Intracellular metabolism and bioactivity of quercetin and its in vivo metabolites

Abstract

Understanding the cellular effects of flavonoid metabolites is important for predicting which dietary flavonoids might be most beneficial in vivo. Here we investigate the bioactivity in dermal fibroblasts of the major reported in vivo metabolites of quercetin, i.e. 3'-O-methyl quercetin, 4'-O-methyl quercetin and quercetin 7-O-beta-D-glucuronide, relative to that of quercetin, in terms of their further metabolism and their resulting cytotoxic and/or cytoprotective effects in the absence and presence of oxidative stress. Uptake experiments indicate that exposure to quercetin led to the generation of two novel cellular metabolites, one characterized as a 2'-glutathionyl quercetin conjugate and another product with similar spectral characteristics but 1 mass unit lower, putatively a quinone/quinone methide. A similar product was identified in cells exposed to 3'-O-methyl quercetin, but not in the lysates of those exposed to its 4'-O-methyl counterpart, suggesting that its formation is related to oxidative metabolism. There was no uptake or metabolism of quercetin 7-O-beta-D-glucuronide by fibroblasts. Formation of oxidative metabolites may explain the observed concentration-dependent toxicity of quercetin and 3'-O-methyl quercetin, whereas the formation of a 2'-glutathionyl quercetin conjugate is interpreted as a detoxification step. Both O -methylated metabolites conferred less protection than quercetin against peroxide-induced damage, and quercetin glucuronide was ineffective. The ability to modulate cellular toxicity paralleled the ability of the compounds to decrease the level of peroxide-induced caspase-3 activation. Our data suggest that the actions of quercetin and its metabolites in vivo are mediated by intracellular metabolites.

Modulation of Pro-survival Akt/Protein Kinase B and ERK1/2 Signaling Cascades by Quercetin and Its in Vivo Metabolites Underlie Their Action on Neuronal Viability

“…In contrast, lower concentrations of quercetin resulted in a transient inhibition of pAkt, with levels of pAkt (Ser 473 ) and total Akt not significantly different from control levels at 6 h, despite a strong initial inhibition of pAkt. This transient effect could be due to reversible inhibition and/or metabolism of quercetin intracellularly, as we have shown previously (40). Although the two O-methylated metabolites of quercetin also induced reductions in basal phospho-Akt (Ser 473 ), the level of inhibition was less than that caused by quercetin and was not accompanied by changes in total Akt.…”

“…This inhibition appears to be directed toward the ATP-binding site of the kinase, and analogues of quercetin such as LY294002 have been developed as potent PI 3-kinase inhibitors (58). However, the inhibition of PI 3-kinase by quercetin in neurons may be influenced by potential intracellular metabolism known to occur in other cell systems (40) and also observed in cortical neurons. 2 Oxidative metabolism and glutathionylation of quercetin and its O-methylated metabolites may act to hinder or enhance the potency of PI 3-kinase inhibition, and consequently our data cannot directly be compared with those studies conducted in a cell-free environment (57,58).…”

“…These metabolic processes lead to relatively low amounts of quercetin and higher levels of the metabolites in the circulation. Furthermore, the cells in culture readily accumulate relatively high amounts of quercetin and its O-methylated metabolites, which result in further intracellular oxidative metabolism, glutathionylation, and conversion of O-methylated metabolites to free quercetin (40). Potential toxicity of flavonoids such as quercetin toward neuronal cells in vivo may be limited by the action of the blood-brain barrier preventing the entry of such compounds to the brain.…”

“…2 Oxidative metabolism and glutathionylation of quercetin and its O-methylated metabolites may act to hinder or enhance the potency of PI 3-kinase inhibition, and consequently our data cannot directly be compared with those studies conducted in a cell-free environment (57,58). The potential inhibition of neuronal PI 3-kinase by quercetin and its O-methylated metabolites and by the novel intracellular metabolite 2Ј-glutathionyl quercetin (40) is the subject of further investigation.…”

“…Quercetin, 3Ј-O-methyl quercetin (isorhamnetin) and 4Ј-O-methyl-quercetin (tamarixetin) were purchased from Extrasynthese (Genay Cedex, France). Quercetin-O-7-␤-Dglucuronide was synthesized as described previously (40). The antibodies used were anti-phospho-Akt (Ser 473 ) pAb and total Akt pAb (Cell Signaling Technology Inc.); anti-phospho-Akt (Thr 308 ) pAb, anti-phospho-BAD (Ser 136 ) pAb, and anti-BAD monoclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY); anti-phospho-CREB (Ser 133 ) pAb (Calbiochem, La Jolla, CA); anti-ACTIVE MAPK (ERK1/2) pAb and anti-ACTIVE JNK pAb (Promega, Madison, WI); total ERK2 pAb and total JNK1 pAb (Santa Cruz Biotechnology, Santa Cruz, CA).…”