Melatonin, xanthurenic acid, resveratrol, EGCG, vitamin C and α‐lipoic acid differentially reduce oxidative DNA damage induced by Fenton reagents: a study of their individual and synergistic actions

Abstract

DNA damage generated by oxygen‐derived free radicals is related to mutagenesis, carcinogenesis and aging. In the last several years, hundreds of publications have confirmed that melatonin is a potent endogenous free radical scavenger. In the present in vitro study, we have examined the efficacy of three polyphenolic antioxidants, i.e. xanthurenic acid, resveratrol (3,4′,5‐trihydroxy‐trans‐stilbene) and (−)‐epigallocatechin‐3‐gallate (EGCG) and two classical non‐polyphenolic antioxidants, i.e. vitamin C (ascorbic acid) and α‐lipoic acid (LA, 1,2‐dithiolane‐3‐pentanoic acid) in inhibiting ^•OH‐induced oxidative DNA damage. We compared the efficacy of these five antioxidants with the effectiveness of melatonin (N‐acetyl‐5‐methoxytryptamine) and we also investigated the possible synergistic effects of melatonin with the other five molecules. Using high performance liquid chromatography (HPLC), the formation of 8‐hydroxy‐2‐deoxyguanosine (8‐OH‐dG) in purified calf thymus DNA treated with the Fenton reagents, chromium(III) (as CrCl₃) plus hydrogen peroxide (H₂O₂) (Cr(III)/H₂O₂), was measured in the presence or absence of the antioxidants alone or in combination with melatonin. 8‐OH‐dG is considered a biomarker of oxidative DNA damage. Among the antioxidants tested, melatonin was the most effective of these with an IC₅₀ = 3.6 ± 0.1 μm. For the other antioxidants the IC₅₀ values were as follows: xanthurenic acid (IC₅₀ = 7.9 ± 0.3), resveratrol (IC₅₀ = 10.9 ± 0.3), EGCG (IC₅₀ = 5.7 ± 0.3), vitamin C (IC₅₀ = 16.9 ± 0.5) and LA (IC₅₀ = 38.8 ± 0.7). These values differ from that of melatonin with a P < 0.01. Melatonin (1 μM) reversed the pro‐oxidant effect of resveratrol (0.5 μM) and vitamin C (0.5 μM), had an antagonistic effect when used in combination with EGCG (1 μM) and it exhibited synergism in combination with vitamin C (0.5 μM) and with LA (5 μM).