Potentially lethal effects of quercetin, ascorbate and hydrogen peroxide on iron transport and intracellular iron redox reactions in bovine aortic endothelial cells. Sofia Pardalaki and Richard J Naftalin Cardiovascular Division, BHF Centre of Research Excellence, King’s College London SE1 9NH, UK   Introduction Quercetin (Q) is a flavonoid that chelates iron and acts as a transmembrane shuttle for iron via the facilitated glucose transporter GLUT1 (1). Phen Green SK (PGSK) (Invitrogen) is an intracellular fluorescent indicator which is quenched by bound Fe(II) ion. Hence changes in the oxidation state of iron to Fe(III) as well as in the concentration of Fe(II) increase the fluorescence signal of intracellular PGSK. Methods Phen Green assay for intracellular Fe(II) ion. Changes in intracellular iron of bovine aortic endothelial cells BAEC incubated in Krebs-HEPES buffer were monitored in 96-well plates after preloading with the metal ion fluorescence probe, Phen Green (PG, Invitrogen). Luminescence Assay - BAEC were incubated with 50μM L-012 applied extracellularly for 60 min at 37⁰C, L-012 reacts with reactive oxygen species leading to production of luminescence which is detected in a Hidex plate reader. . Reference . Vlachodimitropoulou E, Sharp PA, Naftalin RJ. Free Radic Biol Med. 2011;50:934-44. Q (1.0 mM) causes an increase in Fe (1.0 mM)-dependent PG quench (P < 0.001 because it increases iron uptake into cells by > 10 fold. Ascorbate has a variable effect on Fe dependent PG quench Without Q, ascorbate decreases Fe dependent PG quench because it enhances redox cycling between Fe(II) and Fe(III) Figs 2, 3, 4, 9 With Q = 1.0 mM ascorbate reduces Fe(III) – Fe(II) with resulting increase in iron dependent PG quench (p < 0.01). Figs 2, 4. Ascorbate prevents H2O2 oxidation of Fe(II)- Fe(III) P < 0.01). Fig 2, 4. Current Study Aims : To investigate the relationship between Fe2+, ascorbate and quercetin transport and intracellular iron-mediated redox reactions. Figure 1- Diagram showing relationship between iron, quercetin and ascorbate transport and intracellular redox reactions. Figure 2 Figure 3 Figure 6 Figure 9 Figure 8 Figure 5 With low intracellular Fe  100 nM raising intracellular ascorbate to > 100 mM generates ROS from ascorbyl radicals. Figs 3, 9. With raised intracellular Fe  1 mM raising ascorbate > 100 mM reduces ROS by preventing oxidation of Fe(II). Figs 2,4 Quercetin-dependent uptake is a saturable function of Fe. Fig 5 Quercetin independent Fe uptake is blocked by verapamil (Fig 6 ) indicating that it enters via L or T type Ca channels. Cytochalasin B blocks quercetin-dependent Fe uptake as previously shown with Q dependent Fe efflux (1). (Fig 7) Copper ion uptake is also accelerated by Q. After Cu entry it is reduced by ascorbate. To Cu(I). Fig 8 Conclusions The very high ascorbate concentrations in endothelial cells prevent iron induced formation of ROS. Quercetin may lead to raised cytosolic iron and copper within endothelial cells and could therefore be prooxidant when ascorbate is also raised. Figure 7 Figure 4