A HIGH-CONCENTRATION OF MELATONIN INHIBITS IN-VITRO LDL PEROXIDATION BUT NOT OXIDIZED LDL TOXICITY TOWARD CULTURED ENDOTHELIAL-CELLS

The pineal hormone, melatonin, was recently found to be a potent free scavenger for hydroxyl and peroxyl radicals. Melatonin also inhibits n euronal and thymocyte damage due to oxidative stress. Atherosclerosis development is mediated by low-density lipoprotein (LDL) oxidation and the endocytosis of oxidized LDL by resident macrophages in the subend othelial vascular wall. Furthermore, the cytotoxic effect of oxidized LDL increases atherogenicity. The goal of this study was to compare th e antioxidant activities of melatonin and vitamin E against in vitro L DL oxidation and their cytoprotective actions against oxidized LDL-ind uced endothelial cell toxicity. An attempt at loading LDL with melaton in by incubating human plasma with an ethanolic melatonin solution gav e only low protection against Cu2+-induced LDL oxidation in comparison with vitamin E and gave no detectable incorporation of melatonin into LDL, measured by high-performance liquid chromatography (HPLC) couple d to UV detection. High concentrations of melatonin (10-100 mu M) adde d to the oxidative medium induced a clear inhibition of Cu2+-induced L DL oxidation, characterized as an increase in the lag-phase duration o f conjugated diene formation and decreases in the maximal rate of the propagation phase and in the maximal amount of conjugated diene format ion. Determination of the median efficacious dose (ED50) Of melatonin and vitamin E by their ability to increase lag-phase duration showed t hat melatonin was less active than vitamin E (ED50, 79 vs. 10 mu M, re spectively). Melatonin was also less active than vitamin E in limiting the formation of thiobarbituric acid-reactive substances (TBARS) and LDL fluorescence intensity increase in the medium during Cu2+-induced LDL oxidation. Cu2+-induced LDL oxidation in the presence of 100 mu M melatonin produced oxidized LDLs that were less recognizable for the s cavenger receptors of J774 macrophages than were untreated LDLs. Vitam in E, 10 mu M, was more active than 100 mu M melatonin in inhibiting L DL oxidation and the resulting lipoprotein alterations leading to bind ing internalization and degradation by the J774 macrophages. Vitamin E , 100 mu M, inhibited the pursuit of the oxidation of oxidized LDL med iated by bovine aortic endothelial cells (BAECs) in a culture medium c ontaining Cu2+, whereas 100 mu M melatonin had no antioxidant effect, Melatonin, 100 mu M, as well as 100 mu M vitamin E inhibited intracell ular TEARS formation during the incubation of BAECs with highly oxidiz ed LDL but had no influence on the increase in glutathione (GSH) conce ntration during this lengthy exposure (16 h) of BAECs to highly oxidiz ed LDL, During this period, the same dose of vitamin E but not of mela tonin tended to limit the decrease in adenosine triphosphate (ATP) con centration. Vitamin E, 100 mu M, did not significantly reduce cellular lactate dehydrogenase (LDH) release in the culture medium during the incubation of oxidized LDL with BAECs, whereas 100 UM melatonin dramat ically increased this release. These data show that melatonin is less active than vitamin E in inhibiting in vitro LDL oxidation and does no t inhibit the cytotoxicity of oxidized LDL toward cultured endothelial cells. The concentrations necessary to inhibit LDL oxidation are far beyond those found in human plasma (100 mu M vs. 100 pM). Therefore ou r results indicate that the pineal hormone melatonin per se appears to have little antiatherogenic property in the in vitro oxidation of LDL and the cytoprotective action against the toxicity of oxidized LDL. N evertheless, in vivo LDL oxidation takes place in the subendothelium o f the artery wall, and nothing is known about the concentration of mel atonin or its catabolites in this environment.