title: Oxidation pathways underlying the pro-oxidant effects of apigenin.
abstract: Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (H(2)DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H(2)DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H(2)DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H(2)DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.
authors.affiliation: Department of Biochemistry and Genetics, University of Navarra, 31008 Pamplona, Spain.
authors.forename: Aitor
authors.initials: A
authors.lastname: Andueza
authors.affiliation: Department of Biochemistry and Genetics, University of Navarra, 31008 Pamplona, Spain.
authors.forename: Antonia
authors.initials: A
authors.lastname: García-Garzón
authors.affiliation: Department of Biochemistry and Genetics, University of Navarra, 31008 Pamplona, Spain.
authors.forename: Marina
authors.initials: M
authors.lastname: Ruiz de Galarreta
authors.affiliation: Department of Biochemistry and Genetics, University of Navarra, 31008 Pamplona, Spain.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Ansorena
authors.affiliation: Department of Biochemistry and Genetics, University of Navarra, 31008 Pamplona, Spain.
authors.forename: María J
authors.initials: MJ
authors.lastname: Iraburu
authors.affiliation: Department of Biochemistry and Genetics, University of Navarra, 31008 Pamplona, Spain.
authors.forename: María J
authors.initials: MJ
authors.lastname: López-Zabalza
authors.affiliation: Department of Biochemistry and Genetics, University of Navarra, 31008 Pamplona, Spain. Electronic address: jjmirujo@unav.es.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Martínez-Irujo
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000008:administration & dosage*; D047310:Apigenin / Q000008:administration & dosage*; D002460:Cell Line; D005609:Free Radicals / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D055166:Hepatic Stellate Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D005609:Free Radicals; D017382:Reactive Oxygen Species; D047310:Apigenin; D006861:Hydrogen Peroxide; D005978:Glutathione; D010100:Oxygen
keywords: Autoxidation; Dichlorodihydrofluorescein; Flavonoids; Free radicals; Hepatic stellate cells; Peroxidase; Phenoxyl radicals; Xanthine oxidase