A2E-epoxides Damage DNA in Retinal Pigment Epithelial Cells

Abstract

The autofluorescent pigments that accumulate in retinal pigment epithelial cells with aging and in some retinal disorders have been implicated in the etiology of macular degeneration. The major constituent is the fluorophore A2E, a pyridinium bisretinoid. Light-exposed A2E-laden retinal pigment epithelium exhibits a propensity for apoptosis with light in the blue region of the spectrum being most damaging. Efforts to understand the events precipitating the death of the cells have revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds. Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit reactivity toward DNA with oxidative base changes being at least one of these lesions. Mass spectrometry revealed that the antioxidants vitamins E and C, butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis and cell viability studies revealed a corresponding reduction in the incidence of DNA damage and cell death. Vitamin E, a lipophilic antioxidant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both vitamins simultaneously did not confer additional benefit. Studies in which singlet oxygen was generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, resveratrol, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen. Conversely, vitamin C and ginkgolide B were not efficient quenchers of singlet oxygen under these conditions.The di-retinal conjugate A2E forms as a consequence of light related vitamin A cycling in the retina. This orange-emitting fluorophore is formed synthetically as the condensation product of all-trans-retinal and ethanolamine (1-3). NMR and corroborative total chemical synthesis revealed A2E to be a pyridinium bisretinoid consisting of an unprecedented pyridinium polar head group and two hydrophobic retinoid tails (4, 5). A2E, its slightly less polar photoisomer, iso-A2E, and other minor cis-isomers together constitute the most prominent age-related hydrophobic pigments (lipofuscin) in retinal pigment epithelial (RPE) 1 cell extracts assayed by reverse phase HPLC (3, 6). In vivo, A2E is generated by phosphate hydrolysis of the fluorophore phosphatidylpyridinium bisretinoid, the latter precursor forming from reactions between all-trans-retinal and phosphatidylethanolamine in the photoreceptor outer segment membrane (3,7,8).Although certain levels of A2E are clearly tolerated by RPE cells, adverse effects of its accumulation have also been reported. Thus, not only can A2E mediate detergent-like effects on cell membranes (9), its accumulation can also lead to the alkalinization of lysosomes (10) and to the detachment of proapoptotic proteins from mitochondria (11). A2E also bestows a sensitivity to blue light damage (11-13) that is proportional to t...

The all-trans -retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model

“…It is now well known that short-wavelength visible-light irradiation of A2E leads to the formation of singlet oxygen and other reactive forms of oxygen that subsequently add to carboncarbon double bonds along the side arms of the molecule (13,26). The reactive moieties generated by A2E photooxidation and cleavage, including endoperoxides and aldehydes (14), likely account for the adverse effects observed when RPE cells that have accumulated A2E are irradiated at 430 nm (27)(28)(29)(30). In the present work we found that unconjugated atRAL dimer is a more efficient generator of singlet oxygen than A2E when irradiated at 430 nm.…”

The Age Lipid A2E and Mitochondrial Dysfunction Synergistically Impair Phagocytosis by Retinal Pigment Epithelial Cells

“…Oxidized forms of A2E promote cell damage even in the dark (43). Autooxidative changes of A2E in the dark have also been reported (44).…”

Induction of apoptosis in human leukemia cells by the tyrosine kinase inhibitor adaphostin proceeds through a RAF-1/MEK/ERK- and AKT-dependent process

“…To this end, Jurkat cells were exposed to adaphostin (2.0 mM) for 9 h in the presence or absence of the antioxidants L-N-acetylcysteine (L-NAC) (Moldeus and Cotgreave, 1994), Tiron (1 mM) (Wolf et al, 2001) or Trolox (1 mM) (Sparrow et al, 2003). As shown in Table 1, coadministration of L-NAC or Trolox, with adaphostin essentially abrogated adaphostin-mediated induction of the morphologic features of apoptosis, annexin V/PI positivity, and mitochondrial injury (i.e., loss of DC m ).…”