Photochemistry and Photobiology, 1999, 70(2): 172-1 75 zyxwvu Research Note Photophysical Studies of A2-E, Putative Precursor of Lipofuscin, in Human Retinal Pigment Epithelial Cells Rinaldo Cubeddul, Paola Taronil, Dan-Ning zyxwvu Hu2, Naomi Sakais, Koji Nakanishi3 and Joan E. Roberts*4 INFM-Department of Physics and CEQSE-CNR, Politecnico di Milano, Milan, Italy; *Tissue Culture Center, Departments of Pathology and Laboratory Medicine and Ophthalmology, New York Eye and Ear Infirmary, New York, NY, USA; 3Department of Chemistry, Columbia University, New York, NY, USA and 4Fordham University, Department of Natural Sciences, New York, NY, USA Received 1 March 1999; accepted 17 May 1999 ABSTRACT zyxwvutsr With age, human retinal pigment epithelial cells accu- mulate lipofuscin that can absorb photons zyxwvu in the visible range leading to light-induced damage and impaired vi- sion. A putative precursor of lipofuscin, 2-[2,6-dimethyl- 8-(2,6,6-trimethyl-l-cyclohexen- l-yl)-lE, 3E, SE,7E-octa- tetraenyl]-l-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-tri- methyl- 1 -cyclohexen- 1 -yl)- lE, 3E, 5E-hexatrienyl] - pyridi- nium (AZE), has recently been isolated and characterized from aged human retinal pigment epithelial cells. We have found that A2-E inhibits the growth of human ret- inal pigment epithelial cells at concentrations greater than 1 (1LM. Time-resolved fluorescence measurements of 1 (1LM A2-E in solution, performed under 413 nm exci- tation, showed that fluorescence wave forms integrated across the spectrum (450-600 nm) were best-fitted with three decay times in the nanosecond and subnanosecond time scale: 6.6,1.9 and 0.33 ns. Untreated retinal pigment epithelial cells were characterized by three fluorescence lifetimes: 6.3, 1.7 and 0.35 ns. In retinal pigment epithe- lial cells treated with 1 (1LM A2-E, the fluorescence decay was significantly faster, with the marked presence (~30%) of a fourth short lifetime (0.12 ns). These fluo- rescence decay times for A2-E bound to human retinal pigment epithelial cells are similar to those of lipofuscin granules isolated from aged human retinal pigment epi- thelial cells. This similarity supports the hypothesis that A2-E is a precursor of lipofuscin and suggests that A2-E may play a role in the overall light damage associated with age-related retinal diseases. INTRODUCTION Visible light at moderate intensity is benign and functions to direct vision and circadian rhythm (1,2). However, with *To whom correspondence should be addressed at: Fordham Uni- versity, Department of Natural Sciences, 113 West 60th Street, New York, NY 10023, USA. E-mail: jroberts@mary.fordham.edu zyxwvuts 0 1999 American Society for Photobiology 003 1-8655/99 zyxwvutsrq $5.00+0.00 the pioneering work of Ham zyxw et al. (3), Noel1 and coworkers (43) and others (&lo), it has become clear that visible light can damage the eye if it is sufficiently intense and if the subject is aged. The presence of photosensitizing endoge- nous or exogenous agents dramatically enhances light-in- duced ocular damage (1 1). Recent epidemiological studies (12) and clinical observations (1 3) have shown that blue (400-500 nm) light damage to the eye is associated with an increased risk of age-related macular degeneration that can lead to blindness. The chromophore(s) that induces this phototoxic damage to the retina has not been defined (3-11,1416). There is evidence that it may involve lipophilic, fluorescent granules generally termed lipofuscin that accumulate in several or- gans in the body with age (17). A fluorescent material ab- sorbing in the blue visible range (400-500 nm), thought to be lipofuscin (18), is generated in the retinal pigment epi- thelium (WE)? cells at very high concentrations (up to 19% of cytoplasmic volume) (19). The putative precursor of lipofuscin in W E cells, 2-[2,6- dimethyl-8-(2,6,6-trimethyl- 1 -cyclohexen- 1 -yl)- 1 E, 3 E, 5 E, 7 E-octatetraenyll- 1 -(Zhydroxyethyl)-4- [Cmethyl- 6-(2,6,6-trimethyl- 1 -cyclohexen- 1 -yl)- 1 E,3E,SE-hexatrien- yll-pyridinium (A2-E), has been isolated (20) and character- ized (21) and the structure given below in Fig. 1. It is an amphiphilic quaternary nitrogen compound formed from the reaction of retinaldehyde (22,23) (oxygenated product of vi- tamin A) with ethanolamine (a major lipid constituent in the retina) (24). Using time-resolved spectroscopic techniques, we have previously defined the photophysical properties of two major chromophores, melanin and lipofuscin, isolated from human aged retinal cells (25). We report here on the fluorescence Abbreviations: A2-E, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-l-cycloh- exen- 1 -yl)- 1 E, 3E,SE,7E-octatetraenyl]- 1 -(2-hydroxyethyl)-4-[4- methyl-6-(2,6,6-trimethyl- 1 -cyclohexen- I -yl)- 1 E. 3E.SE-hexa- trienyll-pyridinium; DMSO, dimethylsulfoxide; RPE, retinal pig- ment epithelium. 172