Original contributionCooperation of antioxidants in protection against photosensitized oxidation
Introduction
Photodynamic damage occurs when photoexcited molecules activate oxygen to produce reactive oxygen species, such as free radicals (Type I) or singlet oxygen (Type II) 1, 2, 3. This effect has been utilized in photodynamic therapy of cancer or neovascularization in the retina 2, 3, 4. However, photodynamic damage may occur in the skin or in the eye as an undesirable effect of exposure to light 5, 6, 7, 8. Carotenoids have been found to protect effectively against photodynamic damage 8, 9, 10. Oral supplementation with β-carotene is now a part of the standard regimen of treatment for erythropoietic protoporphyria, a disease caused by accumulation of protoporphyrin IX in the skin, which induces severe photosensitized damage during exposure to sunlight [8]. Also in the eye, particularly in the retina, carotenoids are believed to play a protective role against photodamage 7, 11. Carotenoids act as efficient quenchers of excited triplet states of photosensitizers or singlet oxygen 12, 13, 14. The rates of interactions of carotenoids with singlet oxygen are close to diffusion controlled limits and occur via energy transfer followed by thermal deactivation of the carotenoid triplet state. It has been observed that carotenoids may also efficiently protect against free radical-initiated lipid peroxidation 15, 16, 17, 18. It has been shown, however, that interaction of carotenoids with peroxyl radicals leads to the formation of radical adducts and/or hydrogen (or electron) transfer and formation of carotenoid radicals 19, 20. Radical adducts may further interact with oxygen-forming peroxyl radicals, which then, in turn, take part in propagation of lipid peroxidation. The carotenoid-derived peroxyl radicals are believed to be responsible for pro-oxidant behavior of carotenoids observed at high oxygen tensions 21, 22, 23. Carotenoid radical cations may lead to oxidative damage to proteins due to oxidation of tyrosine or cysteine [24].
It has been observed in several systems that antioxidant protection is enhanced in a synergistic way in the presence of a combination of antioxidants such as carotenoid and vitamin E 25, 26, 27, 28. However, in other systems, a combination of these antioxidants exerted less than additive protection against free radical- and photo-induced formation of peroxides 26, 29, 30.
This study is focused on the role of zeaxanthin and α-tocopherol in photosensitized lipid peroxidation mediated by both singlet oxygen and free radicals. Zeaxanthin is a carotenoid containing two hydroxy groups and is therefore classified as a xanthophyll—a carotenoid-containing oxygen. It has been shown in many systems that xanthophylls are similarly effective in singlet oxygen quenching as hydrocarbon carotenoids such as β-carotene but they exhibit a smaller tendency toward degradation or pro-oxidant behavior 16, 31, 32, 33. Zeaxanthin accumulates in many organs, including the eye and the skin, reaching particularly high concentrations in the macula lutea of the primate retina 34, 35, 36, 37, 38.
α-Tocopherol (vitamin E) is a predominant lipophilic antioxidant, which is an efficient scavenger of alkoxyl and peroxyl radicals 39, 40. As a result of hydrogen transfer, lipid radical is reduced to alcohol or hydroperoxide and chromanoxyl radical is formed. Chromanoxyl radical is less efficient in propagation of lipid peroxidation and, therefore, α-tocopherol acts as an efficient chain-breaking antioxidant. Chromanoxyl radical may be reduced by ascorbate so the α-tocopherol molecule becomes regenerated [41]. α-Tocopherol also quenches singlet oxygen with a rate constant, which is almost two orders of magnitude lower compared with carotenoids in solution 12, 42. In case of α-tocopherol, the chemical quenching plays an important role in interaction of α-tocopherol with singlet oxygen [42].
In a system where oxidation occurs via both singlet oxygen and free radical mechanisms, maximum protection would be achieved if zeaxanthin was quenching efficiently singlet oxygen and α-tocopherol–scavengeing free radicals. However, zeaxanthin would also interact with free radicals and α-tocopherol would be oxidized by singlet oxygen. The relative efficiencies of these processes depend on bimolecular rate constants of interactions of the antioxidants with singlet oxygen and lipid radicals and concentrations of the reactants. Even though in solution some of the bimolecular rate constants of the interactions have been determined, it is difficult to extrapolate these data to heterogeneous systems, such as lipid membranes. Several factors need to be considered, such as the site of generation of singlet oxygen and the distribution and mobility of antioxidants in the membrane, for they may affect the resultant rates of interactions of antioxidants with singlet oxygen and lipid radicals. Indeed, it has been determined that the rate constants of singlet oxygen quenching by antioxidants in liposomal membranes are 88 and 640–1230 times lower than that observed in solution for α-tocopherol and carotenoids, respectively [43]. Therefore, it is unclear whether these two micronutrients—zeaxanthin and α-tocopherol—may offer synergistic protection against photodynamic damage to membrane lipids. Such synergistic protection may occur if (i) at least one of the antioxidants offers protection against reactive species that damage the other antioxidant, and/or (ii) one antioxidant recycles the other antioxidant. In systems where photodynamic action takes place, zeaxanthin could protect α-tocopherol from oxidation by singlet oxygen, while α-tocopherol could protect zeaxanthin from free radical damage—either preventing interaction with free radicals (by competitive scavenging) or by reduction and, therefore, repair of carotenoid radical cation. As the accumulation of micronutrients, such as carotenoids or tocopherols, in the tissues may be modulated to a certain degree by the diet 44, 45, better understanding of their metabolic pathways and mutual interactions may help to design appropriate dietary supplements.
The aim of this work was to test the hypothesis that α-tocopherol and zeaxanthin offer synergistic protection against photodynamic damage to membrane lipids mediated by both singlet and free radicals. Liposomes consisting of phosphatidylcholine and cholesterol were used as a model biological membrane. Photosensitized peroxidation of lipids was induced by irradiation with orange light (520–600 nm) in the presence of 20 μM rose bengal. Cholesterol was employed to elucidate the mechanism of photosensitized oxidation in the membrane lipids and the mechanism of protection offered by single antioxidants or their combination [46]. Cholesterol, like all unsaturated lipids, is sensitive to oxidative modifications and, most importantly, can serve as a highly specific mechanistic probe in situ, which provides information about the type of damage, whether it is mediated by free radicals or singlet oxygen [46]. In free radical-mediated reactions, including Type I photodynamic reactions, many different cholesterol hydroperoxides can arise, the most prominent being the epimeric pair 7α-OOH and 7β-OOH [46]. In reactions mediated by singlet state molecular oxygen (1Δg), only three primary species are possible, 5α-OOH, 6α-OOH, and 6β-OOH, each of which arises via ene addition [46]. The 7α- and 7β-OOH cannot be generated directly from singlet oxygen but might arise indirectly via the allylic rearrangement of 5α-OOH [46]. This rearrangement becomes more favorable as the extent of membrane peroxidation increases, but is usually insignificant in lightly oxidized membranes with <5% cholesterol loss [46]. Importantly, iron-mediated decomposition of different cholesterol hydroperoxides, i.e., 5α-OOH, 6α,β-OOH, and 7α-OOH, occurs at about the same intrinsic rate [46].
Section snippets
General chemicals and reagents
Cholesterol, dimyristoyl phosphatidylcholine (DMPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (POPC), sodium perchlorate, α-tocopherol, and Chelex 100 were obtained from Sigma Chemical Co. (St. Louis, MO, USA), rose bengal (95%) was obtained from Aldrich Chem. Co. (Milwaukee, WI, USA). HPLC-grade acetonitrile, methanol, dichloromethanol, chloroform, and isopropanol were obtained from Merck KgaA (Darmstadt, Germany). Zeaxanthin was a gift from Vitamins & Fine Chemicals, Human Nutrition
Rose bengal-mediated lipid photoperoxidation
Photodynamic damage of liposomal membranes was induced by irradiation with 520–600 nm light in the presence of 20 μM rose bengal. Rose bengal is an efficient photosensitizer, photoexcitation of which, under aerobic conditions, results in generation of singlet oxygen. Reaction of singlet oxygen with unsaturated lipids leads to formation of hydroperoxides. Hydroperoxides may undergo decomposition to radicals due to interaction with excited photosensitizers, such as methylene blue [29]. In the
Discussion
Our results clearly indicate that a combination of zeaxanthin and α-tocopherol offers synergistic protection against photosensitized lipid peroxidation in liposomal system. This synergistic effect of zeaxanthin and α-tocopherol may be explained in terms of inhibitory effects of α-tocopherol on carotenoid consumption (Fig. 9A), which would otherwise occur due to free radical-mediated damage. Sparing zeaxanthin allows it to act longer as an efficient singlet oxygen quencher. Thus, zeaxanthin
Abbreviations
Ch-OOH—cholesterol hydroperoxides
DMPC—1,2-dimyristoyl-sn-glycero-3-phospholine
ESR—electron spin resonance
HPLC-EC—high-performance liquid chromatography with electrochemical detection
UV-VIS—ultraviolet-visible
mHCTPO—4-protio-3-carbamoyl-2,2,5,5-tetraperdeuteromethyl-3-pyrroline-1-yloxy
5α-OOH—3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide
6α-OOH—3β-hydroxycholest-4-ene-6-α-hydroperoxide
6β-OOH—3β-hydroxycholest-4-ene-6-β-hydroperoxide
7α-OOH—3β-hydroxycholest-5-ene-7-α-hydroperoxide
Acknowledgements
We thank Professor Halpern, Chicago University for the generous gift of mHCTPO and Hoffmann-La Roche for zeaxanthin. This work was supported by State Committee for Scientific Research (K.B.N.; grant no. PB 6PO4A 06217), the Wellcome Trust, and EU (contract no. ICAT-CT-2000-70012).
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