Chemico-Biological Interactions 160 (2006) 193–203 Consequences of quercetin methylation for its covalent glutathione and DNA adduct formation Hester van der Woude a , Marelle G. Boersma a , Gerrit M. Alink a , Jacques Vervoort b , Ivonne M.C.M. Rietjens a,c,∗ a Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands b Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands c WU/TNO Center for Food Toxicology, P.O. Box 8000, 6700 EA Wageningen, The Netherlands Received 20 September 2005; received in revised form 8 December 2005; accepted 9 December 2005 Available online 3 March 2006 Abstract This study investigates the pro-oxidant activity of 3 ′ - and 4 ′ -O-methylquercetin, two relevant phase II metabolites of quercetin without a functional catechol moiety, which is generally thought to be important for the pro-oxidant activity of quercetin. Oxidation of 3 ′ - and 4 ′ -O-methylquercetin with horseradish peroxidase in the presence of glutathione yielded two major metabolites for each compound, identified as the 6- and 8-glutathionyl conjugates of 3 ′ - and 4 ′ -O-methylquercetin. Thus, catechol-O-methylation of quercetin does not eliminate its pro-oxidant chemistry. Furthermore, the formation of these A-ring glutathione conjugates of 3 ′ - and 4 ′ -O-methylquercetin indicates that quercetin o-quinone may not be an intermediate in the formation of covalent quercetin adducts with glutathione, protein and/or DNA. In additional studies, it was demonstrated that covalent DNA adduct formation by a mixture of [4- 14 C]-3 ′ - and 4 ′ -O-methylquercetin in HepG2 cells amounted to only 42% of the level of covalent adducts formed by a similar amount of [4- 14 C]-quercetin. Altogether, these results reveal the effect of methylation of the catechol moiety of quercetin on its pro-oxidant behavior. Methylation of quercetin does not eliminate but considerably attenuates the cellular implications of the pro-oxidant activity of quercetin, which might add to the mechanisms underlying the apparent lack of in vivo carcinogenicity of this genotoxic compound. The paper also presents a new mechanism for the pro-oxidant chemistry of quercetin, eliminating the requirement for formation of an o-quinone, and explaining why methylation of the catechol moiety does not fully abolish formation of reactive DNA binding metabolites. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Quercetin; Quinone/quinone methide chemistry; Horseradish peroxidase; Glutathione and DNA adducts; Isorhamnetin and tamarixetin; Phase II metabolism Abbreviations: DMSO, dimethyl sulfoxide; EDTA, ethylene- diamine-tetra-acetic acid; EGTA, ethylene-glycol-bis-(-aminoethyl- ether)-NNN ′ N ′ -tetra-acetic acid; FCS, fetal calf serum; GSH, glutathione; HRP, horseradish peroxidase ∗ Corresponding author. Tel.: +31 317 484357; fax: +31 317 484931. E-mail address: ivonne.rietjens@wur.nl (I.M.C.M. Rietjens). 1. Introduction Quercetin (Fig. 1) is one of the most studied flavonoids. It is present, mainly as glycoside, in many fruits, vegetables, nuts and seeds and, as such, a com- ponent of the daily human diet [1]. The average intake is approximately 16 mg/day in the Netherlands [2]. For long, quercetin has been a compound of interest, mainly due to its presumed health promoting effects, reflected in, 0009-2797/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2005.12.005