Fast track paperContrasting influences of glucuronidation and O-methylation of epicatechin on hydrogen peroxide-induced cell death in neurons and fibroblasts
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.
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