HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/12/2294 01-0309fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by TAN, D.-X. Articles by REITER, R. J. Search for Related Content PubMed PubMed Citation Articles by TAN, D.-X. Articles by REITER, R. J.

N¹-acetyl-N²-formyl-5-methoxykynuramine, a biogenic amine and melatonin metabolite, functions as a potent antioxidant¹

DUN-XIAN TAN^*,, LUCIEN C. MANCHESTER^*, SUSANNE BURKHARDT^*,, ROSA M. SAINZ^*, JUAN C. MAYO^*, RONNIE KOHEN, ESTHER SHOHAMI^*, YU-SHU HUO^*, RÜDIGER HARDELAND and RUSSEL J. REITER^*2

^* Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA;
Instit für Zoologie und Anthropologie, und Universität Göttingen, D-37073 Göttingen, Germany; and
Department of Pharmacology, David E. Bloom Center for Pharmacy, Hebrew University, Jerusalem, Israel 91120

²Correspondence: Department of Cellular and Structural Biology, University of Texas Health Science Center, Mail Code 7762, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900, USA. E-mail: reiter{at}uthscsa.edu

SPECIFIC AIMS

N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK), a melatonin metabolite, is a rarely investigated biogenic amine. The physiological role of AFMK in organisms remains unknown. The potential of AFMK as an antioxidant and free radical scavenger was systematically evaluated by examining its capacity to donate electrons, protect against oxidative stress to macromolecules, and reduce toxin-induced death of neurons.

PRINCIPAL FINDINGS

1. AFMK donates two electrons in physiological solution
Typically, low molecular weight antioxidants (LMWA) that act directly to reduce reactive oxygen species (ROS) are capable of donating electrons (e^-) to ROS and, in so doing, destroy them. In evaluating the oxidation potential of these compounds, their ability to act as reducing agents may indicate their potential as antioxidants. An accepted method to evaluate the oxidation potential of molecules is cyclic voltametry (CV). This method was used to detect the e^- donating potential of AFMK. The presence of anodic waves in the cyclic voltamogram indicates the ability of a compound to donate its e^-. The lower the peak potential, the higher the ability of the scavenger to donate its e^-. The position of current wave on the voltage axis (x axis of the voltamogram) can be determined and is referred to as the potential where the peak current [peak potential Ep(a)] or inflection point (half-wave potential, E_1/2) occurs. The oxidation potential of a compound may be defined phenomenologically as the potential where Ep(a) is observed for a given set of conditions. For AMFK, two anodic waves were detected at Ep(a) of 456 and 668 mV, respectively, in the PBS buffer (50 mM, pH 7.4) (Fig. 1 ). The results indicate that AMFK may be a potent antioxidant that donates two e^- to neutralize free radicals.

View larger version (30K): [in this window] [in a new window]   Figure 1. Cyclic voltametry of AFMK. The potential range is from the -0.3 to 1.3 V at a rate of 100 mV/s vs. the Ag/AgCl reference electrode. AFMK was dissolved in PBS; the final concentration was 50 mM. Two distinguishable anodic waves at Ep(a) of 456 and 668 mV are detected as indicated by a and b, respectively.

2. AFMK reduces 8-hydroxy-2-deoxyguanosine formation induced by free radicals
To examine whether AFMK prevents free radical-induced DNA damage, calf thymus DNA (500 µg) was incubated with 500 µM Cr(III) and hydrogen peroxide (H₂O₂). In this system, Cr (III) reduces H₂O₂ to form the highly reactive hydroxyl radical (^.OH). The ^.OH then attacks the DNA to generate 8-hydroxy-2-deoxyguanosine (8-OHdG), a specific biomarker of oxidatively damaged DNA. The addition of AFMK significantly reduced 8-OHdG formation in a dose-dependent manner. The effective concentration of AFMK required to inhibit 8-OHdG formation ranged from 25 nM to 5 µM. The IC₅₀ (the dose required to inhibit 50% of the reaction) of AFMK for inhibiting 8-OHdG formation was roughly 100 nM.

3. AFMK reduces lipid peroxidation induced by H₂O₂ and ferrous iron
Rat liver homogenates were used to test the protective effect of AFMK against lipid peroxidation. Four-month-old Sprague-Dawley rats were purchased from Harlan (Houston, TX) and housed in an air-conditioned room maintained at 22 ± 0.5°C, 12:12 h light:dark cycle with food and water ad libitum. The rats were killed by decapitation. The liver was dissected and immediately frozen at -80°C until assayed. Liver homogenates were incubated with 1 mM H₂O₂ and 15 µM ferrous iron with or without AFMK at 37°C for 2 h to induce membrane lipid peroxidation. The levels of malondialdehyde (MDA) + 4-hydroxyalkenals (4-HDA) were used as indicators of lipid peroxidation. In this study, MDA and 4-HDA were likely initiated by ^.OH, formed via the Fenton reaction. AFMK reduced lipid peroxidation in rat liver homogenates in a dose-dependent manner with an IC₅₀ of 3 mM.

4. AFMK does not chelate transition metals
To identify whether AFMK chelates transition metals, the absorption spectra of both Cr(III) and Fe²⁺, with or without AFMK, were scanned using a Science UV/Vis Spectrophotometer (Beckman, Fullerton, CA) at wavelengths from 600 nm to 190 nm. AFMK did not modify the absorption spectrum of either Cr(III) or Fe²⁺. This indicates that AFMK does not chelate transition metals under the present experimental conditions.

5. AFMK reduces neuronal death due to H₂O₂, glutamate, or amyloid ß_25–35
HT22 cells, a subclone of the HT4 hippocampal neuron line, were cultured with neurotoxins including H₂O₂, glutamate, and amyloid ß_25–35 peptide. Cell viability was measured using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT). AFMK at a concentration of 100 µM improved cell viability by 20% and at a concentration of 1 mM it almost completely prevented cell death resulting from high levels of H₂O₂ (Fig. 2A ). The excitatory amino acid glutamate induced a significant decrease in cell viability in cultured HT22 cells. Both concentrations of AFMK also prevented cell death induced by glutamate (Fig. 2B ). When the concentration of glutamate was 5 mM, 100 µM AFMK reduced HT22 cell death by 15% and 1 mM AFMK completely prevented the decrease of viability in cells incubated with 5 mM glutamate. Even when the glutamate concentration reached 10 mM, both levels of AFMK still significantly protected HT22 cells from death (Fig. 2B ). When HT22 cells were incubated with amyloid ß_25–35 at concentrations of either 0.2, 2.0, or 20 µM, cell viability decreased significantly. AMFK at the concentration of 1 mM significantly reduced cell death by 30 and 15% in cultured HT22 cells induced by 2 or 20 µM amyloid ß_25–35, respectively (Fig. 2C ).

View larger version (29K): [in this window] [in a new window]   Figure 2. A) Protective effect of AFMK against cultured rat hippocampal neuron (HT22 cells) death induced by H2O2. Cells were cultured with 100 µM or 1 mM AFMK for 24 h and incubated with the indicated concentrations of H2O2. For all toxins, MTT assays were repeated three times using 7 samples per group. CON = control; a = P < 0.05 vs. control; b = P < 0.05 vs. 100 µM AFMK. B) Protective effect of AFMK against cultured rat hippocampal neuron (HT22 cells) death induced by glutamate. Cells were cultured with 100 µM or 1 mM AFMK for 24 h, then incubated with either 1, 5 or 10 mM glutamate. CON = control; a = P < 0.05 vs. control; b = P < 0.05 vs. 100 µM AFMK. C) Protective effect of AFMK against neuronal death induced by amyloid ß25–35. Cells were cultured with 100 µM or 1 mM AFMK for 24 h and incubated with either 0.2, 2.0, or 20 µM amyloid ß25–35. CON = control; a = P < 0.05 vs. control.

CONCLUSIONS AND SIGNIFICANCE

AFMK is formed from melatonin through enzymatic and nonenzymatic pathways in vivo and in vitro (Fig. 3 ). AFMK is a sparingly investigated, endogenously occurring molecule. Because so little is known of its physiological or pathological roles or its levels in organisms, the antioxidative capacity of AFMK was systematically investigated.

View larger version (24K): [in this window] [in a new window]   Figure 3. Enzymatic and chemical reaction pathways of AFMK formation from melatonin and its antioxidative mechanism. By donating e-, AFMK detoxifies free radicals (R.), thereby reducing oxidative damage. In the process of an electron donation and addition of a hydrogen ion (H+), a stable, nonreactive molecule (RH) is formed. .OH = hydroxyl radical, O2-· = superoxide anion. H2O2 = hydrogen peroxide.

AFMK exhibits two anodic waves at Ep(a) of 456 and 668 mV, respectively. This property indicates that AFMK donates two e^- at different potentials to function as a reductive molecule. The presence of anodic waves in CV analysis is also a property of other LMWA including vitamin C, vitamin E, melatonin, glutathione, uric acid, and ß-carboline. These antioxidants show only one anodic wave (donate one e^-) upon CV analysis. The two anodic waves of AFMK imply that AFMK donates one electron more than do classical antioxidants, giving it the ability to neutralize additional reactants.

AFMK also protects macromolecules against oxidative damage. 8-OHdG formation induced by the combination of Cr (III) plus H₂O₂ and lipid peroxidation induced by Fe²⁺ plus H₂O₂ were significantly reduced by the addition of AFMK. The DNA and lipid damage involves free radicals since the two transition metals used reduce H₂O₂ to form the highly reactive and cytotoxic ^.OH. The protective effect of AFMK on DNA oxidative damage is more profound than that on membrane lipids. This difference may be a result of its distribution. As deduced from its structure, AFMK appears to be a hydrophilic molecule, which therefore would be more easily associated with DNA than with lipids.

Whether the antioxidative action of AFMK is related to its ability to directly scavenge free radicals or to its ability to chelate metals was also investigated. Metal chelators retard oxidative damage. AFMK did not modify the absorption spectra of either Cr(III) or Fe²⁺, indicating that AFMK likely prevented both DNA and lipid damage by scavenging free radicals rather than by chelating transition metals.

In cell culture, three different neurotoxins were used to induce death of rat hippocampal neuronal cells (TH22 cell line). H₂O₂ is a classical oxidant that, in high concentration or in the presence of transition metals, triggers oxidative damage in macromolecules due to the generation of the ^.OH. Glutamate is an excitatory amino acid. Via excitatory amino acid receptor-mediated actions, high levels of glutamate induce intracellular calcium overloading, thus generating free radicals that result in neuronal death. Amyloid ß-peptide is related to Alzheimer’s pathology. Deposition of the amyloid ß-peptide in neuronal tissue is followed by free radical generation and neuronal cell damage. AFMK provided protective effects to neurons incubated with these free radical-generating neurotoxins. The death rate in TH22 cells incubated with these neurotoxins was significantly reduced by the addition of AFMK.

In this study, we observed that AFMK functioned as a highly efficient LMWA. The antioxidative mechanism of AFMK is due to its e^- donation to oxidants. The AFMK molecule thereby neutralizes free radicals and provides protection to DNA, lipids, and cultured neurons (Fig. 3) . Thus, we can reasonably speculate that one important function of AFMK is to act as an endogenous antioxidant and form part of an antioxidant defense system with other free radical scavengers and antioxidative enzymes in organisms.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0309fje; to cite this article, use FASEB J. (August 17, 2001) 10.1096/fj.01-0309fje

This article has been cited by other articles:

				X. Ma, C. Chen, K. W. Krausz, J. R. Idle, and F. J. Gonzalez A Metabolomic Perspective of Melatonin Metabolism in the Mouse Endocrinology, April 1, 2008; 149(4): 1869 - 1879. [Abstract] [Full Text] [PDF]

				D.-X. Tan, L. C. Manchester, P. Di Mascio, G. R. Martinez, F. M. Prado, and R. J. Reiter Novel rhythms of N1-acetyl-N2-formyl-5-methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation FASEB J, June 1, 2007; 21(8): 1724 - 1729. [Abstract] [Full Text] [PDF]

				T. W. Fischer, T. W. Sweatman, I. Semak, R. M. Sayre, J. Wortsman, and A. Slominski Constitutive and UV-induced metabolism of melatonin in keratinocytes and cell-free systems FASEB J, July 1, 2006; 20(9): 1564 - 1566. [Abstract] [Full Text] [PDF]

				V. F. Ximenes, S. d. O. Silva, M. R. Rodrigues, L. H. Catalani, G. J. Maghzal, A. J. Kettle, and A. Campa Superoxide-dependent Oxidation of Melatonin by Myeloperoxidase J. Biol. Chem., November 18, 2005; 280(46): 38160 - 38169. [Abstract] [Full Text] [PDF]

				R. J. Reiter, D.-x. Tan, J. Leon, U. Kilic, and E. Kilic When Melatonin Gets on Your Nerves: Its Beneficial Actions in Experimental Models of Stroke Experimental Biology and Medicine, February 1, 2005; 230(2): 104 - 117. [Abstract] [Full Text] [PDF]

				R. J. Reiter and D.-X. Tan Melatonin: a novel protective agent against oxidative injury of the ischemic/reperfused heart Cardiovasc Res, April 1, 2003; 58(1): 10 - 19. [Abstract] [Full Text] [PDF]

				R. Kohen and A. Nyska Invited Review: Oxidation of Biological Systems: Oxidative Stress Phenomena, Antioxidants, Redox Reactions, and Methods for Their Quantification Toxicol Pathol, October 1, 2002; 30(6): 620 - 650. [Abstract] [PDF]

				Vijayalaxmi, C. R. Thomas Jr, R. J. Reiter, and T. S. Herman Melatonin: From Basic Research to Cancer Treatment Clinics J. Clin. Oncol., May 15, 2002; 20(10): 2575 - 2601. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 15/12/2294 01-0309fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by TAN, D.-X. Articles by REITER, R. J. Search for Related Content PubMed PubMed Citation Articles by TAN, D.-X. Articles by REITER, R. J.