The production of reactive oxygen species by dietary flavonols

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 quercetin at physiologic pH. Both the addition of iron for the two flavonols and the addition of iron followed by SOD for quercetin at physiologic pH. Both the addistantially. 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.     

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.     

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.     

Quercetin regulates the inhibitory effect of monoclonal non-specific suppressor factor beta on tumor necrosis factor-alpha production in LPS-stimulated macrophages

Monoclonal non-specific suppressor factor beta (MNSFbeta) is a member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta regulates the ERK1/2-MAPK cascade in the macrophage cell line Raw 264.7. In this study, we found evidence that the flavonol quercetin regulates the effect of MNSFbeta on TNFalpha production in LPS-stimulated Raw264.7 cells. Quercetin inhibited MNSFbeta siRNA-mediated enhancement of both TNFalpha production and ERK1/2 phosphorylation in LPS-stimulated Raw264.7 cells. Quercetin decreased the expression of 33.5-kDa MNSFbeta adduct, which is important to the regulation of ERK1/2 activity, in unstimulated Raw264.7 cells. The various flavonoids tested, including other flavonols, did not affect the formation of this adduct. Collectively, MNSFbeta and quercetin might share a common pathway in regulating the ERK1/2 pathway in macrophages. This is the first report describing the involvement of flavonoids in the action of ubiquitin-like proteins.     

Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells

Quercetin is one of the naturally occurring dietary flavonol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.     

Effect of the flavonol quercetin on ion transport in the isolated intestine of the eel, Anguilla anguilla

Flavonoids are phenolic compounds used in fish diet formulations for the control of sex differentiation. Consequently it is of interest to know their effects on fish gastrointestinal mucosa that is the first target of these substances after food intake. We studied the effects of the flavonol quercetin on the transepithelial electrical parameters of the isolated intestine of the eel, Anguilla anguilla, by employing Ussing chamber technique. We showed that luminal quercetin (10(-4) M) reduced the serosa negative I(sc) (short circuit current) due to a net Cl- absorption in the control conditions. The reduction of I(sc) was blocked by luminal glybenclamide (10(-4) M). The I(sc) response to the flavonoid was also observed when HCO3- was omitted from the perfusion solutions. The effect of quercetin was nullified by TMB8, a blocker of Ca2+ release from intracellular stores, and was strongly reduced by trifluoroperazine, an inhibitor of calmodulin actions, while it was unmodified by experimental manoeuvres able to alter cytosolic cAMP concentrations. These results suggest that mucosal quercetin stimulated Cl- but not HCO3- secretion; the secretory activity appears to be dependent on the Ca2+-calmodulin system, independent of the cAMP pathway. In addition, this study showed that flavonol mimics the effect of carbachol on the tissue. This conclusion is supported by the following observations: 1) noradrenalin reversed the reduction of I(sc) produced by both carbachol and quercetin; 2) the flavonol was ineffective in tissues in which the muscarinic agonist had already elicited its maximal reduction of I(sc).     

Contrasting action of flavonoids on phototoxic effects induced in human skin fibroblasts by UVA alone or UVA plus cyamemazine, a phototoxic neuroleptic.

The potential protective effects of the flavanol catechin, the flavonol quercetin, the flavones, luteolin and rutin, and the isoflavones, genistein and daidzein, against the photo-oxidative stress induced by ultraviolet A radiation (UVA) and by phototoxic reactions resulting from the interaction of UVA with drugs and chemicals, has been assessed with cultured human skin fibroblasts. Lipid peroxidation and cell death have been chosen as model photobiological damage induced by UVA alone or photosensitized by cyamemazine (CMZ) and its photoproduct possessing phototoxic properties. Contrasting effects of flavonoids are observed. The flavanol, the flavonol and the flavones may protect against lipid peroxidation and cell death induced by 30 J cm(-2) of UVA alone or CMZ plus 10 J cm(-2) UVA. On the other hand, an amplification of the photodamage may be observed with isoflavones. A concentration-dependence study demonstrates that among the protective flavonoids, quercetin is the most efficient. The very effective protection brought by quercetin may result from its ability to scavenge reactive oxygen species produced by the photo-oxidative stress. However, the modification of membrane properties and the alteration of the lysosomal function by quercetin may not be neglected in these protective effects. The amplification of the photodamage by isoflavones is in sharp contrast with previous literature data demonstrating photoprotection by genistein. As a consequence, it may be concluded that an eventual antioxidant action of genistein may strongly depend on cells and photosensitizers. Furthermore such contrasting pro-versus anti-oxidant effects have to be taken into account when using flavonoid mixtures of plant extracts.     

Quercetin metabolism in vital and apoptotic human leukaemia cells

The metabolism of the flavonol quercetin in human leukaemia (HL-60) cells was investigated. The fluorescence that is elicited by quercetin upon binding to a target protein was quickly attenuated in vital cells, while apoptotic cells showed persistent fluorescence. The dynamics of induction and loss of fluorescence in the cells were quantified by flow cytometry. Several potential metabolites of quercetin, apart from isorhamnetin, had weak or no fluorogenic properties with test proteins. HPLC analysis showed that quercetin was metabolised to several substances, among them glycosylated metabolites. The loss of fluorescence in vital cells offers the unique opportunity to directly observe the metabolic conversion of quercetin in human cells.     

Bioflavonoid quercetin inhibits mitosis and apoptosis of glomerular cells in vitro and in vivo

Bioflavonoids have been regarded as therapeutic agents for a wide range of disease including inflammation. In this report, we investigated effects of bioflavonoid quercetin on mitosis and apoptosis of glomerular cells in vitro and in vivo. Serum-stimulated rat mesangial cells were treated with or without quercetin, and total cell number, percentages of mitotic cells, and incorporation of [(3)H]-thymidine were evaluated. All three assays showed that mitogenic activity of mesangial cells was markedly attenuated by quercetin. To examine the effect of quercetin on apoptosis, mesangial cells were pretreated with or without quercetin and stimulated by hydrogen peroxide or tumor necrosis factor-alpha. Hoechst staining and DNA ladder assay showed that both apoptotic responses were dramatically inhibited by quercetin. We further investigated effects of quercetin on in vivo mitosis and apoptosis of glomerular cells. Rats were administered with or without quercetin intraperitoneally, and nephrotoxic serum nephritis was induced. Immunohistochemical analyses showed that treatment with quercetin significantly reduced the number of proliferating cell nuclear antigen (+) cells and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (+) cells in the glomerulus. These data suggested that quercetin has the potential for inhibiting mitosis and apoptosis of glomerular cells both in vitro and in vivo.