Enhancing effect of melatonin on chemiluminescence accompanying decomposition of hydrogen peroxide in the presence of copper.

Abstract

The oxidation of melatonin (MEL) using the Cu(II) + H(2)O(2) + HO(-) (the Fenton-like reaction) system was investigated by chemiluminescence (CL), fluorescence, spectrophotometric, and EPR spin trapping techniques. The reaction exhibits CL in the 400-730 nm region. The light emission from the Fenton-like reaction was greatly enhanced in the presence of MEL and was strongly dependent on its concentration. The spectrum measured with cut-off filters revealed maxima at around 460, 500, 580-590, 640-650, and 690-700 nm. The band at 460 nm may be due to the excited cleavage product, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, whereas the bands at 500, 580-590, 640-650, and 700 nm were similar to those observed for singlet molecular oxygen ((1)O(2)). The effect of reactive oxygen species (ROS) scavengers on the light emission was studied. The CL was strongly inhibited by the (1)O(2) scavengers in a dose-dependent manner; at concentration 1 mM the potency of (1)O(2) scavenging was 5,5-dimethylcyclohexandione-1,3 > methionine > histidine > hydroquinone. The potency of HO(*) scavenging by thiourea, tryptophan, cysteine at concentration 5 mM was 79-94%, by 1 mM glutathione and trolox 75 and 94%, respectively, and by 10 mM cimetidine 18%. Specific acceptors of O(2)(*)(-) such as p-nitroblue tetrazolium chloride and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) at concentration 5 mM decreased the CL by 51 and 95%, respectively, whereas superoxide dismutase (SOD) does not reduce the emission at concentration 2.8 U/ml. At higher concentration SOD substantially enhanced the light emission. Addition of 1360 U/ml catalase and 100 microM desferrioxamine strongly inhibited CL (96 and 90%, respectively). The increased generation of (1)O(2) from the Cu/H(2)O(2) system in the presence of MEL was confirmed using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline and by trapping experiments with 2,2,6,6-tetramethylpiperidine (TEMP) and subsequent electron paramagnetic (EPR) spectroscopy. These findings suggest the increased production of reactive oxygen species (O(2)(*)(-), HO(*), (1)O(2)) from the Fenton-like reaction in the presence of MEL. This means that the hormone is not able to act as classical chain-breaking antioxidant even at low concentration, and may show clear prooxidant activity at higher concentrations. In addition, long-lived carbonyl product of the MEL transformation in the triplet state can also be toxic by transferring its energy to organelles and causing a photochemical process.