by ferric iron in the retina, purified rod segments and retinal pigment epithelium,16 buthionine sulfoximine-induced oxidative stress in cutaneous tissue-associated cell types,17 and H2O2induced histamine release in human basophils.18 In the present study we have investigated the effect of quercetin on DNA cleavage and cytotoxicity induced by tert-butylhydroperoxide (tB-OOH). The purpose of this study was to find out whether the effects of quercetin were better explained by a radical scavenging or iron chelating mechanism. Our approach was based on the notion that DNA cleavage evoked by tB-OOH is abolished by iron chelators19 –22 and insensitive to antioxidants,19 –22 whereas cell death induced by the hydroperoxide is abolished by both iron chelators and antioxidants.19,22 The results obtained indicate that the effects of quercetin are best explained by an iron chelating mechanism.
INTRODUCTION
A large and steadily growing family of naturally occurring flavonoids has received intensive study owing to their antioxidant activity. The early observation that these agents prolong the shelf-life of fat-containing foodstuff1 was later on extended by showing that flavonoids are efficient inhibitors of lipid peroxidation.2–5 Few studies, however, have investigated the molecular basis for these effects. Indeed, the polyphenol structure of flavonoids allows both the scavenging of free radicals, with concomitant formation of fairly stable aroxyl radicals,6 – 8 and the chelation of transition metals, including iron.6,9 –12 The question therefore arises as to whether the biological activities of flavonoids are primarily due to their radical scavenging or iron chelating properties. Quercetin is an important member of the flavonoid family and can be found in fairly large amounts in fruits, vegetables, olive oil, red wine, tea, and in the propolis of bee hives.13,14 Quercetin was shown to inhibit H2O2-in-
MATERIALS AND METHODS
Cell culture and treatments
Address correspondence to: Prof. Orazio Cantoni, Istituto di Farmacologia e Farmacognosia, Universita` di Urbino, Via S. Chiara 27, 61029 Urbino (PS), Italy; Tel: 39-722-2671; Fax: 39-722-327670; E-mail: [email protected].
U937 cells were grown in RPMI 1640 culture medium (HyClone Laboratories, Logan, UT) supplemented with 196
Quercetin and tB-OOH-induced cyto- and genotoxicity
10% Fetal clone III (HyClone), penicillin (50 units/ml), and streptomycin (50 mg/ml) at 37°C in T-75 tissue culture flasks (Corning, Corning, NY) gassed with an atmosphere of 95% air-5% CO2. Reagent-grade chemicals, tB-OOH, H2O2 and quercetin were obtained from Sigma-Aldrich (Milan, Italy). Desferroxamine was from Ciba-Geigy (Origgio, Italy). Stock solutions of tB-OOH, H2O2 and desferroxamine were freshly prepared in saline A (8.182 g/l NaCl, 0.372 g/l KCl, 0.336 g/l NaHCO3 and 0.9 g/l glucose). Trolox was dissolved in 1 M NaHCO3. Quercetin and o-phenanthroline were dissolved in dimethyl sulfoxide. Butylated hydroxytoluene (BHT), N,N9-diphenyl-1,4-phenylenediamine (DPPD) and a-tocopherol were dissolved in 95% ethanol. At the treatment stage the final dimethyl sulfoxide/ethanol concentration was never higher than 0.05%. Under these conditions ethanol and dimethyl sulfoxide were neither toxic nor DNA-damaging, nor did they affect the cytogenotoxic properties of tB-OOH. Cells (2.5 3 105/ml) were treated for 30 min in saline A (2 ml), washed and either analyzed immediately for DNA damage or postincubated for 6 h in complete medium and then analyzed for cell viability. Iron chelators, antioxidants, and quercetin were added to the cultures 5 min prior to addition of tB-OOH. Cytotoxicity assay After the treatments, the cells were washed with saline A and resuspended in prewarmed RPMI 1640 medium before being plated into 35-mm tissue culture dishes and incubated at 37°C for 6 h. Cytotoxicity was determined using the trypan blue exclusion assay. Briefly, an aliquot of the cell suspension was diluted 1:1 (v/v) with 0.4% trypan blue and the cells were counted using a haemocytometer. Results are expressed as the percentage of dead cells (ratio of stained cells versus the total number of cells). Comet assay DNA single strand breakage in individual cells was detected using the comet assay,23 with minor modifications. After treatments, U937 cells were resuspended at 2.0 3 104 cells/100 ml in 1.0% low-melting agarose in phosphate-buffered saline (8 g/l NaCl, 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4, 0.2 g/l KCl) containing 5 mM ethylenediaminetetraacetic acid (EDTA) and immediately pipetted onto agarose-coated slides. The slides were immersed in ice-cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% sarkosyl, 5% dimethyl sulfoxide, and 1% Triton X100 [pH 10.0]) for 60 min. The slides were then placed on an electrophoretic tray
197
with an alkaline buffer (300 mM NaOH and 1 mM EDTA) for 20 min to allow the DNA to unwind; electrophoresis was then performed at 300 mA for 20 min in the same alkaline buffer maintained at 14°C. The slides were then washed and stained with ethidium bromide. The DNA was visualized using a Bio Rad DVC 250 confocal laser microscope (Bio Rad, Richmond, CA, USA) and the resulting images were taken and processed with a Hamamatsu chilled CCD 5985 camera (Hamamatsu Italy S.p.a., Milan, Italy) coupled with a NIH Image 1.61 software. Quantification of the DNA damage per cell was calculated as the ratio between comet tail area and nucleus area (tail nucleus ratio) according to the method of Mu¨ller et al.24 The measurements from 50 –75 randomly selected cells per treatment condition were averaged.
Measurement of DNA single strand breaks in post-lysed DNA samples by alkaline elution Post-lysed DNA samples from [methyl-14C]-thymidine-labelled cells were prepared and treated as previously described.22 After treatments, the post-lysed samples were analyzed for DNA damage by the alkaline elution assay, using a procedure virtually identical to that described by Kohn et al.25 with minor modifications.26 Strand scission factor values were calculated from the resulting elution profiles by determining the absolute log of the ratio of the percentage of DNA retained in the filters of the drug-treated sample to that retained from the untreated control sample (both after 8 h of elution). RESULTS AND DISCUSSION
The effect of increasing concentrations of quercetin on U937 cell death caused by tB-OOH was investigated. As illustrated in Fig. 1A, treatment with 3 mM tB-OOH for 30 min, followed by posttreatment incubation for 6 h in fresh culture medium, was highly toxic in U937 cells. Addition of quercetin at the time of peroxide exposure mitigated the cytotoxic response and this effect was concentration dependent (IC50 5 12.67 mM 6 0.86). Importantly, quercetin alone was not cytotoxic at any of the concentrations tested (not shown). Under similar experimental conditions, the antioxidants BHT (200 mM), DPPD (10 mM), a-tocopherol (100 mM), and Trolox (1 mM), and the iron chelator o-phenanthroline (25 mM), also promoted cytoprotection (Fig. 1B). These results are consistent with previously published work from our20 as well as from other19 laboratories and indicate that the lethal response evoked by tB-OOH requires a source of iron and the formation of antioxidant-sensitive lipid peroxidation products. Thus, the cy-
198
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Fig. 1. The effect of quercetin, antioxidants and iron chelators on tB-OOH-induced cytotoxicity. U937 cells were plated at a density of 2.5 3 105 cells/ml in saline A and exposed for 30 min to 3 mM tB-OOH in the absence or presence of increasing concentrations of quercetin (A) or various antioxidant and iron chelators (B). The relative number of dead cells was measured after 6 h of post-challenge growth in fresh culture medium using the trypan blue exclusion assay. Results represent the mean 6 SEM of at least three separate experiments, each performed in duplicate.
toprotective effects of quercetin may be the consequence of its radical scavenging and iron chelation capacity, or both of these activities. The effect of quercetin on DNA cleavage produced by tB-OOH was also investigated. In these experiments the cells were exposed for 30 min to 200 mM tB-OOH, in the absence or presence of increasing concentrations of quercetin, and the level of DNA single strand breakage was measured immediately after the treatments using the microgel comet assay. As illustrated in Fig. 2A, the flavonoid effectively reduced DNA strand scission caused by the hydroperoxide and this effect was concentration dependent (IC50 5 2.73 mM 6 0.29). Figure 2B shows representative photomicrographs of ethidium bromide-stained nuclei and indicates that 30 mM quercetin virtually abolished the extensive DNA cleavage caused by the hydroperoxide. Quercetin alone was not DNA damaging at any of the concentrations tested (not shown). The bar graph shown in Fig. 2C demonstrates that o-phenanthroline (25 mM), unlike BHT (200 mM), DPPD (10 mM), a-tocopherol (100 mM), and Trolox (1
Fig. 2. The effect of quercetin, antioxidants and iron chelators on tB-OOH-induced DNA single strand breakage. The cells were treated for 30 min with 200 mM tB-OOH in saline A in the absence or presence of increasing concentrations of quercetin (A) or various antioxidant and iron chelators (C) and immediately assayed for DNA damage with the microgel comet assay. Results are expressed as the ratio of the comet tail versus the comet head area and represent the mean 6 SEM of at least three separate experiments, each performed in duplicate. (B) shows representative photomicrographs of microgel electrophoresed U937 cells treated under experimental conditions similar to those detailed in (A).
mM), prevented the DNA strand scission caused by tB-OOH. These results are also consistent with previously published work from our20 –22 as well as from other19 –21 laboratories and indicate that the DNA cleavage generated by tB-OOH requires a source of iron and is insensitive to radical scavenging antioxidants. Thus, the inhibitory effects of quercetin on DNA strand scission caused by the hydroperoxide can only be explained by an iron chelating mechanism. The final clue indicating that quercetin is an effective iron chelator is provided by experiments in which the effect of this flavonoid, as well as that of selected iron
Quercetin and tB-OOH-induced cyto- and genotoxicity
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support to the notion that the flavonoid is an effective iron chelator. The experimental approach utilized in this study may represent a valid system to discriminate radical scavenging versus iron chelating mechanisms in the antioxidant effects of different drugs and flavonoids. Acknowledgement—This work was supported by a grant from Specchiasol S.r.l. (Verona).
Fig. 3. The effect of quercetin, antioxidants and iron chelators on H2O2-induced DNA single strand breakage in post-lysed DNA. The cells (4 3 105) were lysed onto polycarbonate filters and, after extensive washing, were treated for 20 min in saline at room temperature. After the treatments, the extent of DNA single strand breakage was determined by the alkaline alution technique. The strand scission factor values were calculated from the resulting elution profiles, as detailed in the Methods section. Each point is the mean 6 SEM of three separate experiments.
chelators and antioxidants, was investigated in a preparation of partially purified DNA. In these experiments the cells were lysed onto polycarbonate filters and, after accurate washing, the post-lysed DNA samples were treated for 20 min with 100 mM H2O2. The extent of DNA strand scission was estimated using the alkaline elution technique. H2O2 was used as a DNA-damaging agent because tB-OOH does not cause strand scission in partially purified DNA.22 Indeed, our previous work demonstrated that the mechanism whereby the organic hydroperoxide generates DNA lesions involves calciumdependent mitochondrial formation of tB-OOH-derived DNA-damaging species mainly represented by H2O2.27 Furthermore, desferroxamine was used in place of ophenanthroline because the latter is known to form a complex with cupric ions that can directly generate lesions in preparations of purified DNA.28 The results illustrated in Fig. 3 demonstrate that the DNA cleavage generated by H2O2, although insensitive to 1 mM Trolox or 10 mM DPPD, was prevented by 100 mM quercetin or 10 mM desferroxamine. In conclusion, the results presented in this study demonstrate that iron chelation is a prominent effect of quercetin in U937 cells exposed to tB-OOH. Indeed, whereas the cytoprotective effects may be explained by radical chain-breaking or metal-binding activities, prevention of DNA strand scission in intact cells was entirely based on the iron chelating capacity of the flavonoid. The fact that cleavage of partially purified DNA caused by H2O2 was insensitive to antioxidants Trolox (hydrophilic) and DPPD (lipophilic), but abolished by the iron chelator desferroxamine and by quercetin, provides additional
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ABBREVIATIONS
BHT— butylated hyroxytoluene DPPD—N,N9-diphenyl-1,4-phenylene-diamine EDTA— ethylenediaminetetraacetic acid tB-OOH—tert-butylhydroperoxide