The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) was found to be unstable in air when adsorbed on a thin-layer silica gel chromatography plate, a result that is in good agreement with the relatively high reactivity of this compound. Three novel main products were separated from the reaction mixture and identified by mass spectrometry and nuclear magnetic resonance data as: (i) 3-acetamidomethyl-6-methoxycinnolinone (AMMC), (ii) 3-nitro-AMK (AMNK, N1-acetyl-5-methoxy-3-nitrokynuramine), and (iii) N-[2-(6-methoxyquinazolin-4-yl)-ethyl]-acetamide (MQA). AMMC and AMNK are shown to be nonenzymatically formed also in solution, by nitric oxide (NO) in the first case, and by a mixture of peroxynitrite and hydrogen carbonate, in the second one. The use of three different NO donors, PAPA-NONOate, S-nitroso-N-acetylpenicillamine and sodium nitroprussiate led to essentially the same results, with regard to a highly preferential formation of AMMC; AMNK was not detected in these reaction systems. Competition experiments with the NO scavenger N-acetylcysteine indicate a somewhat lower reactivity compared with the competitor. Peroxynitrite led to AMNK formation in the presence of physiological concentrations of hydrogen carbonate at pH 7.4, but not in its absence, indicating that nitration involves a mixture of carbonate radicals and NO2, formed from the peroxynitrite-CO2 adduct. No AMMC was detected after AMK exposure to peroxynitrite. Both AMNK and AMMC exhibited a much lower reactivity toward 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) cation radicals than did AMK. In a competition assay for hydroxyl radicals, AMMC showed prooxidant properties, whereas AMNK was a moderate antioxidant. AMMC and AMNK should represent relatively stable physiological products, although their rates of synthesis are still unknown and may be low. Formation of these compounds may contribute to the disappearance of AMK from tissues and body fluids.

Toxicity of the pesticide quinalphos may comprise secondary, delayed effects by its main metabolite 2-hydroxyquinoxaline (HQO). We demonstrate that HQO can destroy photocatalytically vitamins C and E, catecholamines, serotonin, melatonin, the melatonin metabolite AMK (N(1)-acetyl-5-methoxykynuramine), and unsubstituted and substituted anthranilic acids when exposed to visible light. In order to avoid HQO-independent ascorbate oxidation by light and to exclude actions by hydroxyl radicals, experiments on this vitamin were carried out in ethanolic solutions. Other substances tested (vitamin E, melatonin, anthranilic acids) were also photocatalytically destroyed by HQO in ethanol. After product analyses had indicated that HQO was not, or only poorly, degraded in the light, despite its catalytic action on other compounds, we followed directly the time course of HQO and ascorbate concentrations in ethanol. While ascorbate was largely destroyed, no change in HQO was demonstrable within 2 h of incubation. Destruction was not prevented by the singlet oxygen quencher DABCO. Obviously, HQO is capable of undergoing a process of organic redox cycling, perhaps via an intermediate quinoxaline-2-oxyl radical. Health problems from HQO intoxication may not only arise from the loss of valuable biomolecules, such as antioxidant vitamins and biogenic amines, but also from the formation of potentially toxic products. Dimerization and oligomerization are involved in several oxidation processes catalyzed by HQO, especially in the indoleamines, in dopamine, and presumably also in vitamin E. Melatonin oxidation by HQO did not only lead to the well-known - and usually protective - metabolite AFMK (N(1)-acetyl-N(2)-formyl-5-methoxykynuramine), but also to a high number of additional products, among them dimers and trimers. DABCO did not prevent melatonin destruction, but changed the spectrum of products. Serotonin was preferentially converted to a dimer, which can further oligomerize. Several indole dimers are known to be highly neurotoxic, as well as oxidation products formed from catecholamines via the adrenochrome/noradrenochrome pathway. Destruction of melatonin may cause deficiencies in circadian physiology, in immune functions and in antioxidative protection.