Cardio-protective and Cytotoxic Effects of Quercetin on H9c2 CardiomyocytesAshley WillsonSchool of Science and Technology, Nottingham Trent University, Nottingham, UK

BACKGROUND AND PURPOSEQuercetin has previously been shown to have cardioprotective effects on cardiomyocytes. In this study we investigated the effect of quercetin on H2O2 treated H9c2 cells, as well as the possible cytotoxic effect of quercetin on H9c2 cells.

EXPERIMENTAL APROACHH9c2 cells were dosed with varying concentrations of quercetin and left to incubate for 24, 48 or 72 hours. Cell viability was assed using an MTT assay as well as morphology changes recorded using Coomassie blue staining. Oxidative stress was induced using 800µM of hydrogen peroxide. H9c2 cells were incubate with either 100µM or 30µM of quercetin for 24 hours and then exposed to the hydrogen peroxide for one hour. Cell viability was assessed using an MTT assay and morphology changes were recorded using Coomassie blue staining.

KEY RESULTSAfter a 48 and 72 hour incubation period, 100µM of quercetin reduced the percentage cell viability significantly when compared to the control. Lower concentrations did not reduce cell viability. 30µM and 100µM showed significant protection during the oxidative stress assay, with 30µM increasing the percentage cell viability significantly more than the high concentration.

CONCLUSIONS AND IMPLICATIONSQuercetin showed cardioprotective effects on hydrogen peroxide dosed cardiomyocytes. High concentrations of quercetin after 48 hours showed a decrease in cell viability. These results suggest quercetin has significant antioxidant properties, however its safety may need to be further assessed.

ABBREVIATIONSMTT, (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan), TCA, Tricyclic Amino Acid cycle, ATP, Adenosine Triphosphate, NAD, Nicotineamide Adenine Dinucleotide, ANT, Adenosine Nucleotide Transporter, DMSO, Dimethyl Sulfoxide, DMEM, Dulbecco’s Modified Eagle’s Medium.

IntroductionQuercetin belongs to a family of chemicals called flavonoids. Flavonoids are an expansive groups of chemicals (>4000) found in many plants, which as well as being involved in the regulation of auxins, also can vary the colour, taste and texture of plants. Quercetin was first isolated by Svent-Gyorgyi in 1936, and was identified to have an important role in capillary wall integrity and capillary resistance (Gabor, 1988). Flavonoids in general have seen a surge in popularity in both lay and scientific communities due to the emergence of certain beneficial effects ascribed to the family of chemicals, particularly to the possible correlation of a high quercetin diet and a low incidence rate of heart disease observed in populations who follow a Mediterranean diet, even though analysis of the diet indicated that it contained a high percentage of fats. One of the most well understood properties of quercetin is its anti-inflammatory effects.Flavonoids interfere with arachidonic acid synthesis by inhibiting the activity of lipoxygenases and cyclooxygenases, with the cyclooxygenase enzyme being responsible for the metabolism of arachidonic acid to prostaglandins, such as Thromboxane, a messenger responsible for stimulating platelet aggregation, and the enzyme lipoxygenase being responsible for the production of leukotrienes, which can act as inflammatory messengers. Overview on Heart Failure and Oxidative StressWithin the last 20 years it has been shown that patients suffering from heart failure have heightened levels of reactive oxidative species within cardiac tissue (Belch 1991, Mallat 1998, McMurry 1993) which is thought to be caused by an increase in cytosolic Na+ during heart

and then will oxidise NADH, whilst Ca2+ will enter the mitochondria via the Ca2+ uniporter and stimulate the tricyclic acid cycle (TCA), therefore increasing the production of NADH. Importantly this activation on the TCA also produces NADPH, which plays an important role in the maintenance of the reduced state of the ROS scavengers’ glutathione and thioredoxin, which play a key role in the scavenging of H2O2. During heart failure the high levels of cytosolic Na+

will impact on the mitochondrial Na+-Ca2+

exchanger, causing more Ca2+ efflux. Without the Ca2+ ions being able to activate the Ca2+

sensitive TCA enzymes, there is less NADH and NADPH being produced, decreasing the amount of antioxidants (Gauthier et al,. 2013). Reactive Oxygen Species can damage the cell by attacking several key parts of the cell including the lipid membrane, cell enzymes and DNA itself, causing apoptosis. Programed apoptosis can also occur via the mitochondrial pathway, due to DNA damage, which causes the release of cytochrome C into the cytosol and triggering programed cell death. (Rang and Dale 2007). Damage to the cell membrane releases arachidonic acid into the cytosol and increases the production of prostaglandins and leukotrienes, which can causing an increase in inflammation and damage the cell, and surrounding tissue (Park 2003).

Overview on Quercetin’s Role in Protection on Oxidative Stress Induced CellsRecent studies have aimed to investigate whether there is a link between diets high in flavonoids and a low incidence of heart disease and, if there is, what the cause is. Park in 2003 studied the effect of quercetin on cardiomyocyte reactive oxidation species induced apoptosis and concluded that quercetin seemed to show protective effects via the mitochondrial dysfunction and caspase pathway (Park 2003). They demonstrated that H2O2 mediated toxicity lead to DNA fragmentation within H9c2 cells,

failure. Rapid changes of heart pacing and rhythm require tight control over levels of ATP and Ca2+ within the mitochondria, in order to oxidise and reduce mitochondrial NADH redox states. ATP will enter the mitochondria via the adenine nucleotide transporter (ANT) and the enzyme ATP synthase

Caspases are a series of proteins formed within a cell that serve primarily as mediators of programed cell death, in event of something going wrong with the cell. There are two main pathways for the activation of caspases during apoptosis, intrinsic and extrinsic. The intrinsic pathway is mediated by mitochondrial dysfunction during cell stress, which releases cytochrome-c a component of the electron transport chain from the mitochondrial intermembrane space into the cytosol (Creagh 2014). During oxidative stress this occurs via the loss of the mitochondrial transmembrane potential (MBT), as well as facilitation via the apoptotic protein Bak. Bak can interact with the permeability transition pore complex as well as forming oligomers that function as channels for the cytosolic release of cytochrome-c (Park et al., 2003). The release of cytochrome-c into the cytosol triggers the rapid oligomerisation of apoptotic protease factor 1, which gathers caspase-9 into the apoptosome. Activation of caspase-9 allows it to cleave the inactive downstream caspases, including caspase-3, resulting in cell death (Creagh 2014). The extrinsic pathway is based off the activation of extracellular receptors, resulting in the activation of caspase-8, followed by caspase-3 (Creagh 2014).

This study was designed to look at quercetin, and specifically its protective

and caused an increase in cytosolic cytochrome c, indicating mitochondrial dysfunction. When tested with quercetin, they showed that quercetin would directly scavenge H2O2. Yokoo however also reported that quercetin inhibited the activator protein 1 (AP-1) pathway, part of the signalling pathway in H2O2 induced apoptosis (Yokoo and Kitamura 1997).

Medium (4.5g Glucose w/L-Glutamine) (DMEM), Foetal Bovine Serum (FBS), Trypsin/EDTA 1x, Penicillin and Streptomycin were purchased from Lonza group LTD (Slough, UK). All other chemicals were of an analytical grade. Quercetin was diluted using DMSO, which was present in all experiments, including the control to a final stock concentration on 100mM.

Cell CultureRat embryonic cardiomyocyte cells H9c2 were obtained from the European Collection of Animal Cell Cultures (Porton Down, Salisbury UK). The Cells were cultured in DMEM 4.5g glucose w/L-Glutamine solution which had been treated with FBS, penicillin and streptomycin. The cells were maintained within a humidified incubator (95% air 5% Co2 at 37◦C) until the cells were around 70-90% confluent. Cells were detached using 1x Trypsin/EDTA and subcultured to a 1:5 split. Every 5 days the cells would be sub-cultured into two flasks in preparation for cell plating after two days. Cells were observed every two days for signs of confluence, morphology changes and infection using an optical microscope at 400x magnification.

Cytotoxic AssayH9c2 Cells were plated into 24 well plates (15,000 cells per well) and left to incubate for 24 hours in 1ml of DMEM 4.5g glucose w/L-glutamine. A series of dilutions of the stock Quercetin (100mM in DMSO) were produced using DMEM as a diluent. 100 µM, 30 µM, 10 µM, 3 µM and 1 µM of Quercetin were

effect on cardiomyocytes during oxidative stress, to see whether the results from papers such as Yokoo and Kitamura are repeatable. The study will also look at any cytotoxic effects of quercetin in order to examine its safety and efficacy, especially given the circumstances that Quercetin supplements (100g-250g) are so readily available from popular high-street health stores.

Materials and MethodsMaterials: Quercetin, 1-(4, 5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), Methanol, Dimethyl Sulfoxide (DMSO) and Coomassie Blue were purchased from Sigma-Aldrich Company LTD (Poole, Dorset, UK). Dulbecco’s Modified Eagle’s

Oxidative Stress AssayH9c2 cells were plated into 24 well plates (15,000 cells per well) and left to incubate for 24 hours with 1ml of DMEM 4.5g glucose w/L-glutamine. 100 µM and 30 µM of Quercetin were diluted from the stock quercetin (100mM in DMSO) using DMEM as a diluent. After 24 hours the media was replaced with 0.5ml of fresh DMEM and two rows were dosed with 0.5 ml of 100 µm of quercetin and two rows were dosed with 0.5ml of 30 µM of quercetin. The other two rows were given 0.5ml of DMEM. The plates were left to incubate for 24 hours. In order to induce oxidative stress in the cells, H2O2 was used at a concentration of 800 µM, diluted from the stock concentration of 10M H2O2.

After the 24 hours one row which contained the 100 µM or 30 µM of Quercetin was dosed with 0.5 ml of media containing 800 µM of H2O2 and one of the

produced. After the 24 hour incubation period, the media was replaced with 0.5ml of fresh DMEM and each row was dosed with 0.5ml of their respective Quercetin concentration, including a control which was given 0.5 ml of DMEM instead of quercetin. The plates were then left to incubate for either 24 hours, 48 hours or 72 hours. In order to quantify cell viability the metabolic reduction of MTT to a coloured formazan product was tested. The cell plates would be then given 50µl of MTT (0.5mg/ml) and left to incubate for another hour. The liquid portion of the wells were then removed and 200 µl of DMSO was used to solubilise the coloured formazan. The 24 well plates were then measured at 570nm in a plate reading spectrophotometer, in order to read the absorbance of the solubilised formazan product.

Data AnalysisData analysis was conducted using one way ANOVA test with a post hoc Dunnett’s test (p < 0.5 was considered statistically significant). All data is presented as ± SEM.

ResultsCytotoxic Effects of QuercetinAs mentioned previously, the first part of this experiment was to look at the possible cytotoxic properties of Quercetin, given the research indicating that Quercetin might act as a pro-oxidant at higher concentrations (Schmalhausen, et al., 2007) (Laughton, et al., 1989). In this study we investigated the concentrations at which quercetin becomes cytotoxic, and whether the incubational period effects the cytotoxic effects. As seen in figure 1, 24 hour incubation had no effect on cytotoxicity, while a 48 and 72 hour incubation period reduced the percentage cell viability of the cells dosed with 100µM of quercetin. Looking at figure 2, it is clear to see that in all three incubational periods there has been a decrease in proliferation between the control group, and the group

rows that was incubated with media alone was dosed with 0.5ml of the hydrogen peroxide media. One row in each plate was only given 0.5 ml of standard media to act as the control. In order to quantify cell viability the metabolic reduction of MTT to a coloured formazan product was tested. The plates were left to incubate for 1 hour, then the plates were dosed with 50 µl of MTT (0.5mg/ml) and left to incubate for an hour. The liquid part of the wells was then removed and 200 µl of DMSO was used to solubilise the coloured formazan product. The 24 well plates were then measured at 570nm in a plate reading spectrophotometer, in order to read the absorbance of the solubilised formazan product.

Coomassie StainingAfter the plates had been measure the DMSO was removed and 500µl of 90% methanol (-20◦C) was added to each well. The plates were left at -20◦C for 10 minutes. After the 10 minutes the methanol was removed and 300µl of Coomassie blue was added to each well. The plates were then left for 5 minutes. The plates were then washed 3 times with distilled water and left to bench dry overnight. Photographs were then taken using an optical microscope at 400x magnification.

dosed with 100µM. It appears then that 100µm can reduce cell viability. As a result of the MTT assay, we also found that 1µM and in the case of 24 hour incubation, 3µM of quercetin, can increase the percentage cell viability.

`

Figure 2 Coomassie blue staining of quercetin cytotoxicity plates. All photos taken at 400x

Figure 1 Cell viability of H9c2 cells after 24, 48 or 72 hour incubation periods, determined with an MTT assay. All results are displayed as a % of the control, with ±SEM of n=3. *p <0.05 versus untreated control cells.

membrane, however there were more viable cells, and the nucleus of the cells were not as condensed.

magnification.

Cardioprotective Effects of Quercetin on Oxidative Stress induced CardiomyocytesThe main aim of this study was to further explore the antioxidant properties of quercetin, and to investigate whether these properties extend to cardiomyocytes as well. To induce oxidative stress on cells, 800µM of H2O2 was used to replicate the effect of heart failure on reactive oxygen species (ROS) production. In order to assess the protective effect on cell viability, an MTT assay was performed. Looking at figure 3, we can see that by pre-dosing the cells with 100µM of quercetin and 30µM of quercetin protected the cells from oxidative stress by a significant amount. The results from this experiment seem to indicate that 30µM is more protective than 100µM. Figure 4 shows the stained cells of both H2O2 treated cells and H2O2 + quercetin. The H2O2 cells showed a definite decrease in the volume of cells within the wells, and also the typical degradation of the plasma membrane, along with the heavily condensed nucleus. H2O2 + quercetin showed that there was still a degradation of the plasma Interestingly however H2O2 also attacks the phospholipid membrane and release arachidonic acid, which when metabolised can lead to inflammation and cell death. H2O2

can also cause DNA condensation and fragmentation, as shown by Park in 2003. Park also showed that H2O2 also lead to Caspase-3 activation. Caspase-3 is well known in its central role in programed cell death (Vagner 2015). Park showed that incubating H9c2 cells with H2O2 caused mitochondrial dysfunction, causing a release of cytochrome c, which activated the cascade of caspases, starting with caspase-9, leading to caspase-3 and ending in cell death. Park showed that quercetin could decrease the cytosolic levels of Bak, a protein known to facilitate the release of cytochrome c from the mitochondria either by interacting with

Figure 3 Cell viability of H9c2 induced into oxidative stress with 800µM of H2O2, when pre-dosed with quercetin for 24 hours, determined with an MTT assay. All results are calculated as a percentage of the control, ±SEM n=3. *p<0.05 versus H2O2 alone.

Figure 4 Coomassie Blue staining of quercetin oxidative stress plates. All photos taken at 400x magnification.

DiscussionCardioprotective Effects of Quercetin In this experiment we used H2O2 oxidative stress. In vivo, H2O2 is produced mainly from the superoxide anion and dismutase of cells (Park et al., 2003), as well as the NADPH oxidase involved in the tricyclic amino acid cycle as mentioned previously. H2O2 is normally easily scavenged by catalase and GSH-PX (Park et al., 2003) although the levels of antioxidants have been reported to be lower than other organs within the body Angeloni et al.,

the permeability transition pore complex, or by forming oligomers which can acts as channels for cytochrome c. With this data Park posited that the cardioprotective effects of quercetin was down not only to its ability to directly scavenge H2O2, but also its ability to down-regulate the expression of apoptotic proteins Bak and Bcl-2 (Park 2013). This data is further reinforced by Angeloni, who found a similar reduction in caspase-3 activity when H2O2 induced cells were exposed to quercetin (Angeloni et al., 2007). However Angeloni went further, and looked at the effect of quercetin on the expression of MAPK and P13K, two super-families which play a critical role in cell growth and death. They found that both P13K and MAPK were cardioprotective in terms of H2O2 induced oxidative stress, and when H9c2 cells were first dosed with quercetin and then H2O2,

there was an increase of Akt, a downstream effector of the P13K superfamily, and an increase in the activation of the ERK1/2 cascade, a subfamily of the MAPK group. Akt is an anti-apoptotic protein that phosphorylates at the nucleus, inhibiting transcription factor activity, thereby reducing the expression of pro-apoptosis agents. Erk1/2, downstream kinases phosphorylated by MAP-kinase 1 and 2, have been shown to become activated during cardiac hypertrophy due to an increase in activation of MEK1 and MEK2. Erk1 and 2 have been shown to have protective effects within cardiomyocytes, although the mechanism of which is still unclear. Some reports have shown that the Erk1 and 2 cascade’s interaction with the cyclooxygenase-2 enzyme might have a possible protective response. alone and with quercetin, there is a definite reduction in the condensation of the nucleus in the cells treated with quercetin when compared to hydrogen peroxide alone. Given that the conversion of MTT to a coloured formazan product is mediated by the mitochondria it is only possible to state that

2007).Given quercetins previously stated relationship with arachidonic acid, this theory is of particular interest, as the connection between Erk1/2 cascades and quercetin is palpable here. Other studies have also shown that Erk1/2 activation has resulted in increased expression of FLICE (FADD-like interleukin 1β-converting enzyme) inhibitory protein, a known inhibitor of the caspase cascade (Bueno et al., 2002). There have also been some studies which have found that during heart failure there is an increase in MAPK activation, however the level or Erk activation remained the same. Given the possible relationship between Erk and anti-caspase activity, this is an interesting area which could be explored further (Bueno et al., 2002). They posited that quercetin’s antioxidant activity is exerted in two ways, the first by its innate scavenging ability of ROS, and the second by its ability to modulate intracellular signalling pathways, which Angeloni suggest is responsible for the decrease in caspase activity (Angeloni et al., 2007).The results from this current experiment reaffirm the results of both Park and Angeloni. We found that quercetin did seem to protect H9c2 cells from oxidative damage in the form of H2O2. Pre-incubation of H9c2 cells with 30µM of Quercetin for 24 hours before the oxidative stress assay gave results of almost triple the cell viability when compared to H2O2 alone, whereas when the cells were pre-incubated with a higher concentration of 100µM of quercetin instead, the increase in cell viability was around double that when compared to H2O2 alone. The reasoning behind the difference in viability is beyond the scope of this investigation, however it is possible to assume it might be due to the somewhat toxic effects of higher concentrations of quercetin. Looking at

quercetin increased cell viability in terms of mitochondria alone, however given the effect of hydrogen peroxide on mitochondria dysfunction and the subsequent release of cytochrome-c, the results gained from this investigation are still relevant. It should be noted that a study in 2005 found that quercetin and other flavonoids were able to reduce MTT themselves without the presence of living cells (Liang et al., 2005), however the amount of was negligible when compared to the conversion of MTT using mitochondria, so the results from this experiment were unlikely to be affected.

Cytotoxic Effects of QuercetinThe other half of this investigation was to first look at the possible cytotoxic effects of quercetin, especially given the circumstances that quercetin is readily and easily available as a supplement from popular high street health stores. We looked at an array of different concentrations of quercetin over 3 different incubation periods. The results showed a decrease in cell viability only after 48 hours and only with concentrations of quercetin at 100µM. There was a similar decrease in cell viability after 72 hours as well. Interestingly 24 hours did not produce any decrease in cell viability at any concentration however at a concentration of 1µM and 3µM quercetin seemed to increase cell viability significantly when compared to the control.It is known that when quercetin reduces a reactive oxygen species, it itself becomes oxidised into a variety of different metabolites, one of with, a two electron oxidation of quercetin produces quercetin-quinone, which also has four tautomeric forms (Jorgensen et al., 1998) (Metodiewa et al., 1999). Quinones are well known to be able to produce toxic effects, specifically against thiols, arylating them. Specifically quercetin-quinone is reported to be particularly toxic towards glutathione, an important antioxidant found within most

the morphology changes in the cells treated with quercetin and hydrogen peroxide, we can see that there is still degradation of the phospholipid bilayer, indicating that the protection caused by quercetin is indeed effected through its ability to reduce the release of mitochondrial cytochrome-c and to reduce the amount of DNA fragmentation, as seen in Park (Park 2003). Indeed looking at the difference in morphology between H2O2

compounding the pre-existing issue. Quercetin-quinone can also affect other protein thiols within the cell, effecting membrane permeability (Yen et al., 2003) and enzymes containing an SH- group (Boots et al., 2002). This data however doesn’t explain why quercetin would decrease the viability in cells that haven’t been exposed to ROS, unless the 48 and 72 hour incubation periods were enough to allow quercetin to scavenge any small amount of ROS already within the cell.There is a second reported toxic effect of quercetin, which is that it is geneotoxic, causing DNA lesions, aberrations, single strand breaks and point mutations (Silva et al., 2000). However there is little research done in this area, and there is some reports that the mutagenic effect of quercetin is also mediated by quinone (Boots et al., 2008).Clearly quercetin does seem to produce some toxic effects in vitro, confirmed by this investigation’s MTT assay, however most research at the moment seems to be focused on quercetin’s protective effects, with little done in regards to its toxicity, however recent interest into quercetin anti-proliferative effects might yield more information in this area.

ConclusionThe aim of this investigation was to look at the emerging interest in flavonoids and their possible cardioprotective effect in

cells (Awad et al., 2002). This leads to a particularly troublesome issue, known as the quercetin paradox. In quercetin’s role as an antioxidant, it will eventually produce toxic metabolites that can act as pro-oxidants, scavenging of ROS and two in quercetin’s ability to activate anti-apoptotic cascades within the cell, reducing mitochondrial dysfunction induced apoptosis. The next steps for quercetin related protections studies would be to look at intercellular mechanisms in relation to quercetin in more detail, particularly how quercetin effects cytochrome-c and whether or not it is dependent on Akt phosphorylation or the Erk cascade. This would easily be done using an ELISA assay for qualitative data, followed by a densitometry analysis for quantities analysis. Furthermore there is a definite lack of in vivo studies when it comes to quercetin and indeed flavonoids in general. An investigation using animal models of heart disease and occlusion might provide invaluable data regarding quercetin’s efficacy within living systems. Given the lack of clear understanding on how Erk1 and 2 activation can be protective, more research is needed in this area as well, especially with the possible connection between COX 2, quercetin and Erk1/2 cascades. Certainly more data is needed in regards to quercetin’s toxicity. The data returned from this assay does indicate that quercetin does seem to effect mitochondrial viability at least at high concentrations and long incubational periods. Again this area would benefit from immunocytochemical assays, in order to look at the specific cascades resulting in cell death.

References

Angeloni, C, Spencer, JPE, Leoncini, E, Biagi, PL & Hrelia, S (2007). Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress. Biochimie 89: 73-82,http://dx.doi.org/10.1016/j.biochi.2006.0

regards to post heart failure oxidative stress. The results found in this experiment concurred with current literature that 30µM of quercetin can protect cardiomyocytes from H2O2

induced oxidative stress. As part of the experiment, we also looked at the possible cytotoxic effects of quercetin and found that after 48 hours of incubation and above, 100µM of quercetin significantly reduced the cell viability of cardiomyocytes, although the mechanism of action for this remains to be understood. The mechanism of action for the cardioprotective effects of quercetin was beyond the scope of this study, however given the recent literature reviewed, and the preliminary qualitative data from this experiment, it would seem that quercetin’s antioxidant effects are two-fold; one in its direct Boots, AW, Haenen, GR, den Hartog, GJ & Bast, A (2002). Oxidative damage shifts from lipid peroxidation to thiol arylation by catechol-containing antioxidants. Biochim Biophys Acta 1583: 279-84, S1388198102002470 [pii].

Boots, AW, Haenen, GRMM & Bast, A (2008). Health effects of quercetin: From antioxidant to nutraceutical. Eur J Pharmacol 585: 325-37, http://dx.doi.org/10.1016/j.ejphar.2008.03.008.

Bueno, OF, Molkentin, JD (2002). Involvement of Extracellular Signal-Regulated Kinases 1/2 in Cardiac Hypertrophy and Cell Death. Circulation Research 91: 776-81, 10.1161/01.RES.0000038488.38975.1A.

Creagh, EM (2014). Caspase crosstalk: integration of apoptotic and innate immune signalling pathways. Trends Immunol 35: 631-40, http://dx.doi.org/10.1016/j.it.2014.10.

9.006.

Awad, HM, Boersma, MG, Boeren, S, van Bladeren, PJ, Vervoort, J & Rietjens, IMCM (2002). The Regioselectivity of Glutathione Adduct Formation with Flavonoid Quinone/Quinone Methides Is pH-Dependent. Chem Res Toxicol 15: 343-51, 10.1021/tx010132l.

Belch, JJ, Bridges, AB, Scott, N & Chopra, M (1991). Oxygen free radicals and congestive heart failure. British Heart Journal 65: 245-8, 10.1136/hrt.65.5.245.

B: Biointerfaces 45: 108-11, http://dx.doi.org/10.1016/j.colsurfb.2005.07.014

kaempferol changes only the flavonoid C-ring. Free Radic Res 29: 339-50, 10.1080/10715769800300381.

Laughton, MJ, Halliwell, B, Evans, PJ, Robin, J & Hoult, S (1989). Antioxidant and pro-oxidant actions of the plant phenolics quercetin, gossypol and myricetin: Effects on lipid peroxidation, hydroxyl radical generation and bleomycin-dependent damage to DNA. Biochem Pharmacol 38: 2859-65, http://dx.doi.org/10.1016/0006-2952(89)90442-5.

Mallat, Z, Philip, I, Lebret, M, Chatel, D, Maclouf, J & Tedgui, A (1998). Elevated Levels of 8-iso-Prostaglandin F2a in Pericardial Fluid of Patients With Heart Failure : A Potential Role for In Vivo Oxidant Stress in Ventricular Dilatation and Progression to Heart Failure. Circulation 97: 1536-9, 10.1161/01.CIR.97.16.1536.

McMURRAY, J, CHOPRA, M, ABDULLAH, I, SMITH, WE & DARGIE, HJ (1993). Eur Heart J 14: 1493-8, 10.1093/eurheartj/14.11.1493.

Metodiewa, D, Jaiswal, AK, Cenas, N,

004.

Duarte Silva, I, Gaspar, J, Gomes da Costa, G, Rodrigues, AS, Laires, A & Rueff, J (2000). Chemical features of flavonols affecting their genotoxicity. Potential implications in their use as therapeutical agents. Chem Biol Interact 124: 29-51, http://dx.doi.org/10.1016/S0009-2797(99)00139-8.

Gábor, M (1988). Szent-Györgyi and the bioflavonoids: new results and perspectives of pharmacological research into benzo-pyrone derivatives. Commemoration on the 50th anniversary of the award of the Nobel Prize. Prog Clin Biol Res 280: 1-15.

Gauthier, L, Greenstein, J, O’Rourke, B & Winslow, R (2013). An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure. Biophys J 105: 2832-42,http://dx.doi.org/10.1016/j.bpj.2013.11.007.

Jørgensen, LV, Cornett, C, Justesen, U, Skibsted, LH & Dragsted, LO (1998). Two-electron electrochemical oxidation of quercetin and

Schmalhausen, EV, Zhlobek, EB, Shalova, IN, Firuzi, O, Saso, L & Muronetz, VI (2007). Antioxidant and prooxidant effects of quercetin on glyceraldehyde-3-phosphate dehydrogenase. Food and Chemical Toxicology 45: 1988-93, http://dx.doi.org/10.1016/j.fct.2007.04.015.

Vagner, T, Mouravlev, A & Young, D (2015). A novel bicistronic sensor vector for detecting caspase-3 activation. J Pharmacol Toxicol Methods 72: 11-8, http://dx.doi.org/10.1016/j.vascn.2014.

Dickancaité, E & Segura-Aguilar, J (1999). Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radical Biology and Medicine 26: 107-16, http://dx.doi.org/10.1016/S0891-5849(98)00167-1.

Ossola, B, Kääriäinen, TM, Raasmaja, A & Männistö, PT (2008). Time-dependent protective and harmful effects of quercetin on 6-OHDA-induced toxicity in neuronal SH-SY5Y cells. Toxicology 250: 1-8,http://dx.doi.org/10.1016/j.tox.2008.04.001.

Park, C, So, H, Shin, C, Baek, S, Moon, B, Shin, S, et al. (2003). Quercetin protects the hydrogen peroxide-induced apoptosis via inhibition of mitochondrial dysfuntion in H9c2 cardiomyoblast cells. Biochem Pharmacol 66: 1287-95, http://dx.doi.org/10.1016/S0006-2952(03)00478-7.

Peng, L, Wang, B & Ren, P (2005). Reduction of MTT by flavonoids in the absence of cells. Colloids and Surfaces

11.006.

YEN, G, DUH, P, TSAI, H & HUANG, S (2003). Pro-oxidative Properties of Flavonoids in Human Lymphocytes. Biosci Biotechnol Biochem 67: 1215-22, 10.1271/bbb.67.1215.

Yokoo, T, Kitamura, M (1996). Dual regulation of IL-1 beta-mediated matrix metalloproteinase-9 expression in mesangial cells by NF-kappa B and AP-1. Am J Physiol 270: F123-30.