Physiologically based kinetic modelling based prediction of oral systemic bioavailability of flavonoids, their metabolites, and their biological effects
Boonpawa, Rungnapa - \ 2017
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Arjen Punt. - Wageningen : Wageningen University - ISBN 9789463430371 - 180
flavonoids - bioavailability - modeling - metabolites - quercetin - physiology - hesperidin - flavonoïden - biologische beschikbaarheid - modelleren - metabolieten - quercetine - fysiologie - hesperidine
Flavonoids, abundantly present in fruits and vegetables, have been reported to exert various positive health effects based on in vitro bioassays. However, effects detected in in vitro models cannot be directly correlated to human health as most in vitro studies have been performed using flavonoid aglycones at high concentration ignoring extensive metabolism of flavonoids in the human body. To better understand positive health effects of flavonoids in humans, it is of importance to gain insight in at which form and concentration flavonoids are present in the systemic circulation after consumption. This insight can be obtained using physiologically based kinetic (PBK) computer modeling. The results obtained show that PBK modeling provides a useful additional research tool for studies on the fate of flavonoids in the human body and can reveal at what oral dose levels of flavonoids in vitro positive health effects can be expected to occur in vivo, presenting opportunities that are not easily provided by other methods.
Dietary epicatechnin and quercetin in cardiovascular health and disease
Dower, J.I. - \ 2016
Wageningen University. Promotor(en): Daan Kromhout; Marianne Geleijnse, co-promotor(en): Peter Hollman. - Wageningen : Wageningen University - ISBN 9789462577862 - 164
cardiovascular disorders - cardiovascular diseases - epicatechin - quercetin - epidemiological surveys - genome analysis - chocolate - hart- en vaatstoornissen - hart- en vaatziekten - epicatechine - quercetine - epidemiologische onderzoeken - genoomanalyse - chocolade
Epidemiological studies showed that the consumption of flavonoid-rich foods such as cocoa and tea is associated with a lower risk of cardiovascular disease (CVD). Randomised controlled trials (RCTs) showed that cocoa and tea improved markers of cardiometabolic health including blood pressure, endothelial function, insulin resistance, arterial stiffness and inflammation.
Cocoa is particularly rich in the flavan-3-ol epicatechin and tea is the main dietary source of epicatechin and of the major flavonol quercetin. However, evidence on the individual roles of epicatechin and quercetin in the health effects of cocoa and tea is still scarce. Therefore, we estimated the strength of the association between epicatechin intake and CVD mortality in a prospective cohort study. Furthermore, we also investigated the effects of epicatechin and quercetin on markers of cardiometabolic health and gene expression, by means of two RCTs.
In Chapter 2, the association between epicatechin intake and CVD mortality was studied using data from the Zutphen Elderly Study, a cohort of 744 elderly Dutch men. During 25 years of follow-up, 329 men died from CVD and 148 from coronary heart disease (CHD). Results from this study showed that men in the highest tertile of epicatechin intake had a 38% lower risk of CHD mortality compared to men in the lowest tertile. For men with prevalent CVD, the risk of CVD mortality was 46% lower for men in the highest tertile of intake, compared to men in the lowest tertile. This is the first epidemiological study to have investigated the association between epicatechin intake and CVD mortality. Hence, more and larger cohort studies are required to confirm this association, possibly with a focus on populations with a high risk of CVD.
In Chapter 3, the chronic effects of pure epicatechin and quercetin on markers of cardiometabolic health were investigated by means of a RCT. Thirty-seven apparently healthy men and women aged 40–80 years consumed (-)-epicatechin (100 mg/d), quercetin-3-glucoside (160 mg/d), or placebo capsules for 4 weeks, in random order. Markers of cardiometabolic health were measured before and after each 4-week intervention. The results of this study showed that epicatechin improved insulin resistance and had a borderline significant effect on endothelial function. This suggests that epicatechin contributes to the cardioprotective effects of cocoa and tea, however, larger long-term RCTs are required to confirm these effects. Pure quercetin supplementation did not affect any of these markers of cardiometabolic health.
Using data from the same study, we investigated the effects of supplementation of pure epicatechin and quercetin on a comprehensive set of biomarkers of endothelial dysfunction and inflammation (Chapter 4). With the exception of sE-selectin (a biomarker of endothelial dysfunction), epicatechin supplementation did not beneficially influence any of the biomarkers, suggesting a lack of evidence for a role of epicatechin in inflammation. Quercetin also lowered sE-selectin as well as the inflammatory biomarker IL-1β and the overall z-score for inflammation. This suggests that quercetin may contribute to the cardioprotective effects of tea by reducing inflammation and possibly by improving endothelial function.
In the same study, the effects of pure epicatechin supplementation on whole genome gene expression profiles of circulating immune cells were also assessed (Chapter 5). Pure epicatechin supplementation modestly reduced gene expression related to inflammation signalling routes in circulating immune cells – routes which are known to play a role in cardiovascular health. However, there was no evidence that epicatechin affected pathways related to insulin resistance or endothelial function.
To directly compare the acute effects of pure epicatechin and epicatechin from dark chocolate on vascular function, we carried out an acute RCT in 20 apparently healthy men aged 40-80 years (Chapter 6). On three separate occasions, subjects consumed: 1) 70g dark chocolate (150 mg epicatechin) with two placebo capsules; 2) two pure epicatechin capsules (100 mg epicatechin) with 75g white chocolate and 3) two placebo capsules with 75g white chocolate (0 mg epicatechin). Endothelial function and arterial stiffness were measured before and two hours after each intervention. To determine epicatechin bioavailability, epicatechin metabolites were measured in blood samples taken at repeated intervals over a period of 8 hours. There was no significant difference in improvement in endothelial function or arterial stiffness between pure epicatechin and dark chocolate. There was also no difference in bioavailability of pure epicatechin and epicatechin from dark chocolate, when standardised per 100 mg of epicatechin. This suggests that epicatechin may contribute to the vascular effects of cocoa and that the bioavailability of pure epicatechin and epicatechin from dark chocolate is similar.
In the general discussion, the main findings of this thesis were first summarised. Methodological considerations related to cohort studies, such as the assessment of flavonoid intake and the possibility of residual confounding were also discussed. Issues related to the relevance of cardiometabolic markers in RCTs and the effect of cocoa flavan-3-ol bioavailability were addressed. Finally, suggestions for future research were put forward.
In conclusion, the results of this thesis suggest that epicatechin contributes to the cardioprotective effects of cocoa and tea. Epicatechin intake was inversely related to CHD mortality in elderly men, and to CVD mortality in men with prevalent CVD. The cardioprotective effects of epicatechin are likely mediated through improvements in insulin resistance and possibly endothelial function. In contrast, quercetin is unlikely to play a major role in the cardioprotective effects of tea. Results for quercetin from cohort studies are inconclusive, and based on the results of our chronic RCT, quercetin did not affect vascular function or insulin resistance, but may help to lower inflammation. Evidence of the role that individual flavonoids play in the aetiology of CVD is still limited. More studies with pure flavonoids are required to elucidate their role.
The influence of phase II conjugation on the biological activity of flavonoids
Beekmann, K. - \ 2016
Wageningen University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): L. Actis-Goretta. - Wageningen : Wageningen University - ISBN 9789462577640 - 171
flavonoids - biological activity - in vitro - biosynthesis - peroxisomes - microarrays - daidzein - genistein - oestrogen receptors - isoflavones - quercetin - kaempferol - serine proteinases - threonine - flavonoïden - biologische activiteit - in vitro - biosynthese - peroxisomen - microarrays - daidzin - genisteïne - oestrogeenreceptoren - isoflavonen - quercetine - kaempferol - serine proteïnasen - threonine
Flavonoid consumption is often correlated with a wide range of health effects, such as the prevention of cardiovascular diseases, neurodegenerative diseases, and diabetes. These effects are usually ascribed to the activity of the parent flavonoid aglycones, even though these forms of the flavonoids generally have a low systemic bioavailability. During uptake, flavonoids undergo phase II metabolism and are present in the systemic circulation nearly exclusively as conjugated metabolites. The aim of this thesis was to study the effect of conjugation on the biological activity of selected flavonoids towards different endpoints relevant for human health. To this end, conjugation with glucuronic acid was taken as the model type of conjugation because this modification is generally observed to be the most important metabolic conjugation reaction for flavonoids in man.
A review of scientific literature published until early 2012 reveals that metabolic conjugation can affect the biological activity of flavonoids in different ways. Conjugation can increase, decrease, inverse or not affect the biological activity, depending on the flavonoid, the type and position of conjugation, the endpoint studied, and the assay system used. Based on the literature reviewed it is concluded that the effect of conjugation has to be studied on a case-by-case basis.
As the research on the biological activity of biologically relevant flavonoid conjugates is often hampered by the generally low commercial availability and high prices of these conjugates, a simple and versatile method for the biosynthesis of metabolically relevant flavonoid conjugates is described. Using this method, relevant conjugates can be prepared from different flavonoid substrates in sufficient quantities for in vitro bioassays. Further, an efficient strategy for the identification of these flavonoid conjugates by LC-MS and 1H-NMR using MetIDB (Metabolite Identification Database), a publicly accessible database of predicted and experimental 1H-NMR spectra of flavonoids, is presented.
To study the effect of conjugation on the biological activities of flavonoids, several different assay systems and endpoints were used to study the activity of different flavonoids and their conjugates. The effects of quercetin, kaempferol, and their main plasma conjugates quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide (K-3G) on different endpoints related to peroxisome proliferator-activated receptor (PPAR)-γ were studied. PPAR-γ activation is reported to have positive health effects related to adipogenesis, insulin resistance and inflammation. The presented results show that the flavonoid aglycones increased PPAR-γ mediated gene expression in a stably transfected reporter gene cell line, and that glucuronidation diminished this effect. These observed increases in reporter gene expression were accompanied by increased PPAR-γ receptor-mRNA expression upon exposure to kaempferol, an effect that was also reduced by glucuronidation. Using the cell-free Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) it was demonstrated that, unlike the known PPAR-γ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the PPAR-γ receptor. Supporting the hypothesis that the tested compounds have a different mode of action from normal LBD agonism, quercetin appeared to synergistically increase the effect of rosiglitazone in the reporter gene assay. The modes of action behind the observed effects remain to be elucidated and might include effects on protein kinase activities affecting expression of the PPAR-γ receptor, or posttranscriptional modifications of PPAR-γ.
Another type of nuclear receptor known to be targeted by certain flavonoids are the estrogen receptor (ER)α- and ERβ. ERs are the main targets of estrogenic compounds, and upon their activation different transcriptional responses with opposite effects on cell proliferation and apoptosis are elicited; ERα activation stimulates cell proliferation, while ERβ activation causes apoptosis and reduces ERα mediated induction of cell proliferation. Using the MARCoNI assay, the intrinsic estrogenic effects of the two main dietary isoflavones daidzein and genistein, and their plasma conjugates daidzein-7-O-glucuronide and genistein-7-O-glucuronide on the ligand induced coregulator binding of ERα- and ERβ-LBD were studied and compared to the effect of the positive control 17β-estradiol (E2). The results show that the tested isoflavone compounds are less potent agonists of ERα- and ERβ-LBD than E2, although they modulate the LBD-coregulator interactions in a manner similar to E2. Genistein is shown to be a more potent agonist than daidzein for both receptor subtypes. While in the MARCoNI assay genistein had a strong preference for ERβ-LBD activation over ERα-LBD activation, daidzein had a slight preference for ERα-LBD activation over ERβ-LBD activation. Glucuronidation reduced the intrinsic agonistic activities of both daidzein and genistein to induce ERα-LBD and ERβ-LBD - coregulator interactions and increased their average half maximal effective concentrations (EC50s) by 8 to 4,400 times. The results presented further show that glucuronidation changed the preferential activation of genistein from ERβ-LBD to ERα-LBD and increased the preferential activation of daidzein for ERα-LBD; this is of special interest given that ERβ activation, which is counteracting the possible adverse effects of ERα activation, is considered one of the supposedly beneficial modes of action of isoflavones.
Many flavonoids are reported to be inhibitors of protein kinases. To study the effect of conjugation on the inhibition of serine/threonine protein kinases by flavonoids, kaempferol and its main plasma conjugate K-3G were selected as model compounds. Protein kinases are involved in a wide range of physiological processes by controlling signaling cascades and regulating protein functions; modulation of their activities can have a wide range of biological effects. The inhibitory effects of kaempferol, K-3G, and the broad-specificity protein kinase inhibitor staurosporine on the phosphorylation activity of recombinant protein kinase A (PKA) and of a lysate prepared from the hepatocellular carcinoma cell line HepG2 were studied using a microarray platform that determines the phosphorylation of 141 putative serine/threonine phosphorylation sites derived from human proteins. The results reveal that glucuronidation reduces the intrinsic potency of kaempferol to inhibit the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively. It is shown that the inhibitory activity of K-3G in the experiments conducted was not caused by deconjugation to the aglycone. Furthermore, the results show that kaempferol and K-3G, unlike the broad-specificity protein kinase inhibitor staurosporine, did not appear to inhibit all protein kinases present in the HepG2 lysate to a similar extent, indicating that kaempferol selectively targets protein kinases, a characteristic that appeared not to be affected by glucuronidation. The fact that K-3G appeared to be only a few times less potent than kaempferol implies that K-3G does not necessarily need to be deconjugated to the aglycone to exert potential inhibitory effects on protein kinases.
The results obtained in the present thesis support the conclusion that glucuronidation of flavonoids does not necessarily abolish their activity and that flavonoid glucuronides may be biologically active themselves, albeit at higher concentrations than the parent aglycones. In line with the conclusions from the earlier literature review, an updated literature review on the effect of conjugation on the biological activity of flavonoids concludes that that the effect of conjugation on the biological activity of flavonoids depends on the type and position of conjugation, the endpoint studied and the assay system used. Based on the results described and the literature reviewed in this thesis, several recommendations and perspectives for future research are formulated. Several methodological considerations are formulated that need to be taken into account when studying the biological activity of flavonoids and their conjugates to avoid confounding results. Further, the relevance of the gut microbiome for flavonoid bioactivity is highlighted, and considerations regarding the pharmacokinetics and pharmacodynamics of flavonoids in vivo are formulated. Altogether, it can be concluded that circulating flavonoid conjugates may exert biological activities themselves, and that understanding these is a prerequisite to successfully elucidate the mechanisms of action behind the biological activities linked to flavonoid consumption.
Unravelling mechanisms of dietary flavonoid-mediated health effects: effects on lipid metabolism and genotoxicity
Hoek-van den Hil, E.F. - \ 2015
Wageningen University. Promotor(en): Ivonne Rietjens; Jaap Keijer, co-promotor(en): Peter Hollman. - Wageningen : Wageningen University - ISBN 9789462573031 - 157
flavanoïden - flavonoïden - vetzuren - quercetine - flavonolen - lichaamsgewicht - lipidenmetabolisme - hart- en vaatziekten - lever - vetweefsel - gezondheid - genotoxiciteit - voeding - muizen - flavanoids - flavonoids - fatty acids - quercetin - flavonols - body weight - lipid metabolism - cardiovascular diseases - liver - adipose tissue - health - genotoxicity - nutrition - mice
Consumption of foods containing flavonoids is associated with a reduced risk of cardiovascular diseases (CVD), possibly by lipid-lowering effects. On the other hand, for one of these flavonoids, quercetin, also genotoxicity was shown especially in in vitro bioassays. Therefore, the first aim of this thesis was to identify mechanisms underlying potential beneficial health effects of flavonoids. The focus was on hepatic lipid metabolism and circulating lipids and a molecular and physiological approach was used. Secondly, we aimed to study the potential in vivo genotoxic effects of quercetin by transcriptome analyses in liver and small intestine, since these represent the tissues of first contact exposed to relatively high levels upon oral intake of flavonoids.
Circulating lipids are important CVD-related risk markers, which are in general determined with commercially available enzyme-based assays. However, the usual enzyme in these assays, peroxidase, has previously been reported to be inhibited by flavonoids. Therefore, we have studied in chapter 2 whether these assays can adequately be used in flavonoid research. We observed that various flavonoid aglycones interfere with peroxidase used in triglycerides (TG) and free fatty acids (FFA) enzymatic assays, reporting incorrect lower TG and FFA levels than actually present. Furthermore, addition of metabolites such as isorhamnetin or quercetin-3-O-glucuronide, the major metabolite of quercetin in human and rat plasma, to murine serum also resulted in a significant reduction of the detected TG levels, while a trend was seen towards reduced FFA levels. It can be concluded that when applying these biochemical assays, vigilance is needed and alternative analytical methods assessing FFA or TG levels should preferably be applied for studying the biological effects of flavonoids on TG and FFA levels.
In chapter 3 mechanistic and physiological effects of quercetin on hepatic lipid metabolism were studied. C57BL/6JOlaHsd male adult mice received a mild high-fat (30 en%) diet without or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Gas chromatography and 1H-NMR were used to quantitatively measure serum lipid profiles. Whole genome microarray analysis of liver tissue was used to identify potential mechanisms underlying altered circulating lipid levels by quercetin supplementation. Body weight, energy intake and hepatic lipid accumulation did not differ significantly between the quercetin and the control group. In serum of quercetin-fed mice, TG levels were decreased by 14% (p<0.001) and total poly unsaturated fatty acids (PUFA) levels were increased by 13% (p<0.01). Levels of palmitic acid, oleic acid, and linoleic acid were all decreased by 9-15% (p<0.05) in quercetin-fed mice. Both palmitic acid and oleic acid can be oxidized by omega-oxidation. Gene expression profiling showed indeed that quercetin increased hepatic lipid metabolism, especially omega-oxidation. At the gene level, this was reflected by the up-regulation of cytochrome P450 (Cyp) 4a10, Cyp4a14, Cyp4a31 and Acyl-CoA thioesterase 3 (Acot3). Two relevant regulators, cytochrome P450 oxidoreductase (Por, rate limiting for cytochrome P450 activities) and the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3) were also up- regulated in the quercetin-fed mice. We concluded that quercetin intake increased hepatic lipid omega-oxidation and lowered corresponding circulating lipid levels, which may contribute to potential beneficial effects of quercetin on CVD.
Subsequently, in chapter 4 effects of quercetin supplementation were studied in mice given a high-fat (40 en%) background diet. The set-up of the experiment was the same as in chapter 3, with the exception of the background diet that was used, which was different in fat content and composition. This high-fat diet-induced body weight gain, and serum and hepatic lipid accumulation, which are all known risk factors for CVD. The aim of this study was to investigate the effects and underlying molecular mechanisms of the effects of the flavonoid quercetin on hepatic lipid metabolism in mice given this high-fat diet background. C57BL/6JOlaHsd male adult mice received the high-fat diet without or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Body weight gain was 29% lower in quercetin fed mice versus control mice (p<0.01), while the energy intake was not significantly different. Quercetin supplementation lowered high-fat diet-induced hepatic lipid accumulation to 29% of the amount present in the control mice (p<0.01). 1H-NMR serum lipid profiling revealed that the supplementation also significantly lowered high-fat diet-induced increases in serum lipid levels. Global gene expression profiling of liver showed that cytochrome P450 2b (Cyp2b) genes, key target genes of the transcription factor Car, were down-regulated. However, the induction of omega-oxidation observed by quercetin supplementation to a mild high-fat (30en%) diet (chapter 3), was not observed this time with the high-fat (40en%) diet. Cumulatively, quercetin decreased high-fat diet-induced body weight gain, hepatic lipid accumulation and serum lipid levels. This was accompanied by regulation of cytochrome P450 2b genes in liver, which are considered to be under transcriptional control of CAR. The quercetin effects are likely dependent on the fat content and composition of the diet.
In chapter 5 we investigated whether flavonoids from other flavonoid subclasses can exert the same effects as we observed for quercetin. Effects of quercetin, hesperetin, epicatechin, apigenin and anthocyanins, in C57BL/6JOlaHsd male adult mice fed a high-fat diet for 12 weeks were compared, relative to a normal-fat diet. High-fat diet-induced body weight gain was significantly lowered by all flavonoids (17-29%), but most by quercetin. Quercetin significantly lowered high-fat diet-induced hepatic lipid accumulation (by 71%). High-fat diet-induced increases of mesenteric adipose tissue weight and serum leptin levels were significantly lowered by quercetin, hesperetin, and anthocyanins. Adipocyte cell size and adipose tissue inflammation were not affected.
The effects on body weight and adiposity could not be explained by individual significant differences in energy intake, energy expenditure, nor by differences in activity. Lipid metabolism was not changed as measured by indirect calorimetry or expression of known lipid metabolic genes in liver and white adipose tissue. Hepatic expression of Cyp2b9 was strongly down-regulated by all flavonoids. Overall, all five flavonoids lowered parameters of high-fat diet-induced adiposity, with quercetin being most effective.
Next to the beneficial health effects of flavonoids, the safety of flavonoids is under discussion, mainly because of potential genotoxic effects found for quercetin in vitro. Therefore, in chapter 6 the in vivo genotoxicity of this flavonoid was studied by transcriptome analyses in two tissues, small intestine and liver, where the highest exposure to quercetin is expected. This is especially of interest in view of high intake by widely available food supplements. Quercetin (0.33%) supplemented to a high-fat diet was administered to C57BL/6JOlaHsd male adult mice during 12 weeks. Serum alanine aminotransferase and aspartate aminotransferase levels revealed no indications for hepatotoxicity. General microarray pathway analysis of liver and small intestinal tissue samples showed no regulation of genotoxicity related pathways. In addition, analysis of DNA damage pathways in these tissues did also not point at genotoxicity. Furthermore, comparison with a published classifier set of transcripts for identifying genotoxic compounds did not reveal any similarities in the regulation of these classifier set by quercetin. Available microarray datasets of known genotoxic liver carcinogens, 2-acetylaminofluorene and aflatoxin B1 in mice were taken along as positive controls for comparison, and indeed showed genotoxic properties (regulation of genotoxic related genes) in the analyses. This transcriptomic analysis showed that supplementation with quercetin at ~350 mg/kg bw/day for 12 weeks did not induce genotoxicity in liver and small intestine.
In conclusion, we have shown in vivo efficacy of flavonoids reflected by effects on metabolic health parameters, including hepatic lipid metabolism. These effects on hepatic lipid metabolism seemed to be related or influenced by the transcription factor CAR. The dietary contexts appeared to modify the health effects. The five studied flavonoids in general showed the same effects, with quercetin being the most effective. No genotoxicity of quercetin was found by transcriptome analyses in liver and small intestine. Overall, we have obtained indications for beneficial health effects of flavonoids in mice, which makes it interesting to study if these effects can be extrapolated to humans to further explore their potential as functional compounds of dietary flavonoid intake.
Direct comparison of health effects by dietary polyphenols at equimolar doses in wildtype moderate high-fat fed C57BL/6JOlaHsd mice
Schothorst, E.M. van; Bunschoten, A. ; Hoevenaars, F.P.M. ; Stelt, I. van der; Janovska, P. ; Venema, D.P. ; Kopecky, J. ; Hollman, P.C.H. ; Keijer, J. - \ 2014
Food Research International 65 (2014)Part A. - ISSN 0963-9969 - p. 95 - 102.
adipose-tissue - body-weight - induced obesity - disease risk - quercetin - bioavailability - expression - flavonoids - leptin - (-)-epigallocatechin-3-gallate
Polyphenols generally show beneficial health effects upon supplementation in diet-induced obese rodent models, including reduced body weight gain and reduced levels of markers for cardiovascular diseases (CVD). However, there appear to be large differences between studies, which might be due to differences in models, strains, dietary background, or even concentration of polyphenol that is used. Therefore, we performed a systematic phenotypic evaluation of the effects of selected polyphenols in wildtype C57BL/6JOlaHsd mice. Epigallocatechin-gallate, quercetin, and resveratrol, representing three different phenolic classes, were each added in equimolar amounts (0.50% (w/w), 0.33%, and 0.25%, respectively) to a purified moderate high fat (30energy%) diet for 12 weeks. We studied the polyphenol-induced physiological and molecular effects between them and relative to the nonsupplemented control group during and at the end of the nutritional intervention. Results showed that these polyphenols were present in circulation, but did not induce beneficial health effects as analysed by oral glucose tolerance testing or serum adipokines and CVD-markers such as vascular adhesion molecules. Remarkably, transcriptomics of white adipose tissue showed overlapping sets of significantly differential transcript levels between these polyphenols; AMPK and Notch signalling were affected by these polyphenols. However, mitochondrial processes and mitochondrial density in this tissue did not differ between the polyphenols, which suggested that there was no direct effect on adipose tissue.
Investigating the Transport Dynamics of Anthocyanins from Unprocessed Fruit and Processed Fruit Juice from Sour Cherry (Prunus cerasus L.) across Intestinal Epithelial Cells
Toydemir, G. ; Boyacioglu, D. ; Capanoglu, E. ; Meer, I.M. van der; Tomassen, M.M.M. ; Hall, R.D. ; Mes, J.J. ; Beekwilder, J. - \ 2013
Journal of Agricultural and Food Chemistry 61 (2013)47. - ISSN 0021-8561 - p. 11434 - 11441.
red grape juice - protein interactions - cellular uptake - absorption - quercetin - dietary - caco-2 - bioavailability - consumption - glucosides
Anthocyanins can contribute to human health through preventing a variety of diseases. The uptake of these compounds from food and the parameters determining uptake efficiency within the human body are still poorly understood. Here we have employed a Caco-2 cell based system to investigate the transport of key antioxidant food components from sour cherry (Prunus cerasus L.) across the intestinal epithelial barrier. Anthocyanins and (-)-epicatechin were supplied in three contrasting matrices: fruit, processed fruit cherry juice, and polyphenolic fractions obtained by solid-phase extraction. Results show that both compound types behave differently. Fruit or juice matrices display comparable transport across the epithelial cell layer. The juice supplements sucrose and citric acid, which are regularly added to processed foods, have a positive effect on stability and transport. Polyphenolic fractions display a lower transport efficiency, relative to that of the fruit or juice, indicating the importance of food matrix components for intestinal absorption of polyphenols
Matrix Modulation of the Bioactivation of Estragole by Constituents of Different Alkenylbenzene-containing Herbs and Spices and Physiologically Based Biokinetic Modeling of Possible In Vivo Effects
Al-Husainy, W.A.A.M. ; Berg, S.J.P.L. van den; Paini, A. ; Campana, A. ; Asselman, M. ; Spenkelink, A. ; Punt, A. ; Scholz, G. ; Schilter, B. ; Adams, T.B. ; Bladeren, P.J. van; Rietjens, I. - \ 2012
Toxicological sciences 129 (2012)1. - ISSN 1096-6080 - p. 174 - 187.
p-form phenolsulfotransferase - potent inhibitors - interaction threshold - drug-metabolism - dna-adducts - mouse-liver - flavonoids - rats - 1'-hydroxyestragole - quercetin
The alkenylbenzene estragole is a constituent of several herbs and spices. It induces hepatomas in rodents at high doses following bioactivation by cytochrome P450s and sulfotransferases (SULTs) giving rise to the ultimate carcinogenic metabolite 1'-sulfooxyestragole which forms DNA adducts. Methanolic extracts from different alkenylbenzene-containing herbs and spices were able to inhibit SULT activity. Flavonoids including quercetin, kaempferol, myricetin, apigenin, and nevadensin were the major constituents responsible for this inhibition with Ki values in the nano to micromolar range. In human HepG2 cells exposed to the proximate carcinogen 1'-hydroxyestragole, the various flavonoids were able to inhibit estragole DNA adduct formation and shift metabolism in favor of glucuronidation which is a detoxification pathway for 1'-hydroxyestragole. In a next step, the kinetics for SULT inhibition were incorporated in physiologically based biokinetic (PBBK) models for estragole in rat and human to predict the effect of co-exposure to estragole and (mixtures of) the different flavonoids on the bioactivation in vivo. The PBBK-model-based predictions indicate that the reduction of estragole bioactivation in rat and human by co-administration of the flavonoids is dependent on whether the intracellular liver concentrations of the flavonoids can reach their Ki values. It is expected that this is most easily achieved for nevadensin which has a Ki value in the nanomolar range and is, due to its methyl ation, more metabolically stable than the other flavonoids.
Chemical Composition and Hepatoprotective Effects of Polyphenol-Rich Extract from Houttuynia cordata Tea
Tian, L. ; Shi, X.L. ; Yu, L.H. ; Zhu, J. ; Ma, R. ; Yang, X.B. - \ 2012
Journal of Agricultural and Food Chemistry 60 (2012)18. - ISSN 0021-8561 - p. 4641 - 4648.
ccl4-induced liver-damage - carbon-tetrachloride - antioxidant activity - lipid-peroxidation - aqueous extracts - free-radicals - in-vitro - quercetin - rats - cells
This study was designed to investigate the antioxidant activity, hepatoprotective effect, and phenolic composition of the ethyl acetate fraction (EAF) extracted from Houttuynia cordata tea. EAF was shown to exhibit strong ferric-reducing antioxidant power (FRAP) and scavenging activity against DPPH radical in vitro, and the antioxidant effects were further verified by suppressing CCl4-induced oxidative stress in mouse liver at three tested doses of EAF (250, 500, and 1000 mg/kg bw). Pretreatment with EAF (1000 mg/kg bw) prior to CCl4 administration significantly (p <0.001) decreased the CCl4-elevated levels of serum AST, ALT, alkaline phosphatase, total bilirubin, and hepatic MDA in mice and prevented the increases in GSH, SOD, and CAT caused by CCl4. HPLC analysis revealed that three predominantly polyphenolic compounds present in EAF were quercitrin (111.7 mu g/mg), quercetin (43.8 mu g/mg), and hyperoside (29.1 mu g/mg). These results combined with liver histopathology indicate that EAF possesses a significant protective effect against acute hepatotoxicity induced by CCl4, which may be due to the strong antioxidant activity of phenolic components.
Interference of flavonoids with enzymatic assays for the determination of free fatty acid and triglyceride levels
Hoek-van den Hil, E.F. ; Beekmann, K. ; Keijer, J. ; Hollman, P.C.H. ; Rietjens, I. ; Schothorst, E.M. van - \ 2012
Analytical and Bioanalytical Chemistry 402 (2012)3. - ISSN 1618-2642 - p. 1389 - 1392.
dietary flavonoids - thyroid peroxidase - quercetin - metabolites - rats - myeloperoxidase - polyphenols - inhibition - oxidation - cells
Flavonoids are bioactive food compounds with potential lipid-lowering effects. Commercially available enzymatic assays are widely used to determine free fatty acid (FFA) and triglyceride (TG) levels both in vivo in plasma or serum and in vitro in cell culture medium or cell lysate. However, we have observed that various flavonoids interfere with peroxidases used in these enzymatic assays, resulting in incorrect lower FFA and TG levels than actually present. Furthermore, addition of isorhamnetin or the major metabolite of the flavonoid quercetin in human and rat plasma, quercetin-3-O-glucuronide, to murine serum also resulted in a significant reduction of the detected TG levels, while a trend was seen for FFA levels. It is concluded that when applying these assays, vigilance is needed and alternative analytical methods, directly assessing FFA or TG levels, should be used for studying the biological effects of flavonoids on FFA and TG levels.
New Insights into an Ancient Antibrowning Agent: Formation of Sulfophenolics in Sodium Hydrogen Sulfite-Treated Potato Extracts
Narvaez Cuenca, C.E. ; Kuijpers, T.F.M. ; Vincken, J.P. ; Waard, P. de; Gruppen, H. - \ 2011
Journal of Agricultural and Food Chemistry 59 (2011)18. - ISSN 0021-8561 - p. 10247 - 10255.
tandem mass-spectrometry - chlorogenic acid - polyphenol oxidase - ascorbic-acid - quercetin - identification - inhibition - phenolics - oxidation - cysteine
The effect of sodium hydrogen sulfite (S), used as antibrowning agent, on the phenolic profile of potato extracts was investigated. This extract was compared to one obtained in the presence of ascorbic acid (A). In the presence of A, two major compounds were obtained, 5-O-caffeoylquinic acid (5-CQA) and 4-O-caffeoyl quinic acid. With S, their 2'-sulfo-adducts were found instead, the structures of which were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Also, for minor caffeoyl derivatives and quercetin glycosides, the corresponding sulfo-adducts were observed. Feruloyl and sinapoyl derivatives were not chemically affected by the presence of S. Polyphenol oxidase (PPO) was thought to be responsible for the formation of the sulfo-adducts. This was confirmed by preparing 2'-sulfo-5-O-caffeoyl quinic acid in a model system using 5-CQA, sodium hydrogen sulfite, and PPO. This sulfo-adduct exhibited a small bathochromic shift (¿max 329 nm) as compared to 5-CQA (¿max 325 nm) and a strong hypochromic shift with an extinction coefficient of 9357 ± 395 M–1 cm–1 as compared to 18494 ± 196 M–1 cm–1, respectively. The results suggest that whenever S is used as an antibrowning agent, the O-quinone formed with PPO reacts with S to produce sulfo-O-diphenol, which does not participate in browning reactions.
Role of Catechin Quinones in the Induction of EpRE-Mediated Gene Expression
Muzolf-Panek, M. ; Gliszczynska-Swiglo, A. ; Haan, L.H.J. de; Aarts, J.M.M.J.G. ; Szymusiak, H. ; Vervoort, J.J.M. ; Tyrakowska, B. ; Rietjens, I.M.C.M. - \ 2008
Chemical Research in Toxicology 21 (2008)12. - ISSN 0893-228X - p. 2352 - 2360.
tea polyphenol (-)-epigallocatechin-3-gallate - activated protein-kinases - green tea - anticancer properties - medicinal benefits - phenolic-acids - in-vitro - keap1 - nrf2 - quercetin
In the present study, the ability of green tea catechins to induce electrophile-responsive element (EpRE)-mediated gene expression and the role of their quinones in the mechanism of this induction were investigated. To this end, Hepa1c1c7 mouse hepatoma cells were used, stably transfected with a luciferase reporter gene under the expression regulation of an EpRE from the human NAD(P)H:quinone oxidoreductase 1 (NQO1) gene. The results obtained show that several, but not all, catechins tested are able to induce EpRE-mediated gene transcription, with epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG), both containing a pyrogallol and a galloyl moiety, being the most powerful inducers. Moreover, it was demonstrated that the EpRE-mediated response to catechins was increased in cells with reduced cellular glutathione (GSH) levels and decreased in cells with increased levels of GSH, corroborating a role for catechin quinones. The intrinsic capacity of catechins to form quinone type metabolites upon their oxidation was demonstrated using incubations of epigallocatechin (EGC) and EGCG with tyrosinase and the GSH-trapping method. Glutathione conjugates formed in these incubations were identified as 2¿-glutathionyl-EGC, 2¿,6¿-diglutathionyl-EGC, 2¿-glutathionyl-EGCG, and 2¿,6¿-diglutathionyl-EGCG, supporting the formation of quinone type metabolites involving especially the pyrogallol moiety of these catechins. Formation of the EGCG-quinone-glutathionyl adducts was also observed in the EpRE-LUX cellular system. This further supports the importance of the pyrogallol moiety for the quinone chemistry of the catechins. Finally, the presence of the pyrogallol moiety in the catechins also results in a relatively lower half-wave oxidation potential (E1/2) and calculated heat of formation (DHF) for conversion of the catechins to their corresponding quinones, pointing at an increased ability to become oxidized. Altogether, our studies reveal that catechins, especially those containing a pyrogallol moiety, induce EpRE-mediated detoxifying gene expression and that this induction is likely to be the result of their quinone chemistry.
Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ER/ER ratio
Sotoca Covaleda, A.M. ; Ratman, D. ; Saag, P. van der; Ström, A. ; Gustafsson, J.A. ; Vervoort, J.J.M. - \ 2008
Journal of Steroid Biochemistry and Molecular Biology 112 (2008)4-5. - ISSN 0960-0760 - p. 171 - 178.
estrogen-receptor-beta - luciferase reporter - nuclear - quercetin - line - recruitment - modulation - expression - genistein - prostate
This study investigates the importance of the intracellular ratio of the two estrogen receptors ER¿ and ERß for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERß has been postulated to play a role in modulating ER¿-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ER¿ to ERß is known to vary between tissues. Using human osteosarcoma (U2OS) ER¿ or ERß reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERß but also a higher maximal potential for activating ERß-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERß expression (T47D-ERß), the effect of a varying intracellular ER¿/ERß ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERß expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERß expression was increased. With increased expression of ERß the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ER¿/ERß ratio for the ultimate effect of (phyto)estrogens on cell proliferation.
In vivo relevance of in vitro detected estrogenic effects of food associated compounds
Veld, M.G.R. ter - \ 2008
Wageningen University. Promotor(en): Ivonne Rietjens; Tinka Murk. - [S.l.] : S.n. - ISBN 9789085852858 - 152
oestrogene eigenschappen - weekmakers - voedselverpakking - ftalaten - quercetine - oestrogenic properties - plasticizers - food packaging - phthalates - quercetin
The aim of the present thesis was to study the in vivo relevance of in vitro detected estrogenic effects of food associated compounds, with emphasis on prenatal exposure. The estrogenic potency of 21 food packaging-associated compounds was studied in ERα or ERβ transfected U2-OS (human osteoblasts devoid of endogenous estrogen receptors) cell lines. Six plasticizers and three anti-oxidants were slightly estrogenic in the ERα cells. BPA, NP, tris (2-ethylhexyl) trimellitate (TEHTM), propyl gallate and butylated hydroxy anisole (BHA) were estrogenic both in ERα and ERβ cells. These compounds appeared to be more estrogenic relative to estradiol (E2) in ERβ than in ERα cells. To study the in vivo relevance of these effects, in vivo biomarker-responses of BPA, NP, DEHA, DEHP, DIHP, p,p’-DDE and quercetin were studied in ER-Luc male mice. Of these seven compounds, BPA, DEHP and quercetin induced estrogenic effects after a single oral dosage at exposure levels 10-104 times higher than the established Tolerable Daily Intakes (TDI’s). It remains to be seen whether these margins are sufficiently high to allow the conclusion that these compounds are unlikely to represent a human health risk. As exposure to estrogenic compounds during developmental stages could be an important risk factor in developing hormone dependent cancers later in life, these seven compounds were studied in pregnant ER-Luc mice as well. After oral exposure of the mother animal the compounds were unable to significantly induce luc-activity in any of the tissues including fetuses. Unexpectedly however, NP, BPA and DIHP significantly lowered the placental luc-activity. The results indicate that at the current levels of exposure to food associated estrogenic compounds, direct estrogenic effects in the fetus are not expected. The mechanism and consequences of the significant luc-reduction in the placenta should be investigated to a further extent to elucidate its possible significance. Because of the absence of significant luc-induction in the fetuses, the fate and distribution of radioactively labeled E2, NP and p,p’-DDE were studied in pregnant C57black mice in order to investigate whether the compounds can actually reach the fetuses. E2 did not reach the fetus at a level above the detection limit, NP and p,p’-DDE levels were above the detection limit in fetuses exposed via the mother from GD8-16. Levels of E2 and NP detected in the placenta were significantly higher than those in the fetuses, up to five fold for E2 and three fold for NP, possibly due to prevention of transport of the compounds to the fetuses by placental binding proteins.
Based on the findings of the present thesis it was concluded that in spite of the in vitro estrogenicity of various food-born estrogens, in vivo estrogenic effects are not likely expected as estrogenicity in vivo is shown at levels 10-104 fold higher than the TDI’s for humans and placental binding proteins may reduce fetal exposure. However, the estrogenic compounds were given as a single acute dose, whereas TDI values and also risks associated with dietary exposure can be expected to result from chronic combined exposure. Therefore, it remains to be seen whether the estimated margins are sufficiently high. Future experiments are therefore needed that focus on long term and combined exposure. Another factor that remains to be solved relates to the fact that only in in vitro assays the relative ability of compounds to differentially activate ERα or ERβ can be quantified.
All together the results of the present thesis reveal that the extent at which in vitro estrogenic effects may result in in vivo estrogenicity may vary with the compound under study and should be evaluated on a case by case basis taking differences in absorption, distribution, metabolism and excretion, the intrinsic estrogenic potency of chemicals for either the ERα or ERβ and the differential levels of expression of these receptors in different tissues in vivo into account.
Biomarkers of quercetin-mediated modulation of colon carcinogenesis
Dihal, A.A. - \ 2007
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): R.H. Stierum; Ruud Woutersen. - [S.l.] : S.n. - ISBN 9789085046783 - 240
biochemische merkers - quercetine - colorectaal kanker - biochemical markers - quercetin - colorectal cancer
Colorectal cancer (CRC) is hypothesized to be prevented by intake of fruits and vegetables that contain anti-carcinogenic compounds, including theflavonoidquercetinthat is found in apples and onions. In this thesis,quercetin'smechanisms of cancer-preventive action were studied both in vitro and in vivo . The in vitro experiments were performed using the human Caco-2 cell line as a model for CRC, andquercetinstabilized byascorbatein the culture medium. Unexpectedly,ascorbate-stabilizedquercetinshowed enhancement of cellular processes involved in CRC-development, including stimulated cell proliferation, reduced cell differentiation and enhancement of pathways that stimulate cell survival. Furthermore,transcriptomicsshowed thatquercetindownregulatedexpression of genes involved intumorsuppression and phase II metabolism, andupregulatedoncogenes. Comparison with Caco-2 cells exposed toquercetinin the absence ofascorbateshowed the opposite, i.e. anti-carcinogenic effects by thisflavonoid. This led to the hypothesis thatquercetin-induced reactive oxygen species that eradicatetumorcells were scavenged by vitamin C, causingtumorcell survival. Withoutascorbate, these reactive oxygen species may be responsible for anti-carcinogenic effects, pointing to beneficial effects of supposed adverse reactive intermediates.Subsequently, the CRC-modulating potency ofquercetinand its conjugaterutinwere investigated in a rat model for CRC.Quercetin, but not its conjugaterutindecreased thetumorincidence, which was associated with the blood plasma levels of this anti-oxidant, but not reflected by the putativepreneoplasticbiomarker lesions, designated aberrant crypt foci. The combination oftranscriptomicsand proteomics showed thatquercetininhibited the potentiallyoncogenicmitogen-activated proteinkinase(Mapk) pathway and enhanced expression oftumorsuppressor genes, cell cycle inhibitors, and genes involved inxenobioticmetabolism. In addition,quercetinaffected the energy production pathways, by increasing mitochondrial fatty acid degradation, and inhibitingglycolysis. This observation provided a new hypothesis pointing at another anti-carcinogenic mechanism forquercetin, based on an alteration in routes for energy metabolism, shifting them infavorof non-tumorlike pathways like mitochondrial fatty acid degradation at the cost of thetumor-likeglycolyticpathway for cellular energy supply.Overall, the studies presented in the present thesis provided new hypotheses for the mode of action ofquercetinas an anti-tumoragent, but it appeared that the actual dose needed to exert this beneficial effect amounted to about 60 - 100 times the already relatively high prescribed dose forquercetinsupplements. Therefore, it is concluded that health claims on the use ofquercetinas an anti-cancer agent need better scientific support.
Towards functional effects of polyphenols : modulation of energy metabolism revealed
Boer, V.C.J. de - \ 2007
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Jaap Keijer; Peter Hollman. - [S.l.] : S.n. - ISBN 9789085046080 - 184
quercetine - weefselverdeling - colorectaal kanker - energiemetabolisme - quercetin - tissue distribution - colorectal cancer - energy metabolism
A diet rich in fruits and vegetables contains high levels of polyphenols (up to 1 gram per day). Epidemiological studies suggest that a high dietary intake of selected polyphenols can be protective against development of cardiovascular heart diseases in humans. In addition, mechanistic studies demonstrate that polyphenols possess beneficial properties ininvitro and animal model systems. Due to the possible beneficial health effects of polyphenols, they are currently being sold extensively as food supplements. However, the basis for most of the health claims attributed to polyphenols in food supplements is often very small. Our objective was to elucidate relevant mechanisms of action of selected polyphenols. We studied the tissue distribution and in vivo physiological effects of quercetin (a polyphenol abundant in the human diet) after chronic dietary exposure, followed by in vitro elucidation of possible biological mechanisms. We revealed lungs as novel tissue target of quercetin and demonstrated that dietary quercetin alters fatty acid catabolism pathways in rats. In addition, dietary quercetin lowered tumor incidence in the colon of rats in a model of colon carcinogenesis. Furthermore, a major in vivo metabolite of quercetin, quercetin 3-O-glucuronide, opposed the effect of quercetin aglycone on SIRT1 activation in vitro , whereas quercetin 3-O-glucuronide attenuated glucose utilization in cultured adipocytes in a similar fashion as quercetin aglycone. Although we used high dietary dosages of quercetin and further studies should elucidate physiological effects of a normal dietary intake of polyphenols, the experiments described in this thesis point to a possible beneficial effect of dietary polyphenols. However, as long as the molecular mechanisms in humans are unknown and the risk of increasing dietary intakes of polyphenols via food supplements is not thoroughly investigated, there is no scientific justification for supplementing the diet with large amounts of polyphenols. Nevertheless, our approach successfully identified modulation of energy metabolism by polyphenols as an important process involved in mediating the possible health effects associated with dietary polyphenol intake.
An in vitro and in silico study on the flavonoid mediated modulation of the transport of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through Caco-2 monolayers
Schutte, M.E. ; Freidig, A.P. ; Sandt, J.J.M. van de; Alink, G.M. ; Rietjens, I.M.C.M. ; Groten, J.P. - \ 2006
Toxicology and Applied Pharmacology 217 (2006)2. - ISSN 0041-008X - p. 204 - 215.
cancer resistance protein - multidrug-resistance - p-glycoprotein - cell-lines - biochanin-a - absorption - carcinogen - expression - quercetin - efflux
The present study describes the effect of different flavonoids on the absorption of the pro-carcinogen PhIP through Caco-2 monolayers and the development of an in silico model describing this process taking into account passive diffusion and active transport of PhIP. Various flavonoids stimulated the apical to basolateral PhIP transport. Using the in silico model for flavone, kaempferol and chrysoeriol, the apparent Ki value for inhibition of the active transport to the apical side was estimated to be below 53 muM and for morin, robinetin and taxifolin between 164 and 268 microM. For myricetin, luteolin, naringenin and quercetin, the apparent Ki values were determined more accurately and amounted to 37.3, 12.2, 11.7 and 5.6 microM respectively. Additional experiments revealed that the apical to basolateral PhIP transport was also increased in the presence of a typical BCRP or MRP inhibitor with apparent Ki values in the same range as those of the flavonoids. This observation together with the fact that flavonoids are known to be inhibitors of MRPs and BCRP, corroborates that inhibition of these apical membrane transporters is involved in the flavonoid-mediated increased apical to basolateral PhIP transport. Based on the apparent Ki values obtained, it is concluded that the flavonols, at the levels present in the regular Western diet, are capable of stimulating the transport of PhIP through Caco-2 monolayers from the apical to the basolateral compartment. This points to flavonoid-mediated stimulation of the bioavailability of PhIP and, thus, a possible adverse effect of these supposed beneficial food ingredients.
The future of toxicology: low-dose toxicology and risk-benefit analysis
Rietjens, I.M.C.M. ; Alink, G.M. - \ 2006
Chemical Research in Toxicology 19 (2006)8. - ISSN 0893-228X - p. 977 - 981.
omega-6 fatty-acids - fish-oil - colon carcinogenesis - cell-proliferation - tumor-growth - f344 rats - quercetin - cancer - hormesis - protein
Toxicology historically has been directed at studying the mechanisms of adverse effects of isolated compounds on living organisms at high levels of exposure, forming the basis for risk and safety assessment. One way to refocus and mobilize new research funds would be to better match the priorities in regulatory issues and direct the research within the field of toxicology more to low-dose toxicology and risk-benefit analysis. Low-dose toxicology can only be developed when taking into account mechanistic insight and will require risk-benefit analysis and a definition of interactions between compounds at realistic doses of exposure, especially in the case of dietary constituents. This is because the biological effects at low levels of exposure not only may be adverse but also can be beneficial depending on the target organ, the actual end point studied, the receptors activated, and/or the gene expression, protein, and metabolite patterns affected. Toxicologists have the tools and knowledge to study mechanisms of biological effects of chemicals on living organisms, and they should redirect their focus from looking only at adverse effects at high levels of exposure to characterizing the complex biological effects, both adverse and beneficial, at low levels of exposure. This may even result in the notion that beneficial effects can be the result of reaction pathways that are generally considered adverse and vice versa. Low-dose toxicology not only will provide a significant research challenge for the years ahead but also should contribute to better methods for low-dose risk assessment for complex mixtures of chemical compounds. This refocusing from high- to low-dose effects turns the field from a science focusing on adverse effects into a science studying the biological effects of chemical compounds on living organisms, taking into account the realization that the ultimate biological effect of a chemical may vary with its dose, the end point or target organ considered, and/or the combined exposure with other chemicals. By defining the effects of chemicals on living organisms at physiologically relevant exposure levels, toxicologists may contribute not only to better risk and safety assessment but also to preventive medicine, generating knowledge on possible adverse and also beneficial effects of chemicals. In addition, it will result in an approach for food safety assessment more in line with that for drug safety assessment taking the risk-benefit balance into consideration.
SIRT1 stimulation by polyphenols is affected by their stability and metabolism
Boer, V.C.J. de; Goffau, L. de; Arts, I.C.W. ; Hollman, P.C.H. ; Keijer, J. - \ 2006
Mechanisms of Ageing and Development 127 (2006)7. - ISSN 0047-6374 - p. 618 - 627.
cerevisiae life-span - calorie restriction - histone deacetylase - cell-survival - transcription factors - protein deacetylases - heart-disease - flavonoids - resveratrol - quercetin
Silent information regulator two ortholog 1 (SIRT1) is the human ortholog of the yeast sir2 protein; one of the most important regulators of lifespan extension by caloric restriction in several organisms. Dietary polyphenols, abundant in vegetables, fruits, cereals, wine and tea, were reported to stimulate the deacetylase activity of recombinant SIRT1 protein and could therefore be potential regulators of aging associated processes. However, inconsistent data between effects of polyphenols on the recombinant SIRT1 and on in vivo SIRT1, led us to investigate the influence of (1) stability of polyphenols under experimental conditions and (2) metabolism of polyphenols in human HT29 cells, on stimulation of SIRT1. With an improved SIRT1 deacetylation assay we found three new polyphenolic stimulators. Epigallocatechin galate (EGCg, 1.76-fold), epicatechin galate (ECg, 1.85-fold) and myricetin (3.19-fold) stimulated SIRT1 under stabilizing conditions, whereas without stabilization, these polyphenols strongly inhibited SIRT1, probably due to H2O2 formation. Using metabolically active HT29 cells we were able to show that quercetin (a stimulator of recombinant SIRT1) could not stimulate intracellular SIRT1. The major quercetin metabolite in humans, quercetin 3-O-glucuronide, slightly inhibited the recombinant SIRT1 activity which explains the lack of stimulatory action of quercetin in HT29 cells. This study shows that the stimulation of SIRT1 is strongly affected by polyphenol stability and metabolism, therefore extrapolation of in vitro SIRT1 stimulation results to physiological effects should be done with caution.
Mechanisms of toxic action of the flavonoid quercetin and its phase II metabolites
Woude, H. van der - \ 2006
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Gerrit Alink. - Wageningen : Ponsen & Looijen - ISBN 9789085043492 - 229
quercetine - werkwijze - metabolische detoxificatie - risicoschatting - quercetin - mode of action - metabolic detoxification - risk assessment
During and after absorption in the intestine, quercetin is extensively metabolised by the phase II biotransformation system. Because the biological activity of flavonoids is dependent on the number and position of free hydroxyl groups, a first objective of this thesis was to investigate the consequences of phase II metabolism of quercetin for its biological activity. For this purpose, a set of analysis methods comprising HPLC-DAD, LC-MS and 1 H NMR proved to be a useful tool in the identification of the phase II metabolite pattern of quercetin in various biological systems. These studies showed that the 3'- and 4'-hydroxyl groups of quercetin, (catechol hydroxyl groups) were important targets for methylation, sulfation and glucuronidation. Methylation of a catechol hydroxyl group of quercetin proved to decrease the pH-dependent radical scavenging capacity of the compound, both by increasing its pK a for deprotonation and by decreasing its electron-donating properties. Methylation of a catechol hydroxyl group had a similar effect as replacement of the hydroxyl group by a hydrogen atom. Regarding the pro-oxidant properties of quercetin, methylation of a catechol hydroxyl group of quercetin did not eliminate the pro-oxidant chemistry of quercetin, reflected in the formation of covalent adducts with glutathione upon oxidation of quercetin by horseradish peroxidase. However, methylated quercetin proved to form only 42% of the level of DNA adducts in exposed cells as compared to a similar amount of unconjugated quercetin, indicating that methylation of quercetin attenuates also this biological reactivity towards DNA.A second objective of this thesis was to obtain more insight into the possible toxic effects of quercetin by studying various mechanisms that might be relevant in the context of carcinogenesis. Quercetin appeared to have a biphasic effect on the proliferation of cancer cell lines expressing the estrogen receptor (ER). The stimulation of cancer cell proliferation was ER-dependent and appeared to occur at concentrations that are physiologically relevant in humans. With respect to the pro-oxidant activity of quercetin, peroxidase- and tyrosinase-type oxidative enzyme activity did not play a major role in the intracellular formation of covalent adducts of quercetin with DNA and protein, indicating that the formation of covalent adducts of quercetin with cellular macromolecules might also be relevant in cell types lacking oxidative enzyme activity. Furthermore, the covalent quercetin DNA adducts were of transient nature, which may either eliminate or attenuate the adverse effects of covalent DNA adduct formation. The studies presented in this thesis provided indications for the dualistic character of quercetin, regarding its role in the process of cancer development.
Radical Scavenging Capacity of Wine Anthocyanins Is Strongly pH-Dependent
Borowski, T. ; Tyrakowska, B. ; Oszmianski, J. ; Rietjens, I.M.C.M. - \ 2005
Journal of Agricultural and Food Chemistry 53 (2005)14. - ISSN 0021-8561 - p. 5526 - 5534.
antioxidant activity - flavonoids - quercetin - chemistry - salts
The radical scavenging capacity of red wine anthocyanins was quantified by the so-called TEAC assay with special emphasis on the influence of pH and conjugation on this activity. The pH appears to be a dominant factor in the radical scavenging capacity of wine anthocyanins, with higher pH values increasing this capacity significantly. On the basis of the pKa values for deprotonation and theoretical calculations, it could be concluded that the effect is due to an increase in intrinsic radical scavenging capacity upon deprotonation. The data also reveal that the reduction in radical scavenging activity of anthocyanins upon their conjugation can, at least in part, be ascribed to an increase in pKa values upon conjugation. Altogether, the results obtained provide molecular insight into factors that influence radical scavenging potential of anthocyanins and reveal that the radical scavenging-mediated supposed beneficial health effects of these wine pigments will be influenced by the pH of the surrounding matrix or tissue