Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells - PubMed (original) (raw)

Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells

Young P Jang et al. Photochem Photobiol. 2005 May-Jun.

Abstract

The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon-carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E.

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Figures

Figure 1

Figure 1

A: Anthocyanins, a subclass of flavonoid, are defined by a three-ring (C6C3C6) structure consisting of two benzene rings (A and B) flanking the oxygen-containing pyran ring (C). Individual differences result from variation in the number and arrangement of the hydroxyl groups as well as the extent of alkylation and/or glycosylation of these groups. B: Anthocyanins undergo reversible structural changes with four structural types existing in equilibrium: flavylium cation 1, quinonoidal anhydro base 2, colorless carbinol bases 3 and pale yellow chalcones 4. The relative concentrations of these species at equilibrium are dependent on acid–base proton transfer, hydration of the pyrilium nucleus and ring-chain tautomeric reactions. Acid–base equilibrium between the flavylium cation and the quinonoidal anhydro base is pH dependent. The diene structure (arrows) on the quinonoidal anhydro base 2 is an excellent substrate for singlet oxygen.

Figure 2

Figure 2

Bilberry extract reduced A2E photooxidation. FAB-MS of A2E (200 μ_M_ in PBS), A2E irradiated at 430 nm (blue light) and A2E irradiated in the presence of bilberry extract. The molecular ion peak at MHz 592 corresponds to the molecular mass of A2E. The photooxidation of A2E is indicated by the presence of additional peaks that differ by mass 16. Illumination in the presence of bilberry extract reduces the intensity of these peaks and eliminates MHz 656, 672 and 688.

Figure 3

Figure 3

Reverse-phase HPLC profile of anthocyanins isolated from water-soluble extracts of bilberry. Monitoring was performed at 520 nm. Nine species were detected: delphinidin 3-galactoside (del-gal), delphinidin 3-glucoside (del-glc), cyanidin 3-galactoside (cya-gal), delphinidin 3-arabinoside (del-ara), cyani-din 3-glucoside (cya-glc), cyanidin 3-arabinoside (cya-ara), petunidin 3-glucoside (pet-glc), malvidin 3-glucoside (mal-glc) and malvidin 3-arabinoside (mal-ara). The structures of these compounds are presented above.

Figure 4

Figure 4

Protective effects of anthocyanin pretreatment on blue light–induced A2E oxidation. The loss of A2E that accompanies A2E oxidation was quantified by HPLC after A2E was irradiated at 430 nm in the presence and absence of glycosidic (galactoside, glucoside and arabinoside) and non-glycosidic (no sugar) forms of delphinidin, cyanidin, petunidin and malvidin as indicated. Anthocyanins were used at a concentration of 100 mM. Data are expressed as percent protection ([A2E + anthocyanins + blue light] − [A2E + blue light]) ÷ ([A2E] − [A2E + blue light]) × 100; mean ± SEM of three experiments. P values are given when significant differences apply.

Figure 5

Figure 5

Anthocyanins inhibit A2E-oxidation by quenching singlet oxygen. A: A2E was oxidized (A2E-oxidized) by singlet oxygen generated from the endoperoxide of 1,4-dimethylnaphthalene (25°C in CD3OD). B: A2E oxidation with and without the addition of delphinidin galactoside (del-gal, 100 m_M_) or cyanidin galactoside (cya-gal, 100 m_M_) was quantified by HPLC as the loss in A2E. The symbols (+) and (−) indicate the presence or absence, respectively, of a compound; P < 0.001 when compared with a mixture of A2E and endoperoxide in the absence of anthocyanin. Data are the mean ± SEM of three experiments.

Figure 6

Figure 6

Anthocyanins protect A2E-laden RPE from blue light–induced death. A: Light microscopic detection of delphinidin galactoside and cyanidin galactoside incorporated by ARPE-19 cells. Phase-contrast images. B: ARPE-19 cells that had accumulated A2E were incubated without anthocyanin (−) or with delphinidin galactoside (del-gal, 100 m_M_) or cyanidin galactoside (cya-gal, 100 m_M_) for 3 days and were then irradiated at 430 nm. Values are mean ± SEM of three experiments; *P < 0.001 when compared with the absence of anthocyanin. C: Fluorescence detection of nuclei of nonviable cells (red) and nuclei of all cells (blue); images representative of experiments in B.

Figure 7

Figure 7

In cultured ARPE-19 cells that have incorporated anthocyanins, the accumulation of A2E is retarded. Cells were incubated with 100 μ_M_ delphinidin 3-galactoside (del-gal) or cyanidin 3-galactoside (cya-gal) for 2 days or were not incubated (−). The cells were then allowed to accumulate A2E (20 μ_M_ for 10 days) after which cell-associated A2E and iso-A2E (photoisomer of A2E) was quantified by HPLC and normalized to cellular protein levels. Percent incorporation of A2E/isoA2E into RPE cells was 71.4/70.5% for del-gal and 54.0/56.9% for cya-gal, respectively. Values are mean ± SEM of three experiments, P < 0.001.

Figure 8

Figure 8

Anthocyanins protect against A2E-induced membrane damage. ARPE-19 cells incorporated delphinidin galactoside (del-gal) and cyanidin galactoside (cya-gal) from a 100 μ_M_ concentration for 3 days before being incubated with 100 μ_M_ A2E for 18 h. Nuclei of membrane-compromised cells were labeled with a membrane-impermeable dye and counted. Values are mean ± SEM of three experiments; *P < 0.001 when compared with A2E without prior incorporation of anthocyanin.

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