Elsevier

Free Radical Biology and Medicine

Volume 31, Issue 9, 1 November 2001, Pages 1139-1146
Free Radical Biology and Medicine

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Contrasting influences of glucuronidation and O-methylation of epicatechin on hydrogen peroxide-induced cell death in neurons and fibroblasts

https://doi.org/10.1016/S0891-5849(01)00704-3Get rights and content

Abstract

The purpose of this study was to examine the comparative mechanisms by which the dietary form of the flavonoid epicatechin and its predominant in vivo metabolite, epicatechin glucuronide, influence oxidative stress-induced cell death in fibroblasts and neurons. The results demonstrate the contrasting influences of in vivo glucuronidation and methylation on the bioactivity of epicatechin.

Introduction

Many studies have demonstrated that flavonoids have mechanisms of action independent of their conventional hydrogen-donating free radical scavenging properties by modulating enzyme activities, interfering with pathways of intermediary metabolism, downregulating the expression of adhesion molecules, acting at various sites within signal transduction cascades, and mimicking substrates for various binding sites [1], [2], [3], [4]. The potential role flavonoids play in intracellular signaling events is becoming increasingly clear, especially with regard to the influence they have on protein kinases, phosphatidylinositol-3 kinase, and nuclear factor κ B [5], [6], [7]. Accumulating evidence also links flavonoids to interactions within mitogen-activated protein kinase (MAPK) signaling cascades [8], [9], [10]. Furthermore, catechins and specific flavonols have been reported to interact with proteins such as the mitochondrial ATPase [11], calcium plasma membrane ATPase [12], protein kinase A [13] and protein kinase C [5] through binding to the ATP binding site [14].

However, the precise mechanisms by which flavonoids exert their cytoprotection in vivo will depend on the extent to which they are conjugated and metabolized during absorption across the small intestine [15], in the liver [16], and in the colon. Flavonoids and their glycosides are subject to phase I and II metabolism in the small intestine and in the liver, and may be substrates for β-glucosidases, UDP-glucuronosyltransferases, and catechol-O-methyl-transferases [17], [18], [19], [20]. Indeed, various publications report the occurrence of glucuronidated, methylated glucuronidated, methylated, and sulphated products circulating in blood plasma and excreted with urine after consumption of flavonoid-rich diets [21], [22], [23], [24].

A clear understanding of potential mechanisms by which flavonoids exert their biological activities in vivo can only result from an investigation of the action of the in vivo flavonoid conjugates and metabolites as well as the commonly used dietary glycosides and aglycones. The purpose of this study was to examine the comparative mechanisms by which the dietary form of the flavonoid epicatechin (major component of wine, tea, cocoa, etc.) and its predominant in vivo metabolite, epicatechin glucuronide, influence oxidative stress-induced cell death in two systems: human dermal fibroblasts and primary cortical neurons.

Section snippets

Synthesis of epicatechin glucuronides and 3′-O-methyl epicatechin

The method employed was developed from reports reviewing the enzyme chemistry, enzyme kinetics, and biological properties of UDP-glucuronosyltransferases and the use of this enzyme for in vitro glucuronidation [25], [26]. Rat livers were washed in ice-cold isolation buffer (pH 7.4) containing PBS (50 mM), sucrose (0.32 M), EDTA (1 mM), and DTT (0.5 mM). Homogenization was then carried out in ice-cold buffer (pH 7.0) containing Tris (0.1 M), MgCl2.6H2O (10 mM) and DTT (0.5 mM) using a Potter

Results

Epicatechin glucuronides and 3′-O-methyl epicatechin, major in vivo metabolites of epicatechin, were synthesized and examined for their protective effects against oxidative stress-induced cell death in cortical neurons and dermal fibroblasts. The analysis of the products obtained was carried out by HPLC and nanoES-MS/MS. HPLC of analysis of the epicatechin glucuronide preparation revealed three major peaks (retention times: 31.9 min, 32.3 min, and 36.4 min). The peak at 36.4 min was identified

Discussion

Very few investigations have studied the influence of metabolism on the bioactivity of flavonoids [8], [27], [32], [33]. Thus most of the effects reported based on in vitro experiments cannot be extrapolated to in vivo situations. For example, one of the most intensively investigated flavonoids, quercetin, is often used as the aglycone in cell culture systems leading to biological activities such as modulation of the multidrug-resistance protein [34], induction of apoptosis [35], inhibition of

Acknowledgements

This research was supported by the Biotechnology and Biological Sciences Research Council (grant no. 18/D14751) and by a European Union Fifth Framework RTD Programme Grant (grant no. QLK4-1999-01590). We thank Professor Rex Tyrell for the human dermal fibroblasts.

References (51)

  • G.J Dutton et al.

    Assays for UDPglucuronyltransferase activities

    Methods Enzymol.

    (1981)
  • H Schroeter et al.

    Phenolic antioxidants attenuate neuronal cell death following uptake of oxidized low-density lipoprotein

    Free Radic. Biol. Med.

    (2000)
  • J.-H Moon et al.

    Identification of quercetin-3-O-β-glucorinoide as an antioxidative metabolite in rat plasma after oral administration of quercetin

    Free Radic. Biol. Med.

    (2001)
  • T Koga et al.

    Effect of plasma metabolites of (+)-catechin on monocyte adhesion to human aortic endothelial cells

    Am. J. Clin. Nutr.

    (2001)
  • G.M Raso et al.

    Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophages J774A.1

    Life Sci.

    (2001)
  • K Ishige et al.

    Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms

    Free Radic. Biol. Med.

    (2001)
  • H Wang et al.

    Structure-activity relationships of quercetin in antagonizing hydrogen peroxide-induced calcium dysregulation in PC12 cells

    Free Radic. Biol. Med.

    (1999)
  • A Saija et al.

    Flavonoids as antioxidant agentsimportance of their interaction with biomembranes

    Free Radic. Biol. Med.

    (1995)
  • J.H Medina et al.

    Neuroactive flavonoidsnew ligands for the Benzodiazepine receptor

    Phytomedicine

    (1998)
  • M.E Gerritsen et al.

    Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression

    Am. J. Pathol.

    (1995)
  • J Panes et al.

    Apigenin inhibits tumor necrosis factor-induced intercellular adhesion molecule-1 upregulation in vivo

    Microcirculation

    (1996)
  • E.J.R Middleton et al.

    The effects of plant flavonoids on mammalian cellsimplications for inflammation, heart disease and cancer

    Pharmacol. Rev.

    (2000)
  • C.A Musonda et al.

    Quercetin inhibits hydrogen peroxide (H2O2)-induced NF-κB DNA binding activity and DNA damage in HepG2 cells

    Carcinogenesis

    (1998)
  • T.H Tsai et al.

    Determination of unbound hesperetin in rat blood and brain by microdialysis coupled microbore liquid chromatography

    J. Food Drug Anal.

    (2000)
  • H Schroeter et al.

    Flavonoids protect neurones from oxidised low-density lipoprotein-induced apoptosis involving JNK, c-jun and caspase-3

    Biochem. J.

    (2001)
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