Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin ROBERTA FERRARESI 1 , LEONARDA TROIANO 1 , ERIKA ROAT 1 , ENRICO LUGLI 1 , ELISA NEMES 1 , MILENA NASI 1 , MARCELLO PINTI 1 , MARIA I. GARCIA FERNANDEZ 2 , EDWIN L. COOPER 3 , & ANDREA COSSARIZZA 1 1 Department of Biomedical Sciences, University of Modena and Reggio Emilia School of Medicine, Modena, Italy, 2 Department of Human Physiology, School of Medicine, University of Malaga, Spain, and 3 Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California at Los Angeles, Los Angeles, CA 90095-1763, USA Accepted by Professor A. Azzi (Received 8 June 2005; in revised form 25 July 2005) Abstract We have analyzed the anti- or pro-oxidant effects of the flavonoid quercetin (QU) by evaluating, in U937 cell line, hydrogen peroxide (H 2 O 2 ), superoxide anion ðO 2 2 Þ; reduced glutathione (GSH) content, mitochondrial membrane potential, DNA content, phosphatidylserine exposure on the outer face of the plasma membrane and cell viability. Polychromatic flow cytometry was used to evaluate in the same cells several functional parameters. For short periods of treatment QU exerted an anti-oxidant effect (decrease in H 2 O 2 levels), whereas for long periods it showed a pro-oxidant activity (increase in O 2 2 ). In these conditions, GSH content was reduced, and this correlated with a lack of anti-oxidant activity of QU, which in turn could be correlated with proapoptotic activity of this molecule. Thus, QU can exert different effects (anti-/prooxidant) depending on exposure times and oxidative balance, and in particular on stores of GSH. Keywords: Quercetin, ROS, GSH, flow cytometry, apoptosis, mitochondrial membrane potential Abbreviations: QU, Quercetin; H 2 O 2 , Hydrogen peroxide; O 2 2 , Superoxide anion; GSH, Reduced glutathione; Dc m , Mitochondrial membrane potential; PS, Phosphatidyilserine; ANX-V, Annexin-V; JC-1, 5,5 0 ,6,6 0 tetrachloro-1,1 0 ,3,3 0 - tetraethylbenzimidazolcarbocyanine iodide; H 2 DCF-DA, 2,7-Dicholorodihydrofluorescein diacetate; MBB, Monobromobimane; HE, Hydroethidine; PI, Propidium Iodide; FSC, Forward scatter; SSC, Side scatter Introduction Reactive oxygen species (ROS) are continuously produced during cell metabolism. Under normal conditions they are scavenged and converted to non- reactive species by different intracellular, enzymatic and non enzymatic, anti-oxidant systems (catalase, peroxidase, superoxide dismutase, vitamins, reduced glutathione (GSH)) [1]. If there is an overproduction or an ineffective elimination of ROS, they can react with proteins, lipids and nucleic acids causing several cell damages and eventually cell death. In fact, ROS are involved in the etiology of degenerative diseases, coronary artery disease, stroke and cancer [2,3]. ROS also acts as signalling molecules important for signal transduction [4,5]. In particular, H 2 O 2 can induce activation of NFkB, a transcription factor involved in several cellular processes [6]. Mitochondria are the most important intracellular sources of ROS. Indeed, nearly 4% of the oxygen used by the respiratory chain is incompletely reduced and can generate ROS [7]. This organelle is particularly vulnerable and therefore exposed to ROS injury. ROS accumulation causes lipid peroxidation and/or an ISSN 1071-5762 print/ISSN 1029-2470 online q 2005 Taylor & Francis DOI: 10.1080/10715760500306935 Correspondence: A. Cossarizza, Department of Biomedical Sciences, University of Modena and Reggio Emilia School of Medicine, via Campi 287, Modena 41100, Italy. Tel: 39 059 2055415. Fax: 39 059 2055426. E-mail: Cossarizza.andrea@unimore.it Free Radical Research, November 2005; 39(11): 1249–1258