Abstract
Background: Melatonin shows antioxidant/prooxidant activity but its mechanism of action remains unknown. Materials and Methods: The radical-scavenging activity of melatonin and various melatonin/co-antioxidant mixtures in a 1:1 molar ratio was evaluated in terms of the length of the induction period (IP) for polymerization of methyl methacrylate (MMA), initiated by thermal decomposition of 2,2′-azobisisobutyronitrile (AIBN) or by benzoyl peroxide (BPO) under nearly anaerobic conditions, which was monitored by differential scanning calorimetry (DSC). Results: The observed IP (A) for a pinoline, L-ascorbyl 2,6-dibutyrate (ASDB), vitamin E (alpha-, beta-, gamma- or delta-T) or 2-mercaptoethanol (2ME) mixture was compared with the calculated total sum of IP (melatonin+each co-antioxidant) (B). For both the AIBN and BPO systems, the A/B for the melatonin/ASDB, beta-T, gamma-T or delta-T mixture was 0.3-0.7, whereas that for the melatonin/2ME mixture was approximately 1. For the AIBN system, the A/B for the melatonin/alpha-T or pinoline mixture was 0.7-0.8. By contrast, for the BPO system, that for the melatonin/alpha-T or pinoline mixture was approximately 1. Conclusion: The prooxidant effect of the melatonin/ascorbate or vitamin E mixtures induced by radical-oxidizing activity may help to explain the anticancer activity of melatonin in biological systems.
- Radical-scavenging activity
- melatonin/co-antioxidant mixtures
- induction period method
- vitamin E
- ascorbate
Melatonin is the major secretory product of the pineal gland and is mostly associated with regulation of the circardian dark/light rhythm of the human body. Recently, melatonin has also been recognized as a potent antioxidant and immunomodulator, and is considered to be an important natural oncostatic agent (1-3).
It has been demonstrated that melatonin acts as a potent scavenger of a variety of radical oxygen species (ROS) and reactive nitrogen species (RNS) (4-5), as well as 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) cation radicals (6) and lipid peroxy radicals (7). We have previously reported the kinetic radical-scavenging activity of melatonin determined using the induction period method (IP) in the radical polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of 2,2′-azobisisobutyronitrile (AIBN) (a source of the alkyl radical, R*) or benzoyl peroxide (BPO) (a source of the benzoyloxy radical, PhCOO*) under nearly anaerobic conditions (8). The model was well able to explain the mechanism of the radical-scavenging activity and to predict the chain-breaking activity of bioactive agents such as polyamines (9), beta-caroteines (10) and vitamin E (11). This model system exploits the high sensitivity of differential scanning calorimetry (DSC), and furthermore, the use of anaerobic conditions makes this system relatively biomimetic, since oxygen is sparse in living cells (12). The radical-scavenging activity of melatonin in vivo does not occur in isolation, but through an intricate antioxidant network together with co-antioxidants such as vitamin C, glutathione (GSH) and vitamin E. Previously, when melatonin was combined with vitamin C, GSH or vitamin E, the protective effects against iron-induced lipid peroxidation were reported to be dramatically enhanced (13). However, the kinetics of the radical-scavenging activity of melatonin with ascorbates, GSH or vitamin E remain unknown under oxygen-insufficient conditions.
In the present study, the radical-scavenging activity of melatonin in combination with vitamin E (alpha-T, beta-T, gamma-T and delta-T), 2-mercaptoethanol (2ME), L-ascorbyl 2,6-dibutyrate (ASDB) or pinoline at a molar ratio of 1:1 was investigated, using IP method for radical polymerization of MMA initiated by AIBN or BPO under nearly anaerobic conditions (8). The comparative radical-scavenging activity of melatonin-related compounds (melatonin, pinoline, 5-methoxytriptamine, 5-methoxyindole, indole) was also investigated. ASDB and 2ME were used because GSH and ascorbate cannot be studied in this system due to their limited solubility in MMA.
Materials and Methods
Materials. Vitamin E (DL-alpha-, DL-beta-, DL-gamma-, DL-delta-tocopherol), N-acetyl-5-methoxytryptamine (melatonin), indole, 5-methoxyindole, 5-methoxytryptamine, pinoline, ASDB, 2ME, MMA, BPO and AIBN were obtained from Tokyo Kasei Chemical Co. (Tokyo, Japan). The AIBN and BPO were recrystallized from methanol and chloroform/methanol (1:2.5 v/v), respectively.
Induction period (IP) and initial rate of polymerization (Rp). The time-conversion curves for melatonin and related compounds are shown in Figure 1. The IP and initial Rp in the presence (Rpinh) or absence (Rpcon) of an antioxidant were determined using the method reported previously (7-10). In brief, the experimental resin consisted of MMA and AIBN (or BPO) with or without melatonin, co-antioxidants or the melatonin/coantioxidant mixture (1:1 molar ratio). AIBN (or BPO) was added at 1.0 mol% and additives at 0.1 mol% (10 mM). Approximately 10 mL of the experimental resin (MMA: 9.12-9.96 mg) was loaded into an aluminum sample container and sealed by applying pressure. The container was placed in a differential scanning calorimeter (model DSC 3100; MAC Science Co., Tokyo, Japan) kept at 70°C, and thermal changes induced by polymerization were recorded for the appropriate periods. The heat due to polymerization of MMA was 13.0 kcal/mole in these experiments. The conversion rate of the samples employed was 94.24-95.70%. Polymerization curves were derived from the DSC thermograms using the integrated heat evoked by the polymerization of MMA. Polymerization curves break when an initiator is consumed (Figure 1). These breaks are sharp and provide a reliable measure of the IP of the inhibitor. The presence of oxygen inhibits polymerization because oxygen reacts with AIBN and BPO radicals, and therefore the polymerization curves break when oxygen is completely consumed. The length of IP due to oxygen in a DSC container was the IP for the controls. The IP was calculated from the difference between the IP of each inhibitor and that of the control. The initial Rpcon and Rpinh of the melatonin, co-antioxidants and melatonin/co-antioxidant mixtures were calculated from the initial slope of the linear plot for the conversion rate of MMA polymerization (tangent drawn at the early polymerization stage) (Figure 1). The initiation rates (Ri) for AIBN and BPO at 70°C were 5.66×10−6 M s−1 and 2.28×10−6 M s−1, respectively.
Results
Radical-scavenging activity of melatonin-related compounds. The results are shown in Table I. The IP value for the AIBN system declined in the order 5-methoxytryptamine> melatonin≈pinoline>5-methoxyindole>indole. The Rpinh/Rpcon value for the melatonin-related compounds was 0.94-1.0 and was similar to that of the control. In contrast, the IP value for the BPO system declined in the order 5-methoxy-tryptamine≈5-methoxyindole≈melatonin>pinoline>indole. The Rpinh/Rpcon value was 0.7-0.8. The melatonin-related compounds in the BPO system largely suppressed the propagation rate in comparison with those in the AIBN system.
Radical-scavenging activity of the melatonin/co-antioxidant mixture. The observed IP (A), calculated IP (B) (the simple sum of the IP for melatonin and that for the co-antioxidant), B-A, the ratio of A to B (A/B) and Rpinh/Rpcon are shown in Table II. For the AIBN system, the A/B value for the melatonin/pinoline mixture at a molar ratio of 1:1 was approximately 0.7, showing that the IP of melatonin with pinoline decreased by approximately 30%. In contrast, for the BPO system, the corresponding A/B value was approximately 1.0, showing that the radical-scavenging activity of melatonin and pinoline was additive. The A/B value for the melatonin/ASDB mixture in the AIBN and BPO systems was approximately 0.3 and 0.6, respectively; the antioxidant activity of melatonin in combination with ASDB was considerably reduced. Namely, for both systems, the intrinsic antioxidant activity of melatonin was markedly counterbalanced by ASDB, and also the decrease in the Rpinh/Rpcon value was evident for the BPO system, but not for the AIBN system. The A/B values of the melatonin/vitamin E (alpha-, beta-, gamma- or delta-T) mixtures were approximately 0.8 for the AIBN system. In contrast, those for the BPO system declined in the order alpha-T (1.0)>beta-T (0.8)>gamma-T (0.7)>delta-T (0.5). The antioxidant activity of melatonin was not affected by alpha-T. The Rpinh/Rpcon value for the melatonin/beta-T, gamma-T or delta-T mixture declined to a greater extent than that for the alpha-T mixture in both systems. By contrast, the value for the melatonin/2ME mixture was approximately 1 in both systems. Also, its Rpinh/Rpcon value was similar to that of the control.
Stoichiometric factor (n). The stoichiometric factor of vitamin E for the AIBN and BPO system was determined from the findings shown in Table II. When the initiating radicals are generating at a constant rate by AIBN or BPO, the n* of vitamin E and the n** of vitamin E in the melatonin/vitamin E mixture are given by equations 1 and 2, respectively: n*=[IP]/[IH]×Ri (1) and n**=([IP] of the melatonin/vitamin E mixture−[IP] of melatonin)/([IH]×Ri) (2), where [IH] is the concentration of vitamin E, an inhibitor.
For the AIBN systems, the n* of alpha-T, beta-T, gamma-T and delta-T was 1.7, 1.9, 2.1 and 2.0, respectively. By contrast, the corresponding n** was 1.2, 1.2, 1.4 and 1.5, respectively. The n** of vitamin E was a roughly half of the corresponding n*. Generally, vitamin E scavenges two radicals (11). Vitamin E with an n value of about 1 indicates dimerization.
By contrast, for the BPO system, the n* of alpha-T, beta-T, gamma-T and delta-T was 0.1, 0.5, 0.6 and 1.3, respectively, whereas the corresponding n** was 0.1, 0.3, 0.3 and 0.5, respectively. The n** of vitamin E was a roughly half of the corresponding n*. From these findings, the radical-scavenging activity of vitamin E for the AIBN system was greater than that for the BPO system. Upon both AIBN- and BPO-radical scavenging, melatonin exerted a suppressive effect on vitamin E. However, for the BPO system, the n* and n** of alpha-T was similar.
Discussion
The radical-scavenging activity of melatonin and the melatonin-related compounds with the co-antioxidants was divisible into three classes: efficient (beta-, gamma-, delta-T), intermediate (melatonin, pinoline, 5-methoxytryptamine, and 5-methoxyindole), and poor (alpha-T, indole, 2ME and ASDB). Interestingly, the antioxidant activity of alpha-T upon R* (AIBN radical) scavenging was similar to the efficient tocopherol antioxidant group, whereas that upon PhCOO* (BPO radical) scavenging was in the poor antioxidant group. Thus, the radical-scavenging activity of alpha-T was dependent on the radical species, which was in agreement with a previous report (11). Alpha-T may be a modulator of the cellular redox status, but does not necessarily act as an intracellular antioxidant upon peroxy (ROO*) radical-scavenging in vivo (14). The present data for the scavenging of alpha-T upon PhCOO* suggested that alpha-T may not necessarily act as an intracellular antioxidant upon peroxy radical scavenging. AIBN in the presence of sufficient oxygen produces ROO*, the peroxy radical, whereas in the presence of insufficient oxygen it produces R*, the alkyl radical. The radical-scavenging activity of extracellular antioxidants for the azoinitiator radical (R*) under anaerobic conditions has been reported previously, indicating that alpha-T is an ineffective scavenger and also that ascorbate is a poor scavenger (15). Since oxygen is sparse in living cells (12), the radical-scavenging activity of antioxidants should be investigated at a low oxygen pressure. The comparative radical-scavenging activity of melatonin with vitamin E (mostly alpha-T) and vitamin C against ROO* has previously been reported to decline in the order vitamin E>melatonin>GSH>vitamin C (12) and also to decline in the order vitamin E>pinoline> melatonin (16). The hydroxyl radical scavenging activity of melatonin has previously been reported to be greater than that of pinoline (17). The present study indicated that the radical scavenging activity of melatonin upon PhCOO* scavenging was similar to, or slightly higher, than that of pinoline. The present results suggested that for ROO* scavenging, the radical-scavenging activity of alpha-T may be lower than that of melatonin.
The difference in reduction potential between melatonin and a co-antioxidant can provide useful information about the antioxidant activity of melatonin/co-antioxidant mixtures. When the reduction potential of co-antioxidants is lower than that of melatonin, co-antioxidants preferentially become radicals in the melatonin/co-antioxidant mixture oxidized by PhCOO* or R*, and thus the generation of co-antioxidant radicals could be involved in some of the antioxidative activity of melatonin. The reduction potentials of melatonin-related compounds and conventional co-antioxidants (A*/A−) at pH 7 have been shown to be 0.64 V for tryptamine, 0.53 V for indole, 0.33 V for ascorbic acid, 1.33 V for 2ME and 0.48 V for trolox C (assuming a similar value for alpha-T) (18). Also, the reduction potential of melatonin, a tryptamine, has been shown to be 0.95 V (19). It was assumed that melatonin might have the highest reduction potential among the indicated vitamin C and vitamin E analogs (assuming that the values for beta-T, gamma-T and delta-T are lower than that for alpha-T), estimated on the basis of the phenolic O-H bond dissociation enthalpy (BDE) (20). When the melatonin/ASDB or beta-T, gamma-T or delta-T mixture is oxidized by both R* and PhCOO*, ASDB and tocopherols, but not melatonin, are initially able to scavenge initiator radicals. The A/B value of <1 for the melatonin/ASDB mixture suggested that ASDB oxidized by R* and PhCOO* suppressed the intrinsic antioxidant activity of melatonin itself, resulting from the finding that upon both R* and PhCOO* scavenging, the observed IP (min) of melatonin was greater than that of the melatonin/ASDB mixture (Table II). The A/B value of <1 for the melatonin/vitamin E mixture suggested that melatonin may have exerted a suppressive effect on the antioxidant activity of vitamin E. For the AIBN system, melatonin probably is associated with s dimerization of vitamin E compounds, as indicated by the finding that the n** of vitamin E was about 1.
The prooxidant activity of melatonin in biological systems (21) may be implicated in its interaction with co-antioxidants. Interestingly, upon PhCOO* scavenging, the melatonin/alpha-T, pinoline or 2ME mixture showed a A/B value of 1, suggesting that the antioxidant activity of melatonin may not be affected by alpha-T, pinoline or GSH upon ROO*scavenging. Alpha-T could act as a modulator of the cellular redox status, and also GSH could act by protecting cells against oxidative damage.
The synergistic action of melatonin with vitamin C, vitamin E and glutathione has been reported previously to exert an increased protective effect against iron-induced lipid peroxidation when melatonin was combined with vitamin E, vitamin C and GSH (13). Also, alpha-T was reported previously to act synergistically with indoleamines such as melatonin (16). However, the present findings under anaerobic conditions were not in accord with those described above. Synergistic action involving regeneration between antioxidants and co-antioxidants has been shown to occur when the BDE for antioxidants is similar to that of co-antioxidants, or slightly less (22). Since the difference in the reduction potential between melatonin and vitamin C or vitamin E is considerable (18), no synergistic action between the antioxidant melatonin and vitamin C or vitamin E could occur. Further studies will be necessary to clarify the antioxidant mechanism of melatonin in the presence of vitamin E, vitamin C or GSH. The great discrepancy between our results and those in the literature (13, 16) may be closely related to oxygen pressure (15), although the experimental procedures were also considerably different. Despite the large disparity between the present experimental conditions and those existing in natural biological systems, the present findings may help to explain the biphasic mechanism of the antioxidant/prooxidant activity of melatonin in the presence of the co-antioxidants ascorbate, GSH and vitamin E.
- Received July 24, 2010.
- Revision received November 12, 2010.
- Accepted November 15, 2010.
- Copyright © 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved