Consequences of quercetin methylation for its covalent glutathione and DNA adduct formation
Woude, H. van der; Boersma, M.G. ; Alink, G.M. ; Vervoort, J.J.M. ; Rietjens, I.M.C.M. - \ 2006
Chemico-Biological Interactions 160 (2006)3. - ISSN 0009-2797 - p. 193 - 203.
low-density-lipoprotein - coronary-heart-disease - plasma metabolites - dietary flavonoids - lipid-peroxidation - cell-proliferation - beta-glucuronidase - quinone methide - inhibition - oxidation
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 [4-14C]-3¿- and 4¿-O-methylquercetin in HepG2 cells amounted to only 42% of the level of covalent adducts formed by a similar amount of [4-14C]-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
Quenching of quercetin quinone/quinone methides by different thiolate scavengers: stability and reversibility of conjugate formation
Awad, H.M. ; Boersma, M.G. ; Boeren, J.A. ; Bladeren, P.J. van; Vervoort, J.J.M. ; Rietjens, I.M.C.M. - \ 2003
Chemical Research in Toxicology 16 (2003)7. - ISSN 0893-228X - p. 822 - 831.
glutathione s-transferases - irreversible inhibition - lipid-peroxidation - mutagenic activity - quinone methide - active-site - o-quinones - oxidation - flavonoids - antioxidant
Oxidation of flavonoids with a catechol structural motif in their B ring leads to formation of flavonoid quinone/quinone methides, which rapidly react with GSH to give reversible glutathionyl flavonoid adducts. Results of the present study demonstrate that as a thiol-scavenging agent for this reaction Cys is preferred over GSH and N-acetyleysteine. The preferential scavenging by Cys over GSH reported in the present study appeared not to provide a basis for detection of thiol-based flavonoid conjugates in biological systems. This is because physiological concentrations of GSH are substantially higher than those of Cys, which was shown to shift the balance of thiol conjugate formation in favor of glutathionyl adduct formation. Furthermore, the cysteinyl quercetin adducts, although not showing the reversible nature of the glutathionyl conjugates, appeared nevertheless to be unstable. Thus, as a biomarker for formation of reactive quercetin quinone/quinone methides in biological systems, detection of the glutathionyl conjugates or the N-acetyleysteinyl conjugates derived from them should still be the method of choice. At GSH levels that dominate the level of other cellular thiol groups, covalent addition of the quinone to other cellular thiol groups may be efficiently prevented. However, various tissues are known to contain higher levels of protein-bound sulfhydryl moieties than of nonprotein sulfhydryl groups, the latter consisting of especially GSH. Thus, the results of the present study indicate that in biological systems covalent addition of quercetin quinone methide to tissue protein sulfhydryl groups can be expected. The transient nature of these adducts, as shown for all three types of thiol quercetin adducts in the present study, will, however, also result in a transient nature of the protein-bound quercetin adducts to be expected. Because stability of the various thiol quercetin adducts appeared a matter of minutes to hours instead of days, this rapid transient nature of possible quercetin quinone methide adducts may also restrict the ultimate toxicity to be expected from the quercetin quinone/quinone methides.
Inhibition of human glutathione S-transferase P1-1 by the flavonoid quercetin
Zanden, J.J. van; Hamman, O. Ben; Iersel, M.L. van; Boeren, J.A. ; Cnubben, N.H.P. ; Bello, M. Lo; Vervoort, J.J.M. ; Bladeren, P.J. van; Rietjens, I.M.C.M. - \ 2003
Chemico-Biological Interactions 145 (2003)2. - ISSN 0009-2797 - p. 139 - 148.
site-directed mutagenesis - human placenta - quinone methide - ethacrynic-acid - active-site - pi - identification - consequences - inactivation - conjugation
In the present study, the inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by the flavonoid quercetin has been investigated. The results show a time- and concentration-dependent inhibition of GSTP1-1 by quercetin. GSTP1-1 activity is completely inhibited upon I h incubation with 100 muM quercetin or 2 h incubation with 25 muM quercetin, whereas 1 and 10 muM quercetin inhibit GSTP1-1 activity to a significant extent reaching a maximum of 25 and 42% inhibition respectively after 2 h. Co-incubation with tyrosinase greatly enhances the rate of inactivation, whereas co-incubation with ascorbic acid or glutathione prevents this inhibition. Addition of glutathione upon complete inactivation of GSTP1-1 partially restores the activity. Inhibition studies with the GSTP1-1 mutants C47S, C101S and the double mutant C47S/C101S showed that cysteine 47 is the key residue in the interaction between quercetin and GSTP1-1. HPLC and LGMS analysis of trypsin digested GSTP1-1 inhibited by quercetin did not show formation of a covalent bond between Cys 47 residue of the peptide fragment 45-54 and quercetin. It was demonstrated that the inability to detect the covalent quercetin-peptide adduct using LGMS is due to the reversible nature of the adduct-formation in combination with rapid and preferential dimerization of the peptide fragment once liberated from the protein. Nevertheless, the results of the present study indicate that quinone-type oxidation products of quercetin likely act as specific active site inhibitors of GSTP1-1 by binding to cysteine 47. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.