Scavenging of hydroxyl radical by catecholamines Aleksandra Kładna, a Paweł Berczyński, b Irena Kruk, b Teresa Michalska b and Hassan Y. Aboul-Enein c * ABSTRACT:The direct effects ofthe four catecholamines (CATs), adrenaline (A),noradrenaline (NA), dopamine (D)and isoproterenol (I), on free radicals were investigated using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) and hydroxyl radial (HO • ). The CATs examined were found to inhibit the ESR signal intensity of DPPH •in a dose-dependent manner over the range 0.1–2.5 mmol/L in the following order: NA > A > I > D, with IC 50 = 0.30 0.03 for noradrenaline and IC 50 = 0.86 0.02 for dopamine. Hydroxyl radicals were produced using a Fenton reaction in the presence of the spin trap 5,5-dimethy 1-pyrroline N-oxide (DMPO), and ESR technique was applied to detect the CATs reactivity toward the radicals. The reaction rates constant (k r ) of CATs with HO • were found to be in the order of 10 9 L/mol/s,and the k r value for noradrenaline was the highest (k r = 8.4 10 9 L/mol/s).The CATs examined exhibited also a strong decrease in the light emission (62–73% at 1 mmol/L concentration and 79–89% at 2 mmol/L concentration) from a Fenton-like reaction. These reactions may be rele to the biological action of these important polyphenolic compounds. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: catechcholamines; 1,1-diphenyl-2-picrylhydrazyl radical; hydroxyl radical; spin trapping; ESR;chemiluminescence Introduction Catecholamines (CATs), including the neurotransmitter dopamine and a synthetic catecholamine, isoproterenol (chemical structures shown in Figure 1) have been found to show pro-oxidant activity, especially in the presence of transition metal ions such as iron and copper(1,2). The pro-oxidant activity of CATs results from generation of potentially toxic oxygen species (free radicals, such as superoxide anion radical O 2 ð Þ and hydroxyl radical (HO • ), hydro- gen peroxide (H 2 O 2 ) and singlet oxygen 1 O 2 Þ. These reactive ox- ygen species (ROS) have been hypothesized to play a crucial role in the progressive loss of dopaminergic neurons (characteristic of Parkinson’s disease and ageing) under oxidative stress (3). Adren- aline is released following general stress and emotional shock. The concentration of noradrenaline is increased during depres- sion, and the level of noradrenaline increases even during prena- talstress in the progeny (4,5). It was found that CATs undergo increased turnover in the metal-rich intercellular regions (1) and also following cancer therapy (6). In good agreement with the ROS aetiology of diseases, high levels of CATs may cause myocar- dial necrosis (7), arrhythmias and sudden death (8). We have undertaken systematic studies of oxidation of CATs with molecular oxygen, by enzymatically generated free radicals and by Fenton-like reagents to verify the existing hypothesis for CAT-induced toxicity with respect to the generation of ROS, es- pecially hydroxyl radicals (2,9,10). Additionally, we investigated the effect of CATs on the ROS generation during oxidation of farmorubicin, an anthracycline antibiotic used in the treatment of advanced human cancers (11). We found that CATs undergo redox cycling reactions and both oxidation and reduction pro- cesses contribute to overall reaction efficiency. In this study we used electron spin resonance (ESR) in conjunc- tion with the spin-trapping technique to calculate rate constants for the reaction of CATs with HO radicals. We also investigated the scavengingactivitiesof CATs against the stable 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH • ) as well the effect of CATs on chemiluminescence (CL) accompaining the Fenton-like reaction. Materials and methods Chemicals Trolox (6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid) and 5, 5-dimethyl-1-pyrroline-1-oxide (DMPO) were from Merck (Darmstadt, Germany). Ammonium ferrous sulphate hexahydrate was from Fluka (Buch, Switzerland). Catecholamines: adrenaline, noradrenaline,dopamine, isoproterenol(b-hydroxy-b(3,4- dihydroxyphenyl)-N-isopropylethylamine), 2,2-diphenyl-1-picryl- hydrazyl (DPPH) and other reagents were purchased from Sigma Aldrich (St. Louis, MO, USA). All reagents were prepared immedi- ately before use and stored in darkness. * Correspondence to: H. Y. Aboul-Enein, Pharmaceutical and Medicinal ChemistryDepartment, Pharmaceutical and Drug IndustriesResearch Division,NationalResearch Centre, Dokki,Cairo 12311, Egypt.E-mail: haboulenein@yahoo.com a Department of Medical History and Ethics, Pomeranian Medical University of Szczecin, Poland b Institute of Physics, Szczecin University of Technology, Poland c Pharmaceutical and MedicinalChemistryDepartment, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo, Egypt Luminescence 2012 Copyright © 2012 John Wiley & Sons, Ltd. Research article Received: 20 September 2011, Accepted: 24 October 2011 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/bio.1377