Mechanisms involved in A2E oxidation

Abstract

A2E is one of the bis-retinoid pyridinium compounds that accumulate as lipofuscin pigments in retinal pigment epithelial (RPE) cells in association with aging and in some inherited forms of retinal degeneration. Here we observed that 430nm irradiation of A2E in the presence of the spin trap DMPO, led to the appearance of a superoxide dismutase-inhibitable electron paramagnetic resonance (EPR) spectrum characteristic of DMPO-OH; this finding was indicative of hydroxyl radical (OH) formation following initial spin trapping of superoxide anion by DMPO. We also observed an increase in dihydroethidium (HEt) fluorescence and luminol-based chemiluminescence that on the basis of inhibition by superoxide dismutase, was indicative of superoxide anion generation when A2E was irradiated at 430nm in cell-free systems. Nevertheless, while A2E was readily oxidized in the presence of a singlet oxygen generator, superoxide anion did not serve to oxidize A2E. Specifically, by HPLC quantitation and FAB-mass spectroscopy, there was no evidence of A2E oxidation when A2E was incubated with a superoxide anion generator (xanthine/xanthine oxidase) in a variety of solvents (100% PBS, 30% DMSO in PBS, 100% MeOH and CHCl3) or in the presence of detergent. On the other hand, however, peroxy-A2E, an oxidized form of A2E with an endoperoxide moiety on the short-arm of the molecule, readily underwent further oxygen addition when incubated with xanthine/xanthine oxidase. Superoxide anion may be generated by irradiation of A2E but is not involved in the early events that oxidize A2E. Superoxide can contribute to the further oxidation of already-oxidized A2E.

Figure 1

Oxidant production associated with 430 nm irradiation of A2E. A. EPR spectrum of an aqueous buffer solution (pH 7.2) of DMPO (90 mM in DMSO) and A2E (35 μM) after 430 nm irradiation for 10 minutes (0.36 mWatt/mm²). The four EPR signal peaks (arrows) correspond to the formation of an adduct of OH-radical with DMPO; typical coupling constants g_N and g_H are presented. DMPO structure, upper left. B. Superoxide dismutase inhibits dihydroethidium (HEt) fluorescence in the presence of A2E and 430 nm irradiation. HEt (50 μM) was incubated at the indicated concentrations of A2E and the mixture was irradiated at 430nm. The HEt fluorescence was measured by spectrofluorometry (Ex, 485 nm; Em, 565 nm). Values plotted are mean ± S.E.M. of 3 experiments, three replicates per experiment. p < 0.01, A2E + 430nm versus A2E + 430nm + SOD (30 Units/ml); p<0.001, A2E + 430nm versus A2E + 430nm + SOD (100 Units/ml). C. Luminol chemiluminescence in the presence of 430 nm-irradiated A2E. A2E (200 μM) or xanthine (0.4 mM)/xanthine oxidase (100 mUnits/ml), a superoxide anion generator was added to the mixture of luminol and enhancer. The mixture containing A2E was exposed to 430-nm illumination (0.36 milliwatt/mm²). Emission was recorded at 30-second intervals (beginning from the time the reactants were combined or beginning at onset of 430 nm irradiation) for 2 minutes. Values are mean ± S.E.M. of 3 experiments, duplicates per experiment.

Figure 2

Photooxidation of A2E in aqueous and methanolic environment. A. Overlay of HPLC chromatographs generated with unirradiated A2E (black trace) or after A2E in methanol is irradiated (430 nm) for 5 mins (blue) and 30 mins (red). B. Chromatographic overlay of unirradiated A2E (black) and A2E in PBS with 0.1% DMSO after irradiation for 2 mins (blue) and 5 (red) mins. Reversed phase C18 column with monitoring at 430 nm. C. FAB-mass spectrometry. Unirradiated A2E (upper), A2E in methanol (MeOH) irradiated at 430 nm for 30 mins (middle) and A2E in phosphate buffered saline (PBS; with 0.01% DMSO) irradiated for 5 minutes (lower). Peaks differing in m/z by 16 (e.g. 608, 624, 640 etc) are indicative of A2E photooxidation.

Figure 3

A2E is oxidized in the presence of a generator of singlet oxygen but not in the presence of a superoxide anion generator. Analysis by FAB-MS. A2E was incubated without (A) and with (B) 1 mM endoperoxide of 1, 4-dimethylnaphthalene, a singlet oxygen generator. In deuterium solvent, the mass of A2E (m/z 592) is shifted by 1 mass unit to 593, due to hydrogen-deuterium exchange. A2E oxidation in B is indicated by the appearance of m/z 593 + n (16). Samples of A2E incubated in the absence (C) and presence (D) of the superoxide anion generator xanthine/100 mUnits/ml xanthine oxidase (0.4 mM).

Figure 4

Chromatographic overlay of HPLC profiles generated with starting sample of A2E (black) and A2E incubated for 6 hours with either the singlet oxygen generator, endoperoxide of 1,4-dimethylnaphthalene (1 mM) (blue trace) or the superoxide anion generator, xanthine/xanthine oxidase (xanthine, 0.4 mM; xanthine oxidase, 100 mUnits/mL; with 0.4% Tween-80) (red trace). Black and red traces overlap; blue trace exhibits reduced height relative to black trace. Reversed phase C18 column with monitoring at 430 nm.

Figure 5

The reactivity of A2E, A1E, furano-A2E, peroxy-A2E and all-trans-retinal with superoxide anion. HPLC quantitation determined as integrated peak areas and expressed as percent of control (untreated sample). Compounds were incubated with three different conditions of xanthine/xanthine oxidase, 0.2 mM xanthine/50 mUnits/ml xanthine oxidase (A); 0.4 mM xanthine/100 mUnits/ml xanthine oxidase (B); 0.8 mM xanthine/200 mUnits/ml xanthine oxidase (C) and for 15, 30 and 60 minutes (A–C). Structures of A2E, A1E, furano-A2E, peroxy-A2E and all-trans-retinal.

Figure 6

Representative HPLC chromatograms of A2E, A1E and all-trans-retinal (ATR) incubated with the superoxide anion generator xanthine/xanthine oxidase (X/XOD). Starting samples (top) and samples incubated with X/XOD for 15, 30 and 60 mins at 25°C were analyzed by HPLC. Inset in top panels, UV-visible absorbance of A2E, A1E and ATR. Oxidation is indicated by a reduction in peak height and by a blue shift in UV-visible absorbance; compare insets in top and bottom panels of B and C.

Figure 7

Representative HPLC chromatograms of furano-A2E and peroxy-A2E, that were incubated with the superoxide anion generator xanthine/xanthine oxidase (X/XOD). Starting samples (top panels) and samples incubated with X/XOD for 15, 30 and 60 mins at 25°C were analyzed by HPLC. Inset in top panels, UV-visible absorbance of furano-A2E and peroxy-A2E. Oxidation is indicated by a reduction in peak height and by a blue shift in UV-visible absorbance; compare insets in top and bottom panels of B.