Metabolites of melatonin, a scavenger cascade, and melatonin as a prodrug


Reactions of melatonin with free radicals and other oxidants are not only a matter of the toxic reactants eliminated, but also of the products formed. It is highly important to distinguish between metabolites formed under physiological or near-physiological conditions from those produced in chemical systems designed for studying reactions with a single radical species in preparations as pure as possible. Disregard of this point has led to several misinterpretations in the past. We have repeatedly emphasized that studies using reaction systems which preferentially generate hydroxyl radicals mainly lead to hydroxylated adducts or their derivatives such as substituted indolinones, whereas biological material usually contains orders of magnitude more superoxide anions than hydroxyl radicals. Therefore, an entirely different product spectrum is obtained as soon as hydroxyl radicals, or other electron-abstracting radicals, act in the presence of an excess of superoxide anions [60,89]. Radicals derived from melatonin by interaction with a first, reaction-initiating radical likely combine with superoxide anions so that the radical reaction chain is readily terminated [15,49]. The product formed by oxidative pyrrole-ring cleavage is a substituted kynuramine, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK; Fig. 1). We have investigated numerous reaction systems and in all those containing sufficient quantities of superoxide anions, AFMK was by far the most abundant product [44,46,58-60,66,89]. Interestingly, a profound and sursprising difference exists between melatonin and other structurally related indoleamines. While substituted kynuramines represent only a limited or small fraction of oxidation products from other indolic compounds, AFMK usually greatly exceeds the total of other substances formed. This indicates a significant contribution not only of the 5-methoxy residue, but also of the N-acetylated side chain to the oxidation chemistry of melatonin, a conclusion corroborated by various scavenging assays and chemiluminescence associated with pyrrole-ring cleavage [52]. Moreover, AFMK was the only melatonin metabolite detected in culture media of various aquatic organisms, unicells and small metazoans, whereas several additional products were found in axenic media incubated for extended periods of time [90]. AFMK formation seems to be a favored pathway of melatonin degradation in these species.

These findings do not represent a peculiarity of non-vertebrates, but rather seem to reflect the non-hepatic melatonin catabolism in vertebrates. Contrary to statements in the earlier literature claiming that almost all melatonin is metabolized in the liver to 6-hydroxymelatonin followed by conjugation and excretion, recent estimations attribute about 30 percent of overall melatonin degradation to pyrrole-ring cleavage [91]. The rate of AFMK formation may be considerably higher in certain tissues, since extrahepatic P450 monooxygenase activities are frequently too low for a high turnover via 6-hydroxylation. The high amounts of gastrointestinal melatonin (see above), as far as they are not released unmetabolized, have to enter a pathway different from monooxygenation. AFMK formation is highly likely.

The significance of pyrrole-ring cleavage in oxidative metabolism of tissue melatonin is particularly illustrated in the central nervous system, where a secondary product, N1-acetyl-5-methoxykynuramine (AMK) derived from AFMK by deformylation, was identified as a main metabolite [92]. When melatonin was injected into the cisterna magna, about 35 percent was recovered as AMK. Under the conditions used, AFMK and AMK were the only products formed from melatonin in the brain and no 6-hydroxymelatonin was detected. In this case, the high turnover in the kynuric pathway of melatonin catabolism is the more remarkable as it cannot be explained on the basis of the enzymes capable of catalyzing the formation of AFMK: (i) indoleamine 2, 3-dioxygenase which uses tryptophan as the main substrate, exhibits sufficiently high activities only after inflammatory stimulation of the microglia [93-95]; (ii) myeloperoxidase, which can also catalyze pyrrole-ring cleavage of melatonin [91,96,97], is again associated with activated phagocytes. To assume free radical reactions as the main cause of kynuric melatonin degradation in the brain is, therefore, highly suggestive. Non-enzymatic AFMK formation in other tissues will be a matter for future research.

It is a remarkable fact that AFMK is formed by many different mechanisms [summarized in refs. [15,41,59,66,89]]. Apart from the enzymes mentioned, pseudoenzymatic catalysis by oxyferrylhemoglobin or by hemin, interactions with free radicals, e.g., combinations of •OH and O2•-, or CO3•- and O2•-, or organic cation radicals and O2•-, oxidation by singlet oxygen, by ozone, or by O2 under photoexcitation of melatonin all lead to AFMK. Even another product formed from melatonin by interactions with free radicals, cyclic 3-hydroxymelatonin [70], can be further metabolized by free radicals to AFMK [68]. All these findings indicate that AFMK is a central metabolite of melatonin oxidation especially in non-hepatic tissues.

As already mentioned, AFMK is easily deformylated to AMK. To date two enzymes capable of catalyzing this reaction have been identified, arylamine formamidase and hemoperoxidase [49,89,98]. The two methoxylated kynuramines, AFMK and AMK, are of particular interest because of their own radical-scavenging and protective properties. In any case, kynuramines, a separate class of biogenic amines, exhibit various biological activities [99], which are, however, rarely investigated. With regard to antioxidative protection, AFMK was shown to reduce 8-hydroxy-2-deoxyguanosine formation [42] and lipid peroxidation, and to rescue hippocampal neurons from oxidotoxic cell death [41]. Although AFMK interacts, not surprisingly, with the highly reactive hydroxyl radicals, it is otherwise relatively inert towards radicals of lower or intermediate reactivity [43,89]. This is convincingly explained by its preference for two-electron transfer reactions as demonstrated by cyclic voltammetry [41].

The deformylated product AMK, easily formed from AFMK [92], appears to be a highly interesting substance, for several reasons: first, it is a radical scavenger of considerably higher reactivity than AFMK because it easily undergoes single-electron transfer reactions [43,89,100] and, second, it acts as a cyclooxygenase (COX) inhibitor that is much more potent than acetylsalicylic acid [101] and has relative specificity for COX-2 (B Poeggeler, pers. commun.). Moreover, AMK was recently shown to downregulate COX-2 expression in macrophages [102]. AMK might, therefore, contribute to the attenuation of oxidative stress both directly and indirectly by interference with inflammatory responses. A third, mitochondrial effect will be discussed below. Unfortunately, the precise tissue levels of AMK are still unknown, partially because of a lack of specific assays, partially due to its high reactivity which readily leads to other products. Since AMK can be recovered from the urine after a melatonin load [92], sufficient amounts may be present in the tissues, at least after administration of pharmacological doses. Therefore, melatonin seems to act not only directly, but, additionally, as a prodrug of AMK.

It is a remarkable fact that the kynuric pathway of melatonin metabolism includes a series of radical scavengers, which may be regarded as a scavenger cascade [68], with a possible sequence of melatonin ? cyclic 3-hydroxymelatonin ? AFMK ? AMK, where melatonin can be alternately converted to AFMK directly. From melatonin to AFMK, up to 4 free radicals can be consumed [68]; recent determinations [Rosen J, Hardeland R, unpubl. data] have shown that even higher numbers of free radicals can be eliminated, and other, previously unknown products are being characterized. The potent scavenger AMK consumes further radicals in primary and secondary reactions. Interestingly, AMK not only interacts with reactive oxygen but also with reactive nitrogen species and several products have been structurally characterized in Göttingen [[103]; manuscript in preparation]. Neither the end of the kynuric pathway of melatonin nor that of the scavenger cascade is in sight.

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