Free Radical Biology & Medicine, Vol. 9, pp. 441-449, 1990 Printed in the USA. All rights reserved.
0891-5849/90$3.00 + .00 Copyright ¢ 1990PergamonPress plc
Original Contribution THE PRODUCTION OF REACTIVE OXYGEN SPECIES BY DIETARY FLAVONOLS
ANDREW T. CANADA,*t ELIANA GIANNELLA,** TOAN D. NGUYEN,~§ and RONALD P. MASON** *Departments of Anesthesiology and *Medicine, Duke University Medical Center Durham, NC 27710; §Durham Veterans Affairs Hospital, Durham, NC 27705; and **the Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, RTP, NC 27709, USA (Received 27 June 1990; Revised and Accepted 9 August 1990)
Abstract--Flavonols are a group of naturally occurring compounds which are widely distributed in nature where they are found glycosylated primarily in vegetables and fruits. A number of studies have found both anti- and prooxidant effects for many of these compounds. The most widely studied because of their ubiquitous nature have been quercetin, a B-dihydroxylated and myricetin, a B-trihydroxylated flavonol. Some of their prooxidant properties have been attributed to the fact that they can undergo autooxidation when dissolved in aqueous buffer. Studying a number of factors affecting autooxidation, we found the rate of autooxidation for both quercetin and myricetin to be highly pH dependent with no autooxidation detected for quereetin at physiologic pH. Both the addition of iron for the two flavonols and the addition of iron followed by SOD for quercetin increased the rate of autooxidation substantially, Neither kaempferol, a monohydroxylated flavonol nor rutin, a glycosylated quercetin showed any ability to autooxidize. The results with rutin differ from what we expected based on the B-ring structural similarity to quercetin. The autooxidation of quercetin and myricetin was further studied by electron spin resonance spectroscopy (ESR). Whereas quercetin produced a characteristic DMPO--OH radical, it was not detected below a pH of 9. However, the addition of iron allowed the signal to be detected at a pH as low as 8.0. On the other hand, myricetin autooxidation yielded a semiquinone signal which upon the addition of iron, converted to a DMPO-OH signal detected at a pH of 7.5. In a microsome-NADPH system, quercetin produced an increase in oxygen utilization and with ESR, an ethanol-derived radical signal which could be completely suppressed by catalase indicating the dependence of the signal on hydrogen peroxide. These studies demonstrate that the extracellular production of active oxygen species by dietary flavonols is not likely to occur in vivo but the potential for intracellular redox cycling may have toxicologic significance. Keywords--Flavonols, Quercetin, Redox cycling, Reactive oxygen species, Free radicals, Iron, ESR, Free radical scavengers
INTRODUC~ON
(quercetin 3-rutinoside), a glycosylated quercetin widely found in food products, are glycosylated at the 3,5, or 7-position. Glycosylated flavonols can be hydrolyzed to aglycones by bacteria found both in the mouth and in the lower intestinal tract. 2-4 A l t h o u g h the majority of the literature has focused on the favorable pharmacologic effects of flavonols including antitumor, 5 antioxidant, 6 and free radical scave n g i n g activities, 7"8 a n u m b e r of other studies suggest that the aglycones m a y also exhibit adverse pharmacologic effects. Quercetin is a m u t a g e n in virtually every in vitro mutagenicity test. The mutagenicity is e n h a n c e d in the presence of a microsomal $9 fraction, 9'1° indicating the potential for m i c r o s o m a l bioactivation. The report that superoxide dismutase (SOD) increased the mutagenicity of quercetin in the A m e s test li further supports the potential for intraceUular bioactivation. H o d n i c k et al.12 observed that a n u m b e r of flavonoids, including the B-dihydroxy and trihydroxy flavonols
F l a v o n o i d s , a class of c o m p o u n d s which are c o m m o n l y found in most fruits and vegetables, exhibit the basic structure of a 2 - p h e n y l - b e n z o (a) pyrane (Fig. 1A) with the pyrane structure n a m e d as the A c o m p o n e n t and the p h e n y l group as the B c o m p o n e n t . F l a v o n o l s , a subgroup, are characterized by a hydroxyl group at position 3 and an o x y g e n at position 4 (Fig. 1B). The daily intake of flavonoids has b e e n estimated to be l g , with the primary dietary source being vegetables.t Kaempferol, quercetin, and myricetin in both aglycone and glycosylated forms are a m o n g the most c o m m o n flavonols found in the diet. These c o m p o u n d s differ structurally only in the n u m b e r of hydroxyl groups on the B ring (Fig. 1 C E). Most naturally occurring flavonols such as rutin tAddress correspondence to Andrew T. Canada, Department of Anesthesiology, Box 3094, Duke University Medical Center, Durham, NC 27710. 441
442
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quercetin and myricetin, impaired mitochondrial respiration by inhibiting succinoxidase. 12 The extent of this inhibition appeared to correlate with the ability of the flavonols to autooxidize and generate hydrogen peroxide and, by implication, superoxide. Employing electron spin response spectroscopy (ESR), these authors further demonstrated that in the presence of iron and at physiological pH, the products of quercetin autooxidation were superoxide, hydrogen peroxide, and the hydroxyl radical. 12,13 In an earlier study we found that kaempferol, quercetin, and myricetin were toxic to epithelial cells isolated from the small intestine of guinea pigs. 14 Toxicity in this system also appeared to correlate with the potential for autooxidation as quercetin and myricetin were more toxic to the enterocytes than kaempferol, a flavonol which did not autooxidize. However, since the dihydroxylated flavonol quercetin was more toxic than the trihydroxylated myricetin, it is possible that toxic oxygen species production was not the sole mediator of the observed enterocyte damage. We decided therefore to systematically investigate the ability of these hydroxylated flavonols, in aglycone and glycosylated forms, to produce toxic oxygen species in vitro at various pHs. Employing both oxygen consumption and ESR techniques, we found that very little oxygen radical production could be expected extracellularly at physiologic pH. However, we also observed that iron, alone or in combination with SOD, could substantially increase the in vitro autooxidation rates at physiologic pH, suggesting potential biologic relevance. Furthermore, we demon-
strated that quercetin aglycone has the potential for intracellular microsomal-mediated redox cycling. MATERIALS AND METHODS
Chemicals and reagents The flavonols (quercetin, myricetin, kaempferol, and rutin), catalase, superoxide dismutase, iron-EDTA, NADPH, and DMPO were all purchased from Sigma. The DMPO was further purified by vacuum distillation. Hepatic microsomes used in the redox cycling experiments were prepared as described elsewhere. ~5
Oxygen consumption For the studies of flavonol autooxidation, 20 p,L of each flavonol dissolved in ethanol was added to 1.7 mL of Tris buffer, pH 7.5-8.5 (final concentration of flavonol 150--450 p,M), and studied at 25°C in a waterjacketed oxygen consumption cell (Gilson Medical Electronics, Middleton, WI) equipped with a Clark electrode attached to an oxygen monitor (YSI Model 5300, Yellow Springs Instrument, Yellow Springs, Ohio). Catalase (400 pJmL), SOD (400 ix/mL), and iron EDTA (50 IxM) were added to evaluate their effect on flavonol autooxidation in both the oxygen utilization and ESR studies.
Electron Spin Resonance (ESR): Flavonols were dissolved in acetone for the ESR experiments and in the redox cycling and oxygen con-
Production of reactive oxygenspecies
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sumption experiments. 50 txL of the dissolved flavonol was added to 3 mL of 50 mM Tris buffer (pH 7.5-9) containing 100 mM DMPO. For studies requiring a pH of 10, a 50 mM phosphate/carbonate buffer was substituted. Iron was added as Fe (III)-EDTA to a final concentration of 50 IzM. Redox cycling experiments were conducted at 37°C for the oxygen consumption study and 25°C for the ESR studies. Redox cycling was determined by adding a mixture of microsomes (2 mg protein/mL) and NADPH (0.7 raM) to previously oxidized quercetin (final concentration of 450 ~M in 1% ethanol) in 0.1 M potassium phosphate buffer (pH 7.5). Quercetin was added to the aqueous buffer 24 h prior to the experiment and allowed to stand in air. Complete oxidation of quercetin was ascertained by the failure to demonstrate oxygen
consumption when added to a Tris buffer, pH 8.5. Spectra from all samples were recorded using a Varian E-109 spectrometer with a TM cavity, a total scan time of 1-2 min, an 80 gauss span, and a receiver gain of 1 × 104-1 × 105 .
RESULTS AND DISCUSSION
Demonstration of flavonol autooxidation by oxygen consumption As shown in Fig. 2, when compared to a control addition of ethanol (C), both quercetin and myricetin consumed oxygen more rapidly when added to an aqueous buffer, pH 8.0. As expected, autooxidation was more
A.T. CANADAet al.
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Fig. 3. pH dependenceof autooxidationrate of the flavonolsquercetin (Q) and myricetin(M) at 7.5 (A), 8.0 (B), and 8.5 (C).
rapid for the B-trihydroxylated myricetin (M) than for the dihydroxylated quercetin (Q). These oxygen consumption data for quercetin and myricetin are similar to those reported by Hodnick et al. 13 Those authors observed that flavonols possessing B-trihydroxyl (pyrogallollike) and ortho-dihydroxy (catechol) substitutions on the B-ring were the strongest inhibitors of mitochondrial succinoxidase. They felt that this indicated that the potential for autooxidation with the subsequent formation of active oxygen species determined the extent of this mitochondrial inhibition. In partial agreement with their hypothesis, we have shown that quercetin and myricetin were toxic to enterocytes isolated from the small intestine of guinea pigs. 14 However, quercetin, which only slowly oxidized at a pH of 8.0, appeared to be more toxic than the more rapidly autooxidizing myricetin. Furthermore, the B-monohydroxylated flavonol kaempferol (K), which did not autooxidize, produced more cell injury than was observed for the controls. These observa-
tions suggest that toxic oxygen species might not be the entire explanation for our previously observed injury to the enterocytes. Surprisingly, we could not detect any autooxidation for rutin (R), a glycosylated quercetin which contains the B-dihydroxy structure of the aglycone. Thus, glycosylation at the 3-position served to prevent autooxidation, and the B-ortho-dihydroxy configuration is, by itself, not a predictor of oxidation potential. Interestingly, there have been no reports of toxicity attributed to rutin. Factors affecting the rate of autooxidation were next studied. The rates of autooxidation indicated by oxygen consumption for quercetin and myricetin were highly pH dependent (Fig. 3) with virtually no autooxidation for quercetin (Q) and a greatly reduced oxidation rate for myricetin (M) at the physiologic pH of 7.5 (A). Autooxidation increased considerably at the higher pHs of 8.0 (B) and 8.5 (C). The addition of 400U/mL of SOD to quercetin or myricetin autooxidizing at a pH of 8.0 reduced the autooxidation rate of both compounds (Fig. 4A); however, the effect on quercetin was minimal. SOD has been shown to inhibit the autooxidation of pyrogallol, and this property has been used in an assay for this enzyme. 16 Thus, it was not unexpected that SOD reduced the rate of autooxidation of the pyrogallol-like flavonol, myricetin. The addition of 50 txM iron (as iron-EDTA) significantly increased the rate of autooxidation for both compounds (Fig. 4B). Furthermore, in the presence of iron, the addition of SOD further increased the oxygen consumption of quercetin but inhibited that of myricetin. This may explain the previously reported augmentation of quercetin mutagenicity by SOD. 11 Although these authors attributed their findings to SOD prevention of quercetin breakdown (presumably reducing autooxidation), it is hard to envision how a decrease in the rate of autooxidation could enhance mutagenicity. Our results provide an alternate explanation for their findings. The products of these autooxidation reactions were further characterized by ESR and spectroscopy.
Identification of active oxygen species produced by autooxidation using ESR Fig. 5 shows that, using DMPO as the spin-trapping agent, quercetin autooxidation produced a DMPO-OH signal. Compared to the DMPO control (Fig. 5A), quercetin produced a strong signal at a pH of 10 (Fig. 5B). However, in agreement with the oxygen consumption data, this signal was strongly pH dependent, being barely detectable at a pH of 9 (Fig. 5C). No DMPO--OH signal was detected when kaempferol was added under the same conditions at a pH of I0 (scan not shown). As would be predicted from the oxygen consumption data,
Production of reactive oxygen species
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the addition of 50 IxM iron (as iron-EDTA) to autooxidizing quercetin greatly enhanced the DMPO-OH signal at pH 9.0, which was then easily detected at a pH of 8.5 (Fig. 5D). However, even with additional iron present, the signal was not detectable at pH 7.5. These results are different from those reported by Hodnick et al., ~3 who were able to detect a DMPO-OH signal at a pH of 7.5. Myricetin produced a semiquinone-type radical at a pH of 10 in the presence of DMPO (Fig. 6A), again duplicating the results of Hodnick et al. t3 This radical signal could be detected at the lower pH of 8.0. The addition of iron to the mixture resulted in the disappearance of the quinone signal and the appearance of the DMPO-OH radical signal (Fig. 6B). Semiquinones are known to reduce ferric iron ions to the ferrous ion that will reduce H202 to the hydroxyl radical (17).
QT + Fe3+ ___> Q + Fe2+ Fe 2+ H202 - - - > Fe 3+ + "OH. + O H -
This iron-amplified DMPO-OH signal could be easily detected at the physiologic pH of 7.5 and at a myricetin concentration of as low as 100 IxM. Under those conditions, the addition of SOD to myricetin undergoing autooxidation in the presence of iron at a pH of 8.5 increased the DMPO-OH signal twofold (Fig. 6C). These data, which are the opposite of what we found in the oxygen utilization studies, are probably the result of SOD preventing the superoxide-mediated breakdown of the DMPO--OH signal. 17 The addition of catalase to the system (Fig. 6D) virtually eradicated the DMPO-OH signal, thus demonstrating the DMPO-OH signal was hydrogen peroxide dependent, and that hydrogen peroxide is a product of the autooxidation of myricetin in the
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Fig. 5. ESR spectra of quercetin during autooxidation with DMPO as spin-trapping agent; control DMPO at pH 10 (A), quercetin and DMPO at pH of 10.0 (B), 9.0 (C), and at pH of 8.5 after addition of 50 IzM iron (as EDTA-iron) (D). 5 × 104 gain.
presence of iron. Similar to the oxygen consumption data, the DMPO--OH signal at a pH of 8.5 produced by quercetin, already enhanced by the presence of iron (Fig. 7A), was further enhanced by SOD (Fig. 7B) and virtually abolished by catalase (Fig. 7C).
Demonstration of microsomal redox cycling by oxygen consumption and ESR An earlier study had reported that redox cycling of quercetin occurred in mitochondria isolated from mammalian cardiac muscle. 13We decided to extend that study and investigate the ability of a hepatic microsomalNADPH system (pH 7.45) to redox cycle previously oxidized quercetin. For these experiments, quercetin was dissolved in ethanol, added to the Tris buffer pH 7.45 to achieve a concentration of 450 IxM, and allowed to completely oxidize overnight in air as described in the Materials andMethods section. Microsomes and NADPH were then added to the previously autooxidized quercetin in the buffer. After air oxidizing for 24 h, no oxygen consumption was observed (Q Fig. 8). The addition of microsomes (M) or NADPH (N) alone had no effect on oxygen consumption (data not shown). The addition of both NADPH and microsomes (M & N) to the buffer alone produced an increase in oxygen consumption (middle). However when both were added to oxidized
quercetin (bottom) there was a 116% increase in oxygen consumption compared with the microsomes plus NADPH control. This indicated reduction of the oxidized quinone to the parent reduced form with subsequent reoxidation. To further identify the presence of free radicals in this reaction, we conducted an ESR study under the same conditions. Redox cycling was demonstrated by the appearance within 1 min of both an ethanol-derived radical and DMPO-OH (Fig 9B), which was not observed with DMPO, 1% ethanol, NADPH, and microsomes alone (Fig. 9A). Again, the addition of catalase resulted in virtual disappearance of the signal (Fig. 9C), showing its dependence on hydrogen peroxide. An ethanolderived signal has also been reported to be detected from a similar microsome-NADPH-ethanol systemm; however, that system required the presence of Fe +3, which was not added in our redox cycling experiments. Recently, Laughton et al. ,2o measuring hydroxyl radical production from flavonol autooxidation by a deoxyribose assay, reported that the addition of iron-EDTA to 100 ~M quercetin and myricetin increased hydroxyl radical production up to eightfold. They also reported that at 75 IxM, both quercetin and myricetin, in the presence of iron, accelerated bleomycin-dependent DNA damage. They suggested that under certain conditions these compounds, particularly in the presence of iron, could be considered prooxidants, and not antioxidants.
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In s u m m a r y , our data s h o w that the toxic o x y g e n species h y d r o g e n p e r o x i d e and h y d r o x y l radical, are p r o d u c e d during the autooxidation o f B-, di-, and trihydroxy flavonols and that glycosylation at the 3-position apparently prevents this reaction. In the presence o f iron, the addition o f S O D results in an increase in the rate o f autooxidation o f quercetin. A l t h o u g h the autooxidation o f both quercetin and myricetin proceeds slowly at p h y s i o l o g i c pH, the increase in production o f active oxy g e n species by quercetin in the presence o f iron and S O D m a y give t o x i c o l o g i c a l significance to the intracellular production o f o x y g e n radicals by specific dietary flavonols. This m e c h a n i s m m a y be responsible for at least s o m e o f the toxicity w e o b s e r v e d in isolated enterocytes. 14 In an organ such as the large intestine where concentrations o f flavonols m a y approach 2 5 - 1 0 0 ~ M and w h e r e m i c r o o r g a n i s m s readily h y d r o l y z e glycosolated flavonols to the reactive o x y g e n species-producing a g l y c o n e s , these o b s e r v a t i o n s m a y take on
Fig. 7. DMPO-OH signal generated during the autooxidation of quercetin at a pH of 8.5 (A), upon the addition of SOD 400 p,/mL (B), and after the addition of catalase 400 p,/mL (C). 1.6 x 10 4 gain.
toxicologic significance that will need to be explored further. Acknowledgements -- This research was supported by a grant from
the National Foundation for Ileitis and Colitis. The authors would like to thank Dr. Irwin Fridovich for his many helpful suggestions during these studies.
REFERENCES
1. Kuhnau, J. The flavonoids. A class of semi-essential food components: Their role in human nutrition. Wld. Rev. Nutr. Diet. 24: 117-191; 1976. 2+ Parisis, D.M.; Pritchard, E.T. Activation of rutin by human oral bacterial isolates to the carcinogen-mutagen quercetin. Arch. Oral Biol. 28:583-590; 1987. 3. Brown, J.P.; Dietrich, P.S. Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test. Activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources. Mutation Res. 66:223-240; 1979. 4. Griffiths, L.A.; Smith, G.E. Metabolism of myricetin and related compounds in the rat. Metabolite formation in vivo and by the intestinal microflora in vitro. Biochem. J. 130:141-151; 1972. 5. Nishino, H.; Naito, E.; Iwashima, A.; Tanaka, K.; Matsuura, T.; Fujiki, H.; Sugimura, T. Interaction between quercetin and Ca2+ - calmodulin complex: possible mechanism for anti-tumor-promoting action of the flavonoid. GANN 74:311-316; 1984. 6. Das, N.P.; Ratty, A.K. Effects of flavonoids on induced nonenzymatic lipid peroxidation. In: Cody, V.; Middleton, Jr. E.; Harborne, J.B., eds. Plant flavonoids in medicine: biochemical, pharmacological, and structure-activity relationships. New York:
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I I l,tl I I tln~ 11ll,kl I I 11111~111 m 0..~lll-:l.l~lI
:
llIlttllttttttt iit t
Alan R. Liss; 1986:234-247. 7. Robak, J.; Gryglewski, R.J. Flavonoids are scavengers of superoxide anions. Biochem. Pharmacol. 37:837-841; 1988. 8. Bors, W.; Saran, M. Radical scavenging by flavonoids antioxidants. Free Rad. Res. Comms. 2:289-294; 1987. 9. Brown, J.P. A review of the genetic effects of naturally occurring flavonoids, anthraquinones and related compounds. Mutation Res. 75:243-277; 1980. 10. Bjeldanes, L.F.; Chang, G.W. Mutagenic activity of quercetin and related compounds. Science 197:577-578; 1977. 11. Ochiai, M.; Nagao, M.; Wakabayashi, K.; Sugimura, T. Superoxide dismutase acts as an enhancing factor for quercetin mutagenesis in rat-liver cytosol by preventing its decomposition. Mutation Res. 129:19-24; 1984. 12. Hodnick, W.F.; Kung, F.S.; Ruettger, W.J.; Bohmont, C.W.; Pardini, R.S. Inhibition of mitochondfial respiration and production of toxic oxygen radicals by flavonoids. Biochem. Pharmacol. 35:2345-2357; 1986. 13. Hodnick, W.F.; Kalyanaraman, B.; Pritsos, C.A.; Pardini, R.S.The production of hydroxyl and semiquinone free radicals during the auto-oxidation of redox active flavonoids. In: Cody, V.; Middleton, Jr., E.; Harbome, J.B., eds. Plantflavonoids in medi-cine: biochemical, pharmacological, and structure-activity relationships. New York: Alan R. Liss; 1986:149-152.
!i IIINIII liitI!ItMtl illi[ltlllltlllllll}llll tiiiltIiitti lil lJlHM[IIII 13 l[ttH~tltltttmt'd
IttHIII !!IMi Fig. 8. Oxygen consumption of autooxidizing quercetin (Q) after addition of microsomes (M) and NADPH (N) (top); microsomes and NADPH (middle); quercetin plus NADPH (bottom).
14. Canada, A.T.; Watkins, W.D.; Nguyen, T.D. The toxicity of flavonoids to guinea pig enterocytes. Toxicol. Appl. Pharmacol. 99:357-361; 1989. 15. Mason, R.P.; Holtzman, J.L. The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates. Biochemistry 14: 1626-1633. 16. Marklund, S.; Marklund, G. Involvement of the superoxide anion radical in the auto-oxidation and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47:469--474; 1974. 17. Winterbourn, C. Evidence for the production of hydroxyl radicals from the adriamycin semiquinone and H202. FEBS Lett. 136:8994; 1981. 18. Samuni, A.; Krishna, C.M.; Riesz, P.; Finkelstein, E.; Russo, A. Superoxide reaction with nitroxide spin-adducts. Free Radic. Biol. Med. 6:141-148; 1989. 19. Lai, C.; Piette, L.H. Hydroxyl radical production involved in lipid peroxidation of rat liver microsomes. Biochem. Biophys. Res. Comm. 78:51-59; 1972. 20. Laughton, M.J.; Halliwell, B.; Evans, P.J.; Hoult, J.R.S. Antioxidant and prooxidant action of the plant phenolics quercetin, gossypol and myricetin. Biochem. Pharmacol. 38:2859-2865; 1989.
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Fig. 9. ESR spectra generated by the addition of microsomes and NADPH to a solution containing 300 I~M of quercetin dissolved in 10% ethanol in buffer pH 7.45. Spectra from combination of microsomes, NADPH, and 10% ethanol (A), oxidized quercetin with added NADPH and microsomes (B), and after the addition of catalase (C), with scan conducted 1-2 min after addition of reagents, 6.3 x l04 gain.
0891-5849/90$3.00 + .00 Copyright ¢ 1990PergamonPress plc
Original Contribution THE PRODUCTION OF REACTIVE OXYGEN SPECIES BY DIETARY FLAVONOLS
ANDREW T. CANADA,*t ELIANA GIANNELLA,** TOAN D. NGUYEN,~§ and RONALD P. MASON** *Departments of Anesthesiology and *Medicine, Duke University Medical Center Durham, NC 27710; §Durham Veterans Affairs Hospital, Durham, NC 27705; and **the Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, RTP, NC 27709, USA (Received 27 June 1990; Revised and Accepted 9 August 1990)
Abstract--Flavonols are a group of naturally occurring compounds which are widely distributed in nature where they are found glycosylated primarily in vegetables and fruits. A number of studies have found both anti- and prooxidant effects for many of these compounds. The most widely studied because of their ubiquitous nature have been quercetin, a B-dihydroxylated and myricetin, a B-trihydroxylated flavonol. Some of their prooxidant properties have been attributed to the fact that they can undergo autooxidation when dissolved in aqueous buffer. Studying a number of factors affecting autooxidation, we found the rate of autooxidation for both quercetin and myricetin to be highly pH dependent with no autooxidation detected for quereetin at physiologic pH. Both the addition of iron for the two flavonols and the addition of iron followed by SOD for quercetin increased the rate of autooxidation substantially, Neither kaempferol, a monohydroxylated flavonol nor rutin, a glycosylated quercetin showed any ability to autooxidize. The results with rutin differ from what we expected based on the B-ring structural similarity to quercetin. The autooxidation of quercetin and myricetin was further studied by electron spin resonance spectroscopy (ESR). Whereas quercetin produced a characteristic DMPO--OH radical, it was not detected below a pH of 9. However, the addition of iron allowed the signal to be detected at a pH as low as 8.0. On the other hand, myricetin autooxidation yielded a semiquinone signal which upon the addition of iron, converted to a DMPO-OH signal detected at a pH of 7.5. In a microsome-NADPH system, quercetin produced an increase in oxygen utilization and with ESR, an ethanol-derived radical signal which could be completely suppressed by catalase indicating the dependence of the signal on hydrogen peroxide. These studies demonstrate that the extracellular production of active oxygen species by dietary flavonols is not likely to occur in vivo but the potential for intracellular redox cycling may have toxicologic significance. Keywords--Flavonols, Quercetin, Redox cycling, Reactive oxygen species, Free radicals, Iron, ESR, Free radical scavengers
INTRODUC~ON
(quercetin 3-rutinoside), a glycosylated quercetin widely found in food products, are glycosylated at the 3,5, or 7-position. Glycosylated flavonols can be hydrolyzed to aglycones by bacteria found both in the mouth and in the lower intestinal tract. 2-4 A l t h o u g h the majority of the literature has focused on the favorable pharmacologic effects of flavonols including antitumor, 5 antioxidant, 6 and free radical scave n g i n g activities, 7"8 a n u m b e r of other studies suggest that the aglycones m a y also exhibit adverse pharmacologic effects. Quercetin is a m u t a g e n in virtually every in vitro mutagenicity test. The mutagenicity is e n h a n c e d in the presence of a microsomal $9 fraction, 9'1° indicating the potential for m i c r o s o m a l bioactivation. The report that superoxide dismutase (SOD) increased the mutagenicity of quercetin in the A m e s test li further supports the potential for intraceUular bioactivation. H o d n i c k et al.12 observed that a n u m b e r of flavonoids, including the B-dihydroxy and trihydroxy flavonols
F l a v o n o i d s , a class of c o m p o u n d s which are c o m m o n l y found in most fruits and vegetables, exhibit the basic structure of a 2 - p h e n y l - b e n z o (a) pyrane (Fig. 1A) with the pyrane structure n a m e d as the A c o m p o n e n t and the p h e n y l group as the B c o m p o n e n t . F l a v o n o l s , a subgroup, are characterized by a hydroxyl group at position 3 and an o x y g e n at position 4 (Fig. 1B). The daily intake of flavonoids has b e e n estimated to be l g , with the primary dietary source being vegetables.t Kaempferol, quercetin, and myricetin in both aglycone and glycosylated forms are a m o n g the most c o m m o n flavonols found in the diet. These c o m p o u n d s differ structurally only in the n u m b e r of hydroxyl groups on the B ring (Fig. 1 C E). Most naturally occurring flavonols such as rutin tAddress correspondence to Andrew T. Canada, Department of Anesthesiology, Box 3094, Duke University Medical Center, Durham, NC 27710. 441
442
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quercetin and myricetin, impaired mitochondrial respiration by inhibiting succinoxidase. 12 The extent of this inhibition appeared to correlate with the ability of the flavonols to autooxidize and generate hydrogen peroxide and, by implication, superoxide. Employing electron spin response spectroscopy (ESR), these authors further demonstrated that in the presence of iron and at physiological pH, the products of quercetin autooxidation were superoxide, hydrogen peroxide, and the hydroxyl radical. 12,13 In an earlier study we found that kaempferol, quercetin, and myricetin were toxic to epithelial cells isolated from the small intestine of guinea pigs. 14 Toxicity in this system also appeared to correlate with the potential for autooxidation as quercetin and myricetin were more toxic to the enterocytes than kaempferol, a flavonol which did not autooxidize. However, since the dihydroxylated flavonol quercetin was more toxic than the trihydroxylated myricetin, it is possible that toxic oxygen species production was not the sole mediator of the observed enterocyte damage. We decided therefore to systematically investigate the ability of these hydroxylated flavonols, in aglycone and glycosylated forms, to produce toxic oxygen species in vitro at various pHs. Employing both oxygen consumption and ESR techniques, we found that very little oxygen radical production could be expected extracellularly at physiologic pH. However, we also observed that iron, alone or in combination with SOD, could substantially increase the in vitro autooxidation rates at physiologic pH, suggesting potential biologic relevance. Furthermore, we demon-
strated that quercetin aglycone has the potential for intracellular microsomal-mediated redox cycling. MATERIALS AND METHODS
Chemicals and reagents The flavonols (quercetin, myricetin, kaempferol, and rutin), catalase, superoxide dismutase, iron-EDTA, NADPH, and DMPO were all purchased from Sigma. The DMPO was further purified by vacuum distillation. Hepatic microsomes used in the redox cycling experiments were prepared as described elsewhere. ~5
Oxygen consumption For the studies of flavonol autooxidation, 20 p,L of each flavonol dissolved in ethanol was added to 1.7 mL of Tris buffer, pH 7.5-8.5 (final concentration of flavonol 150--450 p,M), and studied at 25°C in a waterjacketed oxygen consumption cell (Gilson Medical Electronics, Middleton, WI) equipped with a Clark electrode attached to an oxygen monitor (YSI Model 5300, Yellow Springs Instrument, Yellow Springs, Ohio). Catalase (400 pJmL), SOD (400 ix/mL), and iron EDTA (50 IxM) were added to evaluate their effect on flavonol autooxidation in both the oxygen utilization and ESR studies.
Electron Spin Resonance (ESR): Flavonols were dissolved in acetone for the ESR experiments and in the redox cycling and oxygen con-
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sumption experiments. 50 txL of the dissolved flavonol was added to 3 mL of 50 mM Tris buffer (pH 7.5-9) containing 100 mM DMPO. For studies requiring a pH of 10, a 50 mM phosphate/carbonate buffer was substituted. Iron was added as Fe (III)-EDTA to a final concentration of 50 IzM. Redox cycling experiments were conducted at 37°C for the oxygen consumption study and 25°C for the ESR studies. Redox cycling was determined by adding a mixture of microsomes (2 mg protein/mL) and NADPH (0.7 raM) to previously oxidized quercetin (final concentration of 450 ~M in 1% ethanol) in 0.1 M potassium phosphate buffer (pH 7.5). Quercetin was added to the aqueous buffer 24 h prior to the experiment and allowed to stand in air. Complete oxidation of quercetin was ascertained by the failure to demonstrate oxygen
consumption when added to a Tris buffer, pH 8.5. Spectra from all samples were recorded using a Varian E-109 spectrometer with a TM cavity, a total scan time of 1-2 min, an 80 gauss span, and a receiver gain of 1 × 104-1 × 105 .
RESULTS AND DISCUSSION
Demonstration of flavonol autooxidation by oxygen consumption As shown in Fig. 2, when compared to a control addition of ethanol (C), both quercetin and myricetin consumed oxygen more rapidly when added to an aqueous buffer, pH 8.0. As expected, autooxidation was more
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Fig. 3. pH dependenceof autooxidationrate of the flavonolsquercetin (Q) and myricetin(M) at 7.5 (A), 8.0 (B), and 8.5 (C).
rapid for the B-trihydroxylated myricetin (M) than for the dihydroxylated quercetin (Q). These oxygen consumption data for quercetin and myricetin are similar to those reported by Hodnick et al. 13 Those authors observed that flavonols possessing B-trihydroxyl (pyrogallollike) and ortho-dihydroxy (catechol) substitutions on the B-ring were the strongest inhibitors of mitochondrial succinoxidase. They felt that this indicated that the potential for autooxidation with the subsequent formation of active oxygen species determined the extent of this mitochondrial inhibition. In partial agreement with their hypothesis, we have shown that quercetin and myricetin were toxic to enterocytes isolated from the small intestine of guinea pigs. 14 However, quercetin, which only slowly oxidized at a pH of 8.0, appeared to be more toxic than the more rapidly autooxidizing myricetin. Furthermore, the B-monohydroxylated flavonol kaempferol (K), which did not autooxidize, produced more cell injury than was observed for the controls. These observa-
tions suggest that toxic oxygen species might not be the entire explanation for our previously observed injury to the enterocytes. Surprisingly, we could not detect any autooxidation for rutin (R), a glycosylated quercetin which contains the B-dihydroxy structure of the aglycone. Thus, glycosylation at the 3-position served to prevent autooxidation, and the B-ortho-dihydroxy configuration is, by itself, not a predictor of oxidation potential. Interestingly, there have been no reports of toxicity attributed to rutin. Factors affecting the rate of autooxidation were next studied. The rates of autooxidation indicated by oxygen consumption for quercetin and myricetin were highly pH dependent (Fig. 3) with virtually no autooxidation for quercetin (Q) and a greatly reduced oxidation rate for myricetin (M) at the physiologic pH of 7.5 (A). Autooxidation increased considerably at the higher pHs of 8.0 (B) and 8.5 (C). The addition of 400U/mL of SOD to quercetin or myricetin autooxidizing at a pH of 8.0 reduced the autooxidation rate of both compounds (Fig. 4A); however, the effect on quercetin was minimal. SOD has been shown to inhibit the autooxidation of pyrogallol, and this property has been used in an assay for this enzyme. 16 Thus, it was not unexpected that SOD reduced the rate of autooxidation of the pyrogallol-like flavonol, myricetin. The addition of 50 txM iron (as iron-EDTA) significantly increased the rate of autooxidation for both compounds (Fig. 4B). Furthermore, in the presence of iron, the addition of SOD further increased the oxygen consumption of quercetin but inhibited that of myricetin. This may explain the previously reported augmentation of quercetin mutagenicity by SOD. 11 Although these authors attributed their findings to SOD prevention of quercetin breakdown (presumably reducing autooxidation), it is hard to envision how a decrease in the rate of autooxidation could enhance mutagenicity. Our results provide an alternate explanation for their findings. The products of these autooxidation reactions were further characterized by ESR and spectroscopy.
Identification of active oxygen species produced by autooxidation using ESR Fig. 5 shows that, using DMPO as the spin-trapping agent, quercetin autooxidation produced a DMPO-OH signal. Compared to the DMPO control (Fig. 5A), quercetin produced a strong signal at a pH of 10 (Fig. 5B). However, in agreement with the oxygen consumption data, this signal was strongly pH dependent, being barely detectable at a pH of 9 (Fig. 5C). No DMPO--OH signal was detected when kaempferol was added under the same conditions at a pH of I0 (scan not shown). As would be predicted from the oxygen consumption data,
Production of reactive oxygen species
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the addition of 50 IxM iron (as iron-EDTA) to autooxidizing quercetin greatly enhanced the DMPO-OH signal at pH 9.0, which was then easily detected at a pH of 8.5 (Fig. 5D). However, even with additional iron present, the signal was not detectable at pH 7.5. These results are different from those reported by Hodnick et al., ~3 who were able to detect a DMPO-OH signal at a pH of 7.5. Myricetin produced a semiquinone-type radical at a pH of 10 in the presence of DMPO (Fig. 6A), again duplicating the results of Hodnick et al. t3 This radical signal could be detected at the lower pH of 8.0. The addition of iron to the mixture resulted in the disappearance of the quinone signal and the appearance of the DMPO-OH radical signal (Fig. 6B). Semiquinones are known to reduce ferric iron ions to the ferrous ion that will reduce H202 to the hydroxyl radical (17).
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This iron-amplified DMPO-OH signal could be easily detected at the physiologic pH of 7.5 and at a myricetin concentration of as low as 100 IxM. Under those conditions, the addition of SOD to myricetin undergoing autooxidation in the presence of iron at a pH of 8.5 increased the DMPO-OH signal twofold (Fig. 6C). These data, which are the opposite of what we found in the oxygen utilization studies, are probably the result of SOD preventing the superoxide-mediated breakdown of the DMPO--OH signal. 17 The addition of catalase to the system (Fig. 6D) virtually eradicated the DMPO-OH signal, thus demonstrating the DMPO-OH signal was hydrogen peroxide dependent, and that hydrogen peroxide is a product of the autooxidation of myricetin in the
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presence of iron. Similar to the oxygen consumption data, the DMPO--OH signal at a pH of 8.5 produced by quercetin, already enhanced by the presence of iron (Fig. 7A), was further enhanced by SOD (Fig. 7B) and virtually abolished by catalase (Fig. 7C).
Demonstration of microsomal redox cycling by oxygen consumption and ESR An earlier study had reported that redox cycling of quercetin occurred in mitochondria isolated from mammalian cardiac muscle. 13We decided to extend that study and investigate the ability of a hepatic microsomalNADPH system (pH 7.45) to redox cycle previously oxidized quercetin. For these experiments, quercetin was dissolved in ethanol, added to the Tris buffer pH 7.45 to achieve a concentration of 450 IxM, and allowed to completely oxidize overnight in air as described in the Materials andMethods section. Microsomes and NADPH were then added to the previously autooxidized quercetin in the buffer. After air oxidizing for 24 h, no oxygen consumption was observed (Q Fig. 8). The addition of microsomes (M) or NADPH (N) alone had no effect on oxygen consumption (data not shown). The addition of both NADPH and microsomes (M & N) to the buffer alone produced an increase in oxygen consumption (middle). However when both were added to oxidized
quercetin (bottom) there was a 116% increase in oxygen consumption compared with the microsomes plus NADPH control. This indicated reduction of the oxidized quinone to the parent reduced form with subsequent reoxidation. To further identify the presence of free radicals in this reaction, we conducted an ESR study under the same conditions. Redox cycling was demonstrated by the appearance within 1 min of both an ethanol-derived radical and DMPO-OH (Fig 9B), which was not observed with DMPO, 1% ethanol, NADPH, and microsomes alone (Fig. 9A). Again, the addition of catalase resulted in virtual disappearance of the signal (Fig. 9C), showing its dependence on hydrogen peroxide. An ethanolderived signal has also been reported to be detected from a similar microsome-NADPH-ethanol systemm; however, that system required the presence of Fe +3, which was not added in our redox cycling experiments. Recently, Laughton et al. ,2o measuring hydroxyl radical production from flavonol autooxidation by a deoxyribose assay, reported that the addition of iron-EDTA to 100 ~M quercetin and myricetin increased hydroxyl radical production up to eightfold. They also reported that at 75 IxM, both quercetin and myricetin, in the presence of iron, accelerated bleomycin-dependent DNA damage. They suggested that under certain conditions these compounds, particularly in the presence of iron, could be considered prooxidants, and not antioxidants.
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In s u m m a r y , our data s h o w that the toxic o x y g e n species h y d r o g e n p e r o x i d e and h y d r o x y l radical, are p r o d u c e d during the autooxidation o f B-, di-, and trihydroxy flavonols and that glycosylation at the 3-position apparently prevents this reaction. In the presence o f iron, the addition o f S O D results in an increase in the rate o f autooxidation o f quercetin. A l t h o u g h the autooxidation o f both quercetin and myricetin proceeds slowly at p h y s i o l o g i c pH, the increase in production o f active oxy g e n species by quercetin in the presence o f iron and S O D m a y give t o x i c o l o g i c a l significance to the intracellular production o f o x y g e n radicals by specific dietary flavonols. This m e c h a n i s m m a y be responsible for at least s o m e o f the toxicity w e o b s e r v e d in isolated enterocytes. 14 In an organ such as the large intestine where concentrations o f flavonols m a y approach 2 5 - 1 0 0 ~ M and w h e r e m i c r o o r g a n i s m s readily h y d r o l y z e glycosolated flavonols to the reactive o x y g e n species-producing a g l y c o n e s , these o b s e r v a t i o n s m a y take on
Fig. 7. DMPO-OH signal generated during the autooxidation of quercetin at a pH of 8.5 (A), upon the addition of SOD 400 p,/mL (B), and after the addition of catalase 400 p,/mL (C). 1.6 x 10 4 gain.
toxicologic significance that will need to be explored further. Acknowledgements -- This research was supported by a grant from
the National Foundation for Ileitis and Colitis. The authors would like to thank Dr. Irwin Fridovich for his many helpful suggestions during these studies.
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1. Kuhnau, J. The flavonoids. A class of semi-essential food components: Their role in human nutrition. Wld. Rev. Nutr. Diet. 24: 117-191; 1976. 2+ Parisis, D.M.; Pritchard, E.T. Activation of rutin by human oral bacterial isolates to the carcinogen-mutagen quercetin. Arch. Oral Biol. 28:583-590; 1987. 3. Brown, J.P.; Dietrich, P.S. Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test. Activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources. Mutation Res. 66:223-240; 1979. 4. Griffiths, L.A.; Smith, G.E. Metabolism of myricetin and related compounds in the rat. Metabolite formation in vivo and by the intestinal microflora in vitro. Biochem. J. 130:141-151; 1972. 5. Nishino, H.; Naito, E.; Iwashima, A.; Tanaka, K.; Matsuura, T.; Fujiki, H.; Sugimura, T. Interaction between quercetin and Ca2+ - calmodulin complex: possible mechanism for anti-tumor-promoting action of the flavonoid. GANN 74:311-316; 1984. 6. Das, N.P.; Ratty, A.K. Effects of flavonoids on induced nonenzymatic lipid peroxidation. In: Cody, V.; Middleton, Jr. E.; Harborne, J.B., eds. Plant flavonoids in medicine: biochemical, pharmacological, and structure-activity relationships. New York:
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Alan R. Liss; 1986:234-247. 7. Robak, J.; Gryglewski, R.J. Flavonoids are scavengers of superoxide anions. Biochem. Pharmacol. 37:837-841; 1988. 8. Bors, W.; Saran, M. Radical scavenging by flavonoids antioxidants. Free Rad. Res. Comms. 2:289-294; 1987. 9. Brown, J.P. A review of the genetic effects of naturally occurring flavonoids, anthraquinones and related compounds. Mutation Res. 75:243-277; 1980. 10. Bjeldanes, L.F.; Chang, G.W. Mutagenic activity of quercetin and related compounds. Science 197:577-578; 1977. 11. Ochiai, M.; Nagao, M.; Wakabayashi, K.; Sugimura, T. Superoxide dismutase acts as an enhancing factor for quercetin mutagenesis in rat-liver cytosol by preventing its decomposition. Mutation Res. 129:19-24; 1984. 12. Hodnick, W.F.; Kung, F.S.; Ruettger, W.J.; Bohmont, C.W.; Pardini, R.S. Inhibition of mitochondfial respiration and production of toxic oxygen radicals by flavonoids. Biochem. Pharmacol. 35:2345-2357; 1986. 13. Hodnick, W.F.; Kalyanaraman, B.; Pritsos, C.A.; Pardini, R.S.The production of hydroxyl and semiquinone free radicals during the auto-oxidation of redox active flavonoids. In: Cody, V.; Middleton, Jr., E.; Harbome, J.B., eds. Plantflavonoids in medi-cine: biochemical, pharmacological, and structure-activity relationships. New York: Alan R. Liss; 1986:149-152.
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IttHIII !!IMi Fig. 8. Oxygen consumption of autooxidizing quercetin (Q) after addition of microsomes (M) and NADPH (N) (top); microsomes and NADPH (middle); quercetin plus NADPH (bottom).
14. Canada, A.T.; Watkins, W.D.; Nguyen, T.D. The toxicity of flavonoids to guinea pig enterocytes. Toxicol. Appl. Pharmacol. 99:357-361; 1989. 15. Mason, R.P.; Holtzman, J.L. The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates. Biochemistry 14: 1626-1633. 16. Marklund, S.; Marklund, G. Involvement of the superoxide anion radical in the auto-oxidation and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47:469--474; 1974. 17. Winterbourn, C. Evidence for the production of hydroxyl radicals from the adriamycin semiquinone and H202. FEBS Lett. 136:8994; 1981. 18. Samuni, A.; Krishna, C.M.; Riesz, P.; Finkelstein, E.; Russo, A. Superoxide reaction with nitroxide spin-adducts. Free Radic. Biol. Med. 6:141-148; 1989. 19. Lai, C.; Piette, L.H. Hydroxyl radical production involved in lipid peroxidation of rat liver microsomes. Biochem. Biophys. Res. Comm. 78:51-59; 1972. 20. Laughton, M.J.; Halliwell, B.; Evans, P.J.; Hoult, J.R.S. Antioxidant and prooxidant action of the plant phenolics quercetin, gossypol and myricetin. Biochem. Pharmacol. 38:2859-2865; 1989.
Production of reactive oxygen species
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Fig. 9. ESR spectra generated by the addition of microsomes and NADPH to a solution containing 300 I~M of quercetin dissolved in 10% ethanol in buffer pH 7.45. Spectra from combination of microsomes, NADPH, and 10% ethanol (A), oxidized quercetin with added NADPH and microsomes (B), and after the addition of catalase (C), with scan conducted 1-2 min after addition of reagents, 6.3 x l04 gain.
