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 neuronal 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 subendothelial vascular wall. Furthermore, the cytotoxic effect of oxidized LDL increases atherogenicity. The goal of this study was to compare the antioxidant activities of melatonin and vitamin E against in vitro LDL oxidation and their cytoprotective actions against oxidized LDL-induced endothelial cell toxicity. An attempt at loading LDL with melatonin by incubating human plasma with an ethanolic melatonin solution gave 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) coupled to UV detection. High concentrations of melatonin (10-100 microM) added to the oxidative medium induced a clear inhibition of Cu2+-induced LDL oxidation, characterized as an increase in the lag-phase duration of conjugated diene formation and decreases in the maximal rate of the propagation phase and in the maximal amount of conjugated diene formation. Determination of the median efficacious dose (ED50) of melatonin and vitamin E by their ability to increase lag-phase duration showed that melatonin was less active than vitamin E (ED50, 79 vs. 10 microM, respectively). 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 microM melatonin produced oxidized LDLs that were less recognizable for the scavenger receptors of J774 macrophages than were untreated LDLs. Vitamin E, 10 microM, was more active than 100 microM melatonin in inhibiting LDL oxidation and the resulting lipoprotein alterations leading to binding internalization and degradation by the J774 macrophages. Vitamin E, 100 microM, inhibited the pursuit of the oxidation of oxidized LDL mediated by bovine aortic endothelial cells (BAECs) in a culture medium containing Cu2+, whereas 100 microM melatonin had no antioxidant effect. Melatonin, 100 microM, as well as 100 microM vitamin E inhibited intracellular TBARS formation during the incubation of BAECs with highly oxidized LDL but had no influence on the increase in glutathione (GSH) concentration during this lengthy exposure (16 h) of BAECs to highly oxidized LDL. During this period, the same dose of vitamin E but not of melatonin tended to limit the decrease in adenosine triphosphate (ATP) concentration. Vitamin E, 100 microM, did not significantly reduce cellular lactate dehydrogenase (LDH) release in the culture medium during the incubation of oxidized LDL with BAECs, whereas 100 microM melatonin dramatically increased this release. These data show that melatonin is less active than vitamin E in inhibiting in vitro LDL oxidation and does not 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 microM vs. 100 pM). Therefore our 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. Nevertheless, in vivo LDL oxidation takes place in the subendothelium of the artery wall, and nothing is known about the concentration of melatonin or its catabolites in this environment.
Département d'Athérosclérose, INSERM U325, Institut Pasteur et Faculté de Pharmacie, Lille, France.
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E
Walters-Laporte
C
C
Furman
S
S
Fouquet
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F
Martin-Nizard
S
S
Lestavel
A
A
Gozzo
D
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Lesieur
J C
JC
Fruchart
P
P
Duriez
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Teissier
D000975:Antioxidants / Q000494:pharmacology*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000494:pharmacology; D004347:Drug Interactions; D004727:Endothelium / Q000187:drug effects; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects*; D008077:Lipoproteins, LDL / Q000009:adverse effects / Q000187:drug effects* / Q000378:metabolism; D008550:Melatonin / Q000494:pharmacology*; D017392:Thiobarbituric Acid Reactive Substances / Q000493:pharmacokinetics; D014810:Vitamin E / Q000494:pharmacology*
D000975:Antioxidants; D008077:Lipoproteins, LDL; D017392:Thiobarbituric Acid Reactive Substances; D014810:Vitamin E; D003300:Copper; D008550:Melatonin