Reaction of Melatonin With Lipoperoxyl Radicals in Phospholipid Bilayers

Free Radical Biology & Medicine, Vol. 23, No. 5, pp. 706–711, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00

PII S0891-5849(97)00018-X

Original Contribution REACTION OF MELATONIN WITH LIPOPEROXYL RADICALS IN PHOSPHOLIPID BILAYERS MARIA A. LIVREA, LUISA TESORIERE, DANIELE D’ARPA, and MASSIMILIANO MORREALE Istituto di Farmacologia e Farmacognosia, Facolta’ di Farmacia, Universita’ di Palermo, 90134 Palermo, Italy (Received 13 September 1996; Revised 17 January 1997; Accepted 28 January 1997)

Abstract—Melatonin, at 5 to 500 mM was incorporated in unilamellar soybean phosphatidylcholine (PC) liposomes, the peroxidation of which was induced by 2,2 *-azobis(2-amidinopropane-hydrochloride) (AAPH), and measured as production of conjugated diene lipid hydroperoxides. Concentration as low as 5 and 10 mM were poorly effective in reducing lipid peroxidation. Melatonin at 30 to 500 mmM caused short inhibition periods, increasing with, but not linearly related to concentration, with a concurrent net decrease of the propagation rate. The time course of melatonin oxidation, measured as loss of fluorescence, was studied during the AAPH-stimulated peroxidation of soybean PC liposomes, or when melatonin was incorporated in nonperoxidable unilamellar dimirystoyl phosphatidylcholine (DMC) liposomes. Consumption kinetics of 30 mM melatonin were linear with time in DMC liposomes and disappearance of melatonin occurred at a rate of 0.058 M 08s 01 . On the other hand, the consumption of melatonin during the oxidation of soybean PC liposomes, was not linear with time. The rate of disappearance was calculated as 0.19 M 08s 01 at the beginning of the propagation phase, then it slowed down to reach the same rate observed in DMC liposomes. This evidence suggests a reaction with lipid-derived peroxyl radicals, possibly in addition to reaction with peroxyl radicals derived from AAPH. Scavenging of lipoperoxyl radicals by melatonin was also evident in experiments where melatonin was incorporated in multilamellar soybean PC liposomes and peroxidation was initiated by 2,2 *-azobis(2,4-dimethyl-valeronitrile). The antioxidant activity of melatonin in soybean PC liposomes is much lower than that of a-tocopherol, under comparable assay conditions. However, a combination of melatonin and a-tocopherol, at 5 mM, resulted in a synergistic antioxidant effect. Time course of a-tocopherol consumption, monitored in the absence and in the presence of melatonin, showed a significant decrease of the consumption rate when compounds were combined, indicating some protection by melatonin. Regeneration mechanisms were not evident and depletion of a-tocopherol was coincident with the inhibition time. q 1997 Elsevier Science Inc. Keywords—Melatonin, Lipid peroxidation, Liposomes, Antioxidants, Vitamin E, Synergism, In vitro

glutathione peroxidase 6 and by raising the mRNA for superoxide dismutase.7 Cell life and function are largely committed to membrane integrity. Melatonin, which is both hydrophilic and hydrophobic and easily travels accross cell membranes, has been investigated for a possible role in the antioxidant protection of membrane lipids. Several reports, in which animals 8,9 or tissue homogenates 10 – 14 were subjected to lipid peroxidation, demonstrated that a substantial protection was attained in the presence of melatonin. All these studies unequivocally demonstrate the ability of melatonin to reduce lipid peroxidation, an effect that in most cases could be related to the scavenging of the initiator oxygen radi-

INTRODUCTION

Melatonin, the hormonal product of the pineal gland, has recently been reported as an effective hydroxyl radical scavenger, 1 an ability that shed some light on several of its nonreceptor-related actions. Other in vivo and in vitro studies gave evidence that the indoleamine may protect DNA from peroxidative damages by chemical carcinogens, 2,3 avoid cytogenetic damage by ionizing radiations, 4,5 and also protect the whole cell antioxidant defense system by increasing the activity of Address correspondence to: Maria A. Livrea, Istituto di Farmacologia e Farmacognosia, Universita’ di Palermo, Via C. Forlanini, 1, 90134 Palermo, Italy. 706

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Melatonin as antioxidant in liposomes

cals employed in the in vitro systems or produced in vivo. A modest activity in inhibiting lipid peroxidation stimulated by FeCl3 and ascorbate in ox-brain liposomes has recently been reported.15 However, it is not clear if melatonin can react with lipid-derived peroxyl radicals. In this article we report preliminary evidence that melatonin interacts with and scavenges lipoperoxyl radicals in soybean phosphatidylcholine liposomes. Moreover, a synergistic antioxidant effect with a-tocopherol is observed. MATERIALS AND METHODS

Melatonin, phosphatidylcholine (PC) from soybean, dimirystoyl phosphatidylcholine (DMC), a-tocopherol, were from Sigma Chemical Company (St. Louis, MO). 2,2 *-Azobis(2-amidino-propane)hydrochloride (AAPH) and 2,2 *-azobis(2,4-dimethyl-valeronitrile) (AMVN) were from Polyscience, Inc. (Warrington, PA), Chelex-100 ion-exchange resin was from Bio Rad, and Sephadex G-25 (Fine) was from Pharmacia. All other reagents and chemicals were of the highest purity or HPLC grade. Buffers used throughout this study were chromatographed over Chelex-100, and suitable plastic labware was used to avoid the effects of adventitious metals. Unless otherwise stated, all operations described herewith were carried out under red light to avoid possible photo-oxidation of fatty acids and to preserve lightsensitive a-tocopherol. Peroxidation assays Large unilamellar liposomes, which incorporated variable amounts of melatonin and/or a-tocopherol, were prepared as previously described 16 by the aid of an Avestin Liposofast (Avestin, Inc. Ottawa, Canada) small volume extrusion device provided with a polycarbonate membrane of 100 nm pore size. Peroxidation was stimulated by 2.0 mM AAPH, added to the suspensions in a small volume of 0.9% NaCl in 5 mM phosphate buffer pH 7.4 (PBS), and incubation was at 377C, under air. To evaluate lipid peroxidation, aliquots of liposomes (20 ml) were taken at 10-min intervals, and dissolved in 50 vol of absolute ethanol. Spectra were then recorded in the range 200 to 300 nm, and the conjugated diene hydroperoxide production was measured by the increase in absorbance at 234 nm, using a molar absorption coefficient of 27,000.17 In other experiments, soybean PC multilamellar liposomes incorporating melatonin were prepared as described 18 and peroxidation was stimulated by 2.5 mM AMVN. Incubation and analysis of conjugated dienes was as above.

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The consumption of melatonin during lipid peroxidation was assayed by spectrofluorimetric measurements, with excitation at 227 nm and emission at 336 nm, on aliquots of the incubation mixture, taken at 15 min intervals and suitably diluted with PBS. The consumption of a-tocopherol was evaluated by extracting aliquots of liposomes diluted to 1.0 ml with PBS, with 2 vol of absolute ethanol and 8 vol of petroleum ether. The organic extract was dried under nitrogen, resuspended in suitable solvent, and analyzed by a Supelco Supelcosily LC-18 column ( 0.46 1 25 cm) , eluted with methanol at 1.5 ml min 01 . Detection was at 290 nm. Kinetic measurements A computer-assisted analysis (Table Curve 2D, Jandel, CA) of the experimental peroxidation curves provided the propagation rate of the uninhibited reaction, Rp , the initiation rate, Ri , as well as the inhibition period (tinh ). Rp is measured as the amount of lipid hydroperoxides formed per second in the absence of any antioxidant or after the inhibition period. Ri is calculated by the inhibition period caused by a-tocopherol, 19 assuming a stoichiometric factor of 2.0. tinh , is calculated by the x-coordinate of the intercept of the tangents to the parts of the curve representing the inhibition and propagation phases. Partitioning studies After 10 mM either soybean PC or DMC unilamellar liposomes incorporating 30 mM melatonin were prepared, they were allowed to remain at 377C in a water bath, under nitrogen, for 150 min. Then 20 ml of either mixture were diluted to 4 ml with PBS and a fluorimetric analysis was carried out as reported above, to evaluate ‘‘total’’ melatonin (i.e., membrane-bound and aqueous phase). Liposome suspensions were then submitted to gel filtration on a Sephadex G-25 column (0.8 1 10 cm) eluted with PBS. Fractions (1 ml) were collected and 20 ml of each fraction were analyzed fluorimetrically as above. A 30 mM solution of melatonin in PBS was processed similarly. Melatonin recovered in the fractions eluting with the void volume of the column was considered liposome bound. Free melatonin eluted with the included volume. RESULTS

Peroxidation of unsaturated lipids is a self-propagation process, in which a single initiating free radical abstracts a hydrogen atom from a methylene carbon of the lipid, giving a carbon-centered radical (initiation

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phase). The carbon centered radical reacts with oxygen, thus forming a very reactive lipoperoxyl radical that may easily attack another lipid molecule, thereby propagating the radical chain reactions (propagation phase). Finally, the lipoperoxyl radicals combine to form nonradical products (termination phase). Radical scavenging antioxidants may block this process either by eliminating the initiator radicals or by interacting with the chain-carrying lipoperoxyl radicals. In the absence of antioxidant, the AAPH-induced production of conjugated diene hydroperoxides in soybean PC liposomes proceeded at a constant rate, and Rp was measured as 15.2 M 08s 01 . The initiation rate, measured by the inhibition period produced by 5 mM a-tocopherol, was 0.52 M 08s 01 , and the kinetic chain length (kcl), as expressed by the ratio Rp /Ri , was 29.2. Concentration dependence of the suppression of lipid peroxidation by melatonin was investigated by evaluating peroxidation rates when 5 to 500 mM melatonin were incorporated into liposomes. Amounts of melatonin as low as 5 and 10 mM did not cause any inhibition period, although slightly reduced the production of conjugated diene hydroperoxides (Fig. 1). Short inhibition periods, that were not linearly related

to the concentration of antioxidant, were observed at 30 to 500 mM melatonin (Fig. 1), although no linear relationship was evident between the duration of the inhibition period and the antioxidant concentration. Moreover, melatonin at 30 to 500 mM slowed down the kinetic chain length during the propagation phase. The propagation rate after the inhibition period, was markedly lower than in the absence of antioxidant, decreased in a concentration-dependent manner, but was not linearly related to melatonin concentration (Fig. 1, inset). Melatonin is both hydrophilic and lipophilic, so it can be supposed to interact either with radicals formed in the aqueous phase and/or with lipid-derived peroxyl radicals inside the bilayer. To distinguish between a reaction of melatonin with AAPH-derived peroxyl radicals, or an interaction with chain-carrying lipid peroxyl radicals, experiments were carried out in which 30 mM melatonin was incorporated in either DMC or soybean PC liposomes and incubated at 377C in the presence of 2 mM AAPH. Oxidation of melatonin was then determined by loss of fluorescence. This analysis revealed that consumption of melatonin was linear with time, with a rate of 0.058 M 08s 01 , when it was incorporated in DMC liposomes (Fig. 2). On the other hand,

Fig. 1. Concentration-dependent antioxidant effect of melatonin incorporated in 10 mM unilamellar soybean PC liposomes, in the presence of 2 mM AAPH. Reaction mixtures were incubated at 377C, under air and contained no melatonin (*) or 5 mM ( h ), 10 mM ( j ), 30 mM ( l ), 100 mM ( s ), 250 mM ( n ), or 500 mM ( m ) melatonin. Conjugated diene hydroperoxides (LOOH) were quantified as reported in Materials and Methods. Inset: relationship between propagation rate and melatonin concentrations. Each point represents the mean of four determinations, carried out with different incubation mixtures. SD ° 10%.

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Melatonin as antioxidant in liposomes

Fig. 2. Time course of consumption of 30 mM melatonin incorporated in either DMC liposomes ( s ) or soybean PC liposomes ( l ), in the presence of 2.0 mM AAPH. Values are corrected for the loss of fluorescence of 30 mM melatonin, incorporated in the relevant liposome preparation, in the absence of AAPH. Dotted line represents the difference curve. Points in each curve represent the mean of three separate experiments with SD ° 8%.

consumption kinetics of melatonin in soybean PC liposomes were not linear (Fig. 2). A higher consumption rate, calculated as 0.19 M 08s 01 from the curve describing the molar decrease, was measured in 15 min, which covered the inhibition period observed for 30 mM melatonin. Then the rate of melatonin disappearance slowed down to reach that measured in DMC liposomes. When corrected for the reaction with AAPHderived peroxyl radicals, the consumption rate of melatonin in soybean PC liposomes may approach the apparent rate at which melatonin reacts with lipoperoxyl radicals. This analysis revealed that melatonin was consumed at a rate of 0.13 M 08s 01 during the inhibition period. As a comparison, the rate of disappearance of 30 mM a-tocopherol incorporated in liposomes, under comparable conditions, was evaluated as 0.17 M 08s 01 . A different mobility and accessibility of melatonin in liposomes from different phosphatidyl cholines could involve a different consumption of the molecule by AAPH-derived peroxyl radicals. Results from partitioning studies showed that, within the experimental time, 35% of melatonin incorporated into unilamellar liposomes diffuses into the aqueous phase, both in soybean PC and in DMC liposomes. Therefore, accessibility of melatonin to aqueous radicals generated from the azo compound may be considered comparable in both systems. Antioxidant activity of melatonin was also investigated when free radicals were generated in the lipid phase. Reaction of 10 to 250 mM melatonin, incorporated in 10 mM multilamellar soybean PC liposomes, is depicted in Fig. 3. Melatonin was poorly effective at a 10 mM, whereas concentrations as high as 50 and 250 mM caused distinct inhibition periods. Differently from

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the lipid system in which the water soluble AAPH was used, melatonin did not show any effect on the propagation rate after the inhibition period (Fig. 3). Alpha-tocopherol is the most important chain-breaking antioxidant in lipid systems. Under the applied assay conditions, melatonin was an antioxidant much less effective than a-tocopherol, on a concentration/effect basis. However, we evaluated the antioxidant activity of a combination of melatonin and a-tocopherol, both at 5 mM, incorporated in soybean PC liposomes, in comparison with the activity of the individual compounds (Fig. 4). Because 5 mM melatonin was an inefficient inhibitor when used alone (tinh Å 0), the data in Fig. 4 provide evidence for synergistic interactions. The time course of consumption of a-tocopherol, in the absence and in the presence of melatonin, is also shown in Fig. 4. Consumption of a-tocopherol did not appear to be prevented for any time by the concurrent presence of melatonin in the liposome, however, it was slowed down (Fig. 4). Depletion of a-tocopherol was coincident with the inhibition time.

DISCUSSION

Studies with pure chemical systems unequivocally gave evidence that melatonin is a radical scavenger. The work of Tan et al.1 demonstrated that it can scavenge the very toxic hydroxyl radical, when generated by photolysis of H2O2 . It has been suggested that de-

Fig. 3. Antioxidant effect of melatonin incorporated in 10 mM multilamellar soybean PC liposomes in the preence of 2.5 mM AMVN. Reaction mixtures were incubated at 377C, under air and contained no melatonin (*), or 10 mM ( l ), 50 mM ( m ), or 250 mM ( j ) melatonin. Each point represents the mean of four determinations, carried out with different incubation mixtures. SD ° 10%.

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Fig. 4. Inhibition of the oxidation of 10 mM soybean unilamellar PC liposomes (*) by 5 mM melatonin ( l ), 5 mM a-tocopherol ( m ), or by their combination ( j ) and consumption of a-tocopherol in the absence ( s ) or in the presence ( n ) of melatonin. Peroxidation was induced by 2 mM AAPH, at 377C, under air. Conjugated diene hydroperoxides (LOOH) were measured as reported in Materials and Methods. Points are the mean of three separate experiments with SD ° 10.4%.

toxification of electrophile free radicals occurs by oneelectron transfer reaction from the indoleamine, that is rapidly converted to the corresponding kynuramine metabolite.20 One-electron transfer reactions have also been ascertained by assaying the ability of melatonin to inhibit the hydroxyl radical-stimulated formation of ABTS cation radical, in aqueous systems, in the presence or in the absence of brain homogenates.21 In addition, laser flash photolysis studies showed that melatonin can donate a hydrogen atom to alkoxyl radicals.22 This reactivity may also explain the observed ability of melatonin to quench AAPH-derived peroxyl radicals in a cell-free system23 and possibly the lipoperoxyl radical scavenging activity observed in the present work. In this article antioxidant behavior of melatonin has been analyzed in terms of a chain breaking antioxidant, acting in a biomimetic liposomal system. Melatonin does not appear a conventional chain-breaking antioxidant in this system. Although concentration dependent, the antioxidant effect, in terms of inhibition period and decrease of the kinetic chain length, was not linearly related to the concentration, rather a decrease of relative efficiency was observed by increasing melatonin amounts. The time course of melatonin consumption in DMC liposomes demonstrates that it easily reacts with AAPH-derived peroxyl radicals. Melatonin depletion is not preserved by the presence of peroxidable lipids. Rather, the rate of consumption increases in the presence of soybean phosphatidylcholine, at the same time

that lipid oxidation is slowed down, which suggests reaction with lipoperoxyl radicals. Interaction of melatonin with lipoperoxyl radicals was confirmed by the reaction of melatonin in multilamellar liposomes stimulated with a lipophilic azoinitiator. The apparent rate of disappearance of melatonin during peroxyl radical oxidation has been calculated as only slightly lower than the rate of disappearance of a-tocopherol under comparable conditions; however, the antioxidant effect in terms of inhibition rate and inhibition period is much lower. This evidence may point out that the rate constant for the reaction of melatonin with lipoperoxyl radicals is much smaller than the constant of a-tocopherol in our liposomal system. Very high rate constants, of the order of 10 11 , have been calculated for the reaction of melatonin with peroxyl radicals in aqueous solutions.21 Incorporation of melatonin in membrane-mimicking systems could largely vary melatonin reactivity, limiting its antioxidant activity. On the other hand, if donation of a hydrogen atom22 can occur to quench lipoperoxyl radicals, the fate of the radical derived from melatonin, which in turn can be reactive, is essential to determine the overall antioxidant activity. Prooxidant effects, already described, 24 would perturb the radical scavenging action. The IC50 measured for the inhibition of peroxidation of ox-brain phospholipid liposomes, incubated with FeCl3 and ascorbate 15 in the presence of ethanol solutions of melatonin, is also indicative of a limited antioxidant activity. Synergistic interactions between melatonin and antioxidants such as trolox, glutathione, and ascorbate have been reported in aqueous systems.25 Here, we gave evidence of a synergistic antioxidant protection with a-tocopherol in a lipid bilayer. The mechanism of this synergistic effect is far from clear. The time course of a-tocopherol consumption in the presence of melatonin does not give evidence for recycling, although its consumption rate is slowed down. Possibly, an effective defense by reaction of melatonin with aqueous radicals generated from AAPH will reduce the amount of attacking radicals on the lipid system, which in turn, would result in elongation of the inhibition period by a-tocopherol and a decrease of its consumption rate. At any instance, because depletion of a-tocopherol in the presence of melatonin was coincident with the observed inhibition time, it is suggested that scavenging of lipoperoxyl radicals was almost entirely supported by a-tocopherol.

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ABTS — 2,2 *azino-bis ( 3-ethylbenzthiazoline-6-sulfonic acid) AAPH — 2,2 *-azobis ( 2-amidinopropane-hydrochloride) AMVN—2,2 *-azobis(2,4-dimethyl-valeronitrile) DMC—dimirystoyl phosphatidylcholine kcl—kinetic chain length PC—phosphatidylcholine Ri —initiation rate Rp —propagation rate tinh —inhibition period

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