Melatonin and its derivatives cyclic 3-hydroxymelatonin, N¹-acetyl-N²-formyl-5-methoxykynuramine and 6-methoxymelatonin reduce oxidative DNA damage induced by Fenton reagents

Free radicals are generated in vivo and they oxidatively damage DNA because of their high reactivities. In the last several years, hundreds of publications have confirmed that melatonin is a potent endogenous free radical scavenger. Some of the metabolites produced as a result of these scavenging actions have been identified using pure chemical systems. This is the case with both N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK), identified as a product of the scavenging reaction of H₂O₂ by melatonin, and cyclic 3-hydroxymelatonin (C-3-OHM) which results when melatonin detoxifies two hydroxyl radicals (^.OH). In the present in vitro study, we investigated the potential of two different derivatives of melatonin to scavenger free radicals. One of these derivatives is C-3-OHM, while the other is 6-methoxymelatonin (6-MthM). We also examined the effect of two solvents, i.e., methanol and acetonitrile, in this model system. As an endpoint, using high-performance liquid chromatography we measured the formation of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) in purified calf thymus DNA treated with the Fenton reagents, chromium(III) [Cr(III)] plus H₂O₂, in the presence and in the absence of these molecules. The 8-OH-dG is considered a biomarker of oxidative DNA damage. Increasing concentrations of Cr(III) (as CrCl₃) and H₂O₂ was earlier found to induce progressively greater levels of 8-OH-dG in isolated calf thymus DNA because of the generation of ^.OH via the Fenton-type reaction. We found that C-3-OHM reduces ^.OH-mediated damage in a dose-dependent manner, with an IC₅₀ = 5.0 ± 0.2 μM; melatonin has an IC₅₀ = 3.6 ± 0.1 μM. These values differ statistically significantly with P < 0.05. In these studies, AFMK had an IC₅₀ = 17.8 ± 0.7 μM (P < 0.01). The 6-MthM also reduced DNA damage in a dose-dependent manner, with an IC₅₀ = 4.2 ± 0.2 μM; this value does not differ from the ICs for melatonin and C-3-OHM. We propose a hypothetical reaction pathway in which a mole of C-3-OHM scavenges 2 mol of ^.OH yielding AFMK as a final product. As AFMK is also a free radical scavenger, the action of melatonin as a free radical scavenger is a sequence of scavenging reactions in which the products are themselves scavengers, resulting in a cascade of protective reactions.