IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sparrow, J. R.
Right arrow Articles by Nakanishi, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sparrow, J. R.
Right arrow Articles by Nakanishi, K.
(Investigative Ophthalmology and Visual Science. 2002;43:1222-1227.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Involvement of Oxidative Mechanisms in Blue-Light-Induced Damage to A2E-Laden RPE

Janet R. Sparrow1, Jilin Zhou1, Shimon Ben-Shabat2, Heidi Vollmer2, Yasuhiro Itagaki2 and Koji Nakanishi2

1 From the Departments of Ophthalmology and 2 Chemistry, Columbia University, New York, New York.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The lipofuscin fluorophore A2E is known to be an initiator of blue-light-induced apoptosis in retinal pigment epithelial cells (RPE). The purpose of this study was to evaluate the role of oxidative mechanisms in mediating the cellular damage.

METHODS. Human RPE (ARPE-19) cells that had accumulated A2E were exposed to blue light in the presence and absence of oxygen, and nonviable cells were quantified. Potential suppressors (histidine, azide, 1,4-diazabicyclooctane [DABCO], and 1,3-dimethyl-2-thiourea [DMTU]) and enhancers (deuterium oxide [D2O] and 3-aminotriazole [3-AT]) of oxidative damage, were also screened for their ability to modulate the frequency of nonviable cells. A2E in PBS, with and without an oxygen-depleter or singlet-oxygen quencher and A2E-laden RPE, were exposed to 430-nm light and examined by reversed-phase high performance liquid chromatography (HPLC) and fast atom bombardment mass spectrometry (FAB-MS).

RESULTS. The death of blue-light-illuminated A2E-laden RPE was blocked in oxygen-depleted media. When A2E-laden RPE were transferred to D2O-based media and then irradiated (480 nm), the number of nonviable cells was increased, whereas the latter was decreased in the presence of histidine, DABCO, and azide. Conversely, no affect was observed with 3-AT and DMTU. When A2E, in either acellular or cellular environments, was irradiated at 430 nm, FAB-MS revealed the generation of a series of higher molecular mass derivatives of A2E. The sizes of these species increased by increments of mass 16. The generation of these photo-products was accompanied by the consumption of A2E, the latter being diminished, however, when illumination was performed after oxygen depletion and in the presence of a singlet-oxygen quencher.

CONCLUSIONS. The augmentation of cell death in the presence of D2O and the protection afforded by quenchers and scavengers of singlet oxygen, indicates that the generation of singlet oxygen may be involved in the mechanisms leading to the death of A2E-containing RPE cells after blue light illumination. The finding that irradiation also produces oxygen-dependent photochemical changes in A2E, indicates that the effects of singlet oxygen may be mediated either directly or through the generation of reactive photo-products of A2E.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The propensity for retinal pigment epithelial cells to be damaged or destroyed by excessive exposure to visible light may be of significance to retinal disorders characterized by enhanced accumulation of the autofluorescent pigments that constitute lipofuscin. Lipofuscin has long been considered capable of eliciting photodynamic damage.1 Indeed, the major hydrophobic fluorophore of RPE lipofuscin, the diretinal adduct A2E, when accumulated within the lysosomal compartment of cultured RPE,2 has been shown to confer a susceptibility to blue-light-mediated death,3 4 which is proportional to the A2E concentration in the cultures and which is not manifest in cells devoid of A2E.3 The wavelength dependency of the insult is consistent with the excitation spectra of A2E3 and with the known susceptibility of RPE cells to blue light exposure in animal models.5 6 7 8 9 10 Moreover, the photochemical events triggered by blue light in the context of intracellular A2E initiate a cell death pathway3 that involves the activation of caspases and is modulated by the mitochondrial protein Bcl-2.11

A chromophore such as A2E mediates light damage, because it is capable of absorbing photons of specific energy by electron excitation. The photoexcited molecule can then either react directly with a substrate, or it can transfer the excitation energy onto an adjacent oxygen molecule, converting it to the singlet state, while the photosensitizer itself is returned to the ground state.12 Subsequently, the singlet oxygen produced on illumination has the option of attacking the photosensitizer (photobleaching) and/or of reacting with other molecules present. Although less efficiently, some photosensitizers can also transfer electrons to oxygen to produce superoxide (O·2) and then hydroxyl radical (OH·). Thus, all told, photoinduced damage can arise either through a direct reaction of the photoactivated molecule with cellular constitutents and/or through the formation of reactive oxygen species.

Previous studies have shown that isolated human RPE cells exhibit substantial light-dependent uptake of oxygen that appears to be related to their content of lipofuscin.13 Photophysical studies have demonstrated that intact lipofuscin granules or chloroform-methanol extracts of lipofuscin can serve as photosensitizers for the generation of singlet oxygen and possibly superoxide and hydrogen peroxide.13 14 15 16 In addition, in the presence of light and isolated lipofuscin, suspensions of RPE cells and rod outer segments undergo lipid peroxidation by mechanisms that can be inhibited by the antioxidants 1,4-diazabicyclooctane (DABCO) and superoxide dismutase.17 Nevertheless, in these studies the identity of the reacting fluorophore was not known.

To begin to track the events that occur from the time that blue light is absorbed by intracellular A2E until the cell death program is initiated, we used a cell culture model that provides for populations of cells with and without intracellular A2E.2 The results of our search for the intermediaries involved in the cellular damage not only implicate singlet oxygen, they also indicate that A2E undergoes photochemical changes that are novel and that may be significant to our understanding of A2E-associated injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Deuterium oxide (D2O; 99.96%) was obtained from ICN Biomedicals, (Aurora, OH); 3-aminotriazole (3-AT), L-histidine, and DABCO were obtained from Sigma Chemical Co. (St. Louis, MO); 1,3-dimethyl-2-thiourea (DMTU) was purchased from Aldrich Chemical Co. (Milwaukee WI) and 1,2,2,6,6-pentamethyl-4-piperidinol (NMP) was from Acros Organics (Geel, Belgium). Sodium azide was obtained from Fisher Scientific (Fairlawn, NJ).

Preparation of A2E-Laden RPE Cultures
Human adult RPE cells (ARPE-19; American Type Culture Collection, Manassas, VA), which are devoid of endogenous A2E,2 were grown in Dulbecco’s modified Eagle’s medium (DMEM) and at confluence were incubated with A2E to allow for intracellular accumulation of the fluorophore, as previously documented by microscopy and HPLC.2 3

Illumination and Treatment
Confluent cultures were transferred to phosphate-buffered saline with calcium, magnesium and glucose (PBS-CMG) and were exposed either to a single spot of blue light delivered from a 100-W mercury lamp (480 ± 20 nm; 35 mW/mm2; 60 seconds) or to a light line delivered from a tungsten halogen source (470 ± 20 nm; 0.4 mW/mm2; 20-minute exposure),3 11 as indicated.

For illumination in the presence of heavy water, D2O was prepared at a final concentration of 90% in PBS-CMG. The D2O-based solution was substituted for culture media just before light exposure, and the cells were returned to culture media immediately afterward. Blue-light-exposed, A2E-laden RPE incubated in H2O-based PBS-CMG served as the control. To screen potential suppressors (histidine, azide, DABCO, and DMTU), and an enhancer (3-AT) of oxidative damage, stock solutions of histidine (200 mM), sodium azide (1 M), DMTU (1 M), and 3-AT (1 M) were prepared in PBS-CMG. A stock solution of DABCO (0.5 M) was prepared in 95% ethanol. Before illumination, cells were incubated for 1 hour (37°C) with each reagent diluted in culture media to the final concentrations indicated. Illuminations were subsequently performed in the presence of the reagent diluted in PBS-CMG. Control cultures were blue-light-exposed, vehicle-treated, A2E-laden RPE.

Oxygen-Depleted Medium
To remove oxygen from the media before illumination, cultures were preincubated at 37°C for 20 minutes with an oxygen depleter (Oxyrase; Oxyrase Inc., Mansfield, OH), diluted 1:50 in media with the addition of 20 mM sodium lactate. After light exposure, Oxygen-depleter-containing media were replaced with fresh media, and the cells were returned to the incubator.

Assaying Cell Viability
For illuminations performed in the presence of D2O or oxygen depleter, cell viability was determined either 6 (D2O) or 18 (oxygen depleter) hours after blue light exposure by labeling the nuclei of dead cells with a membrane-impermeable dye (Dead Red, 1:500 dilution, 15-minute incubation; Molecular Probes, Eugene, OR). Cell death was determined as labeled nuclei per zone of illumination (0.5-mm diameter). In all other experiments, the nonviable cells were assayed 8 hours after blue light exposure by labeling the nuclei of nonviable cells with the red dye, together with 4'6'-diamidino-2-phenylindole (DAPI) labeling of all nuclei. Counting was performed from digital images, and nonviable cells were expressed as a proportion of the total number of cells in a photographic field. In all experiments, replicates were assayed as indicated in the figure legends. Mean results were compared using the unpaired t-test, the Mann-Whitney nonparametric test, and ANOVA followed by the Student-Newman-Keuls multiple-comparison test.

Photo-Derivatives of A2E
Preparations of A2E in PBS (200 µM) were exposed to 430-nm light at intensities of 0.075, 0.095, 0.15, and 0.19 mW/mm2 for 10 minutes After extraction in chloroform-methanol (2:1), separation of the chloroform layer, and evaporation, the samples were examined by fast atom bombardment mass spectrometry (FAB-MS). In some experiments, illumination was also performed in the presence of oxygen depleter (as described earlier) or the singlet-oxygen quencher NMP (100 mM). Subsequently, A2E was quantitated by reversed-phase HPLC (C18 column, 250 mm x 4.6 mm; Cosmosil 5C18; Nacalai Tesque, Kyoto, Japan) using the area of the A2E peak normalized to the internal standard, A2-propylamine, the latter being synthesized from all-trans-retinal and propylamine. To examine for photooxidation of intracellular A2E, A2E-laden ARPE-19 cells were irradiated (430 nm) at an intensity of 0.095 mW/mm2 for 6 minutes. The pelleted cells were homogenized in chloroform-methanol (2:1) and water (vol/vol 3:1), filtered through a column (RP-C18 Sep-Pak; Millipore, Bedford, MA) followed by washing with methanol and 0.1% trifluoroacetic acid (TFA). After drying, the sample was redissolved in methanol and analyzed by FAB-MS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine whether the death of blue-light-illuminated, A2E-containing RPE occurs through oxygen-associated mechanisms, oxygen was removed from the media before illumination using an enzyme system (oxygen depleter) derived from Escherichia coli, which reduces dissolved oxygen to water in the presence of sodium lactate as hydrogen donor. Subsequent blue light illumination revealed that cell death was blocked by an average of 98% in the oxygen-depleted media (0.6 nuclei/illumination zone versus 40 nuclei/illumination zone, oxygen depleter-treated and control respectively; mean of 4 experiments, five to eight replicates/experiment). The numbers of nonviable cells in nonilluminated zones of oxygen-depleted A2E-laden cultures were not different from those in non-oxygen depleted cultures.

Given the observed oxygen-dependence of blue-light-induced apoptosis in our model, we began to evaluate the role of oxygen-derived species in mediating apoptosis by examining the capacity of enhancers (D2O) and quenchers (histidine, DABCO, azide) of singlet oxygen to affect the frequency of nonviable cells after blue light illumination.12 In D2O, the lifetime of singlet oxygen is significantly prolonged; thus, when cells are placed in D2O-based media, cellular responses to intracellularly generated singlet oxygen are potentiated.18 19 20 21 22 23 24 Correspondingly, when A2E-laden cultures were blue light illuminated in the presence of D2O-based buffered salts, we observed an increase of 66% in the numbers of nonviable cells compared with control cultures illuminated in H2O-based buffered solution (P = 0.001; Fig. 1 ).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Agents capable of modulating oxygen derivatives: effects on the incidence of nonviable A2E-laden RPE after blue light illumination in culture. Concentrations (in millimolar) of the compounds are presented. Cultures were incubated with the compounds. Cell death was assayed in 5 to 10 fields of illumination per condition in each experiment. Data are presented as a percentage in relation to the control (blue-light-exposed, vehicle-treated, A2E-laden RPE) and are the mean ± SEM of the number of experiments indicated in parentheses.

 
In the presence of sodium azide,18 19 20 25 26 histidine,21 24 26 and DABCO,27 28 29 all of which act as efficient quenchers and scavengers of intracellularly generated singlet oxygen, consistent decreases in the numbers of nonviable cells were observed after blue light illumination (Fig. 1) . Thus, azide at a concentration of 1 mM had no effect, whereas 5 mM azide reduced cell death by 43% (P < 0.01). Inclusion of 1 mM DABCO produced a difference of 25% compared with the control (P = 0.06). At higher concentrations, DABCO was toxic to the cells. A dose-dependent attenuation of cell death was also observed in the presence of histidine, with no effects observed at 0.1 and 1 mM concentrations (P > 0.05), but a reduction of 23% being observed in the presence of 10 mM histidine (P < 0.01) compared with the level of cell death in its absence.

To assay for participation of other oxygen-derived species in mediating blue light damage to A2E-containing cells, we also pretreated cultures with 3-AT, an inhibitor of the cellular antioxidant enzyme catalase, that can be expected to increase the susceptibility of cells to potential H2O2-induced damage.24 30 31 32 However, the frequency of nonviable A2E-laden cells after blue light exposure was not increased when cultures were preincubated with 10, 50, and 100 mM concentrations of 3-AT for 1 hour (P > 0.05; Fig. 1 ), nor when pretreatment was extended to 6 hours (not shown). Indeed, at 100 mM 3-AT, there appeared to be a decrease in nonviable cells, although the difference between this level and the control level was not significant (P > 0.05). Similarly, DMTU, a strong hydroxyl radical (OH-) scavenger with additional capability for eliminating superoxide anion radical (O2·-) and H2O2,12 33 34 35 , had no effect on the incidence of cell death when used at concentrations of 10 and 50 mM (P > 0.05; Fig. 1 ).

Although these results suggest that the modulation of singlet oxygen has an impact on the frequency of nonviable, A2E-laden RPE after blue light illumination, we were also intrigued by the proclivity for intracellular A2E to undergo fluorescence quenching under blue light (data not shown). To begin to investigate this observation, A2E in aqueous media was exposed to 430 nm illumination (0.095 mW/mm2, 10 minutes), and A2E was quantified by HPLC. The HPLC profile revealed a decrease in the absorbance of the A2E peak, denoting a loss of A2E after blue light illumination, compared with the control nonilluminated sample (Fig. 2) . This decrement in A2E was attenuated, however, when the sample was incubated in oxygen depleter to reduce oxygen before 430-nm illumination (Fig. 2) . The FAB-MS spectra of blue-light-illuminated A2E revealed not only a molecular ion peak at a mass-to-charge ratio (m/z) of 592, corresponding to the molecular mass of A2E (C42H58ON),36 but also a series of additional molecular ion peaks (e.g., m/z 608, 624, 640, 656, 672, 688), each of which differed from its neighbors by mass 16 (Fig. 3) . Moreover, the extent to which these derivatives of A2E were formed was dependent on the intensity of illumination. Thus, when the spectra derived from irradiances of 0.075, 0.095, and 0.15 mW/mm2 were compared, it was apparent that as the irradiance increased, the higher-mass peaks became prominent, and additional peaks at m/z 656, 672, and 688 appeared. Concomitant with these spectral differences, the intensity of the A2E peak at m/z 592 was diminished (Figs. 3C 3D) , consistent with the light associated reduction of A2E observed by quantitative HPLC (Fig. 2) and fluorescence microscopy (not shown).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 2. HPLC quantitation of A2E after blue light irradiation. The content of A2E in a sample was reduced after 430-nm illumination. The loss of A2E was diminished in the presence of an oxygen-depleter. Data are the mean ± SEM of three experiments.

 


View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. FAB-MS of blue-light-illuminated A2E. For nonirradiated A2E (A), the molecular ion peak at m/z 592 corresponded to the molecular mass of A2E. Illumination (430 nm) of A2E in PBS (200 µM) for 10 minutes at irradiances of 0.075 mW/mm2 (B), 0.095 mW/mm2 (C), and 0.15 mW/mm2 (D) generated a series of molecular ion peaks, each of which differed from the previous peak by mass 16. (A, inset) Structure of A2E.

 
These photochemical changes in A2E were not limited, however, to preparation of A2E in aqueous media. When A2E-containing RPE cells were irradiated at an intensity of 0.095 mW/mm2, extracted in chloroform-methanol as for the detection of A2E,36 and analyzed by FAB-MS, prominent molecular ion peaks at m/z 608, 624, and 640 were observed in addition to the peak at m/z 592 attributable to A2E (Fig. 4) . That these higher m/z peaks correspond to the photoproducts generated in aqueous media is supported by the observation that they were not present in unirradiated cells and by the detection of not just one, but the three peaks in series with A2E.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. FAB-MS of intracellular A2E after 430 nm illumination. Irradiation at 0.095 mW/mm2 yielded, in addition to the peak at m/z 592 attributable to A2E, a series of molecular ion peaks that differed by increments of 16.

 
Finally, to investigate whether singlet oxygen was involved in the photooxidation and consumption of A2E, samples of A2E were irradiated (430 nm) in the presence of the singlet-oxygen quencher NMP. The latter compound is efficiently oxidized by singlet oxygen to an N-demethylated product.37 Accordingly, although the amount of A2E in samples irradiated at 0.19 mW/mm2 for 10 minutes was decreased by 97%, NMP (100 mM) reduced this loss to 75% (Fig. 5) .



View larger version (17K):
[in this window]
[in a new window]
  		Figure 5. HPLC quantitation of blue-light-illuminated A2E in the presence of a singlet-oxygen quencher. The amount of A2E was reduced after 430-nm illumination. This loss of A2E was diminished in the presence of the singlet-oxygen quencher NMP (100 mM). Data are the mean ± SEM of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated that the illumination of A2E with blue light leads to the formation of a specific pattern ofA2E-derived products and is accompanied by fluorescence quenching and consumption of A2E. That these photochemical changes in A2E involve the acceptance of oxygen, is supported by the finding that the sizes of the A2E-derived species exhibit mass increments of 16 and by the observation that the loss of A2E is decreased when oxygen is withdrawn from the media and when a singlet-oxygen quencher is added. The insertion of oxygen molecules into A2E is additive, because both the mass of the light-derived species and the abundance of these products progress with the intensity of the light exposure. Because an A2E molecule bears two chains of conjugated carbon-carbon double bonds (Fig. 3 , insert), it is likely that oxygen molecules are inserted at these double bonds. We are currently performing structural studies to confirm this. It is important to note that these photooxidative changes in A2E also occur in a cellular environment.

The enhancement of cell death in the presence of D2O and the protection afforded by quenchers and scavengers of singlet oxygen are both consistent with singlet-oxygen generation’s being a contributing factor in blue-light-mediated death of A2E-containing RPE cells. In the presence of the singlet-oxygen quencher NMP, the photoxidative changes in A2E were diminished. Thus, singlet oxygen may not only mediate the cellular damage directly, it may also serve in the photooxidation of A2E with the products generated from the photochemical changes in A2E, being the ravaging agents. This is an important issue and one we are currently investigating.

Although our observations with respect to D2O and quenchers and scavengers of singlet oxygen do not exclude the possibility that other reactive oxygen species are involved in the photooxidation of A2E, we did not obtain evidence for this prospect. Neither an inhibitor of catalase (3-AT) nor a scavenger of hydroxyl radical and superoxide (DMTU) had an effect on the frequency of cell death after blue light illumination of A2E. Conversely, DMTU has been shown to reduce the loss of 22:6 fatty acids from photoreceptor outer segments of light-exposed rats.38

The results of the present study are consistent with reports of blue light damage in animal models. Several investigators have observed that in monkeys5 6 and rats10 exposed to damaging levels of blue light the primary injury occurs in the RPE cells and is severe. Moreover, blue light damage was accentuated as arterial partial oxygen tensions were increased, whereas a diet that increased plasma levels of ß-carotene, a singlet-oxygen scavenger, was protective.5 6 Repeated illumination of monkey retina leads to RPE atrophy that investigators have described as being similar to that occurring in atrophic AMD.39 Photochemical damage may emanate from a given amount of light, regardless of whether that amount of light is absorbed over a brief or extended period. In other words, over a range of exposure durations, the product of irradiance and exposure duration is constant.1 40 Indeed, it is only cellular repair mechanisms that permit a deviation from this relationship, with long-duration exposure at low irradiances allowing repair processes to neutralize damage.40

In vivo fundus spectrophotometry,41 laser scanning ophthalmoscopy,42 and counts of lipofuscin granules43 and measurements of fluorescence in histologic sections44 45 all support the concept that the content of lipofuscin in RPE cells increases up to approximately age 70 years. An additional finding of some of these studies was, however, that lipofuscin fluorescence declines41 or reaches a plateau45 within the eighth decade. Although the death of lipofuscin-containing RPE cells is suggested to be a cause of the declining fluorescence in this older age group,41 perhaps the change in fluorescence associated with photooxidation of A2E is an additional contributing factor.


   Footnotes
 
Supported by National Eye Institute Grants EY-12951 (JRS) and GM-36564 (KN), Fight for Sight, the Charina Foundation, and Macula Vision Research Foundation. JRS is the recipient of a Research to Prevent Blindness Lew R. Wasserman Merit Award.

Submitted for publication September 24, 2001; revised December 11, 2001; accepted December 21, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Janet R. Sparrow, Department of Ophthalmology, Columbia University, 630 W. 168th Street, New York, NY 10032; jrs88{at}columbia.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Young, RW. (1988) Solar radiation and age-related macular degeneration Surv Ophthalmol 32,252-269[Medline][Order article via Infotrieve]
  2. Sparrow, JR, Parish, CA, Hashimoto, M, Nakanishi, K. (1999) A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture Invest Ophthalmol Vis Sci 40,2988-2995[Abstract/Free Full Text]
  3. Sparrow, JR, Nakanishi, K, Parish, CA. (2000) The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells Invest Ophthalmol Vis Sci 41,1981-1989[Abstract/Free Full Text]
  4. Schutt, F, Davies, S, Kopitz, J, Holz, FG, Boulton, ME. (2000) Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin Invest Ophthalmol Vis Sci 41,2303-2308[Abstract/Free Full Text]
  5. Ham, WTJ, Allen, RG, Feeney-Burns, L, et al (1986) The involvement of the retinal pigment epithelium Waxler, M Hitchins, VM eds. CRC Optical Radiation and Visual Health CRC Press Boca Raton, FL.
  6. Ham, WTJ, Mueller, HA, Ruffolo, JJJ, et al (1984) Basic mechanisms underlying the production of photochemical lesions in the mammalian retina Curr Eye Res 3,165-174[Medline][Order article via Infotrieve]
  7. Ham, WTJ, Ruffolo, JJJ, Mueller, HA, Clarke, AM, Moon, ME. (1978) Histologic analysis of photochemical lesions produced in rhesus retina by short-wavelength light Invest Ophthalmol Vis Sci 17,1029-1035[Abstract/Free Full Text]
  8. Putting, BJ, Van Best, JA, Vrensen, GFJM, Oosterhuis, JA. (1994) Blue-light-induced dysfunction of the blood-retinal barrier at the pigment epithelium in albino versus pigmented rabbits Exp Eye Res 58,31-40[Medline][Order article via Infotrieve]
  9. Busch, EM, Gorgels, TGMF, van Norren, D. (1999) Temporal sequences of changes in rat retina after UV-A and blue light exposure Vision Res 39,1233-1247[Medline][Order article via Infotrieve]
  10. Busch, EM, Gorgels, TGMF, Roberts, JE, van Norren, D. (1999) The effects of two stereoisomers of N-acetylcysteine on photochemical damage by UVA and blue light in rat retina Photochem Photobiol 70,353-358[Medline][Order article via Infotrieve]
  11. Sparrow, JR, Cai, B. (2001) Blue light-induced apoptosis of A2E-containing RPE: involvement of caspase-3 and protection by Bcl-2 Invest Ophthalmol Vis Sci 42,1356-1362[Abstract/Free Full Text]
  12. Halliwell, B, Gutteridge, JMC. (1999) Free Radicals in Biology and Medicine Oxford University Press Oxford, UK.
  13. Rozanowska, M, Jarvis-Evans, J, Korytowski, W, Boulton, ME, Burke, JM, Sarna, T. (1995) Blue light-induced reactivity of retinal age pigment: in vitro generation of oxygen-reactive species J Biol Chem 270,18825-18830[Abstract/Free Full Text]
  14. Gaillard, ER, Atherton, SJ, Eldred, G, Dillon, J. (1995) Photophysical studies on human retinal lipofuscin Photochem Photobiol 61,448-453[Medline][Order article via Infotrieve]
  15. Reszka, K, Eldred, GE, Wang, RH, Chignell, C, Dillon, J. (1995) The photochemistry of human retinal lipofuscin as studied by EPR Photochem Photobiol 62,1005-1008[Medline][Order article via Infotrieve]
  16. Rozanowska, M, Wessels, J, Boulton, M, et al (1998) Blue light-induced singlet oxygen generation by retinal lipofuscin in non-polar media Free Radic Biol Med 24,1107-1112[Medline][Order article via Infotrieve]
  17. Wassell, J, Davies, S, Bardsley, W, Boulton, M. (1999) The photoreactivity of the retinal age pigment lipofuscin J Biol Chem 274,23828-23832[Abstract/Free Full Text]
  18. Wlaschek, M, Wenk, J, Brenneisen, P, et al (1997) Singlet oxygen is an early intermediate in cytokine-dependent ultraviolet-A induction of interstitial collagenase in human dermal fibroblasts in vitro FEBS Lett 413,239-242[Medline][Order article via Infotrieve]
  19. Wlaschek, M, Briviba, K, Stricklin, GP, Sies, H, Scharffetter-Kochanek, K. (1995) Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase J Invest Dermatol 104,194-198[Medline][Order article via Infotrieve]
  20. Berneburg, M, Grether-Beck, S, Kurten, V, et al (1999) Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion J Biol Chem 274,15345-15349[Abstract/Free Full Text]
  21. Polo, L, Presti, F, Schindl, A, Schindl, L, Jori, G, Bertoloni, G. (1999) Role of ground and excited singlet state oxygen in the red light-induced stimulation of Escherichia coli cell growth Biochem Biophys Res Commun 257,753-758[Medline][Order article via Infotrieve]
  22. Matroule, JY, Carthy, CM, Granville, DJ, Jolois, O, Hunt, DW, Piette, J. (2001) Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization Oncogene 20,4070-4084[Medline][Order article via Infotrieve]
  23. Runnels, JM, Chen, N, Ortel, B, Kato, D, Hasan, T. (1999) BPD-MA-mediated photosensitization in vitro and in vivo: cellular adhesion and beta1 integrin expression in ovarian cancer cells Br J Cancer 80,946-953[Medline][Order article via Infotrieve]
  24. Vile, GF, Tyrrell, RM. (1995) UVA radiation-induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen Free Radic Biol Med 18,721-730[Medline][Order article via Infotrieve]
  25. Chan, W-H, Yu, J-S. (2000) Inhibition of UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermal carcinoma A431 cells by genistein J Cell Biochem 78,73-84[Medline][Order article via Infotrieve]
  26. Grossman, N, Schneid, N, Reuveni, H, Halevy, S, Lubart, R. (1998) 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species Lasers Surg Med 22,212-218[Medline][Order article via Infotrieve]
  27. Ohnishi, Y, Murakami, M, Wakeyama, H. (1990) Effects of hematoporphyrin derivative and light on Y79 retinoblastoma cells in vitro Invest Ophthalmol Vis Sci 31,792-797[Abstract/Free Full Text]
  28. Mol de NJ, Beijersbergen, von Henegouwen, GMJ, Mohn, GR, Glickman, BW, van Kleef, PM. (1981) On the involvement of singlet oxygen in mutation induction by 8-methoxypsoralen and UVA irradiation in Escherichia coli K-12 Mutat Res 82,23-30[Medline][Order article via Infotrieve]
  29. Hadjur, C, Richard, MJ, Parat, MO, Favier, A, Jardon, P. (1995) Photodynamically induced cytotoxicity of hypericin dye on human fibroblast cell line MRC5 J Photochem Photobiol B 27,139-146[Medline][Order article via Infotrieve]
  30. Chuang, S-M, Liou, G-Y, Yang, J-L. (2000) Activation of JNK, p38 and ERK mitogen-activated protein kinases by chromium (VI) is mediated through oxidative stress but does not affect cytotoxicity Carcinogenesis 21,1491-1500[Abstract/Free Full Text]
  31. Hiraishi, H, Terano, A, Ota, S, et al (1991) Antioxidant defenses of cultured gastric cells against oxygen metabolites: role of GSH redox cycle and endogenous catalase Am J Physiol 261,G921-G928[Abstract/Free Full Text]
  32. Masaki, H, Okano, Y, Sakurai, H. (1998) Differential role of catalase and glutathione peroxidase in cultured human fibroblasts under exposure to H2O2 or ultraviolet B light Arch Dermatol Res 290,113-118[Medline][Order article via Infotrieve]
  33. Collard, CD, Agah, A, Stahl, GL. (1998) Complement activation following reoxygenation of hypoxic human endothelial cells: role of intracellular reactive oxygen species, NF-kappaB and new protein synthesis Immunopharmacology 39,39-50[Medline][Order article via Infotrieve]
  34. Michael, JR, Markewitz, BA, Kohan, DE. (1997) Oxidant stress regulates basal endothelin-1 production by cultured rat pulmonary endothelial cells Am J Physiol 273,L758-L774
  35. Killilea, DW, Hester, R, Balczon, R, Babal, P, Gillespie, MN. (2000) Free radical production in hypoxic pulmonary artery smooth muscle cells Am J Physiol Lung Cell Mol Physiol 279,L408-L412[Abstract/Free Full Text]
  36. Parish, CA, Hashimoto, M, Nakanishi, K, Dillon, J, Sparrow, JR. (1998) Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium Proc Natl Acad Sci USA 95,14609-14613[Abstract/Free Full Text]
  37. Monroe, BM. (1977) Quenching of singlet oxygen by aliphatic amines J Physical Chem 81,1861-1864
  38. Organisciak, DT, Darrow, RA, Bicknell, IR, Jiang, Y-L, Pickford, M, Blanks, JC. (1991) Protection against retinal light damage by natural and synthetic antioxidants Hollyfield, JG Anderson, RE La Vail, MM eds. Retinal Degenerations ,189-201 CRC Press Boca Raton, FL.
  39. Borges, J, Li, Z-Y, Tso, MO. (1990) Effects of repeated photic exposures on the monkey macula Arch Ophthalmol 108,727-733[Abstract]
  40. Mellerio, J. (1994) Light effects on the retina Albert, DM Jakobiec, FA eds. Principles and Practice of Ophthalmology: Basic Science ,1326-1345 WB Saunders Philadelphia.
  41. Delori, FC, Goger, DG, Dorey, CK. (2001) Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects Invest Ophthalmol Vis Sci 42,1855-1866[Abstract/Free Full Text]
  42. von Rückmann, A, Fitzke, FW, Bird, AC. (1997) Fundus autofluorescence in age-related macular disease imaged with a laser scanning ophthalmoscope Invest Ophthalmol Vis Sci 38,478-486[Abstract/Free Full Text]
  43. Feeney-Burns, L, Hilderbrand, ES, Eldridge, S. (1984) Aging human RPE: morphometric analysis of macular, equatorial, and peripheral cells Invest Ophthalmol Vis Sci 25,195-200[Abstract/Free Full Text]
  44. Wing, GL, Blanchard, GC, Weiter, JJ. (1978) The topography and age relationship of lipofuscin concentration in the retinal pigment epithelium Invest Ophthalmol Vis Sci 17,601-607[Abstract/Free Full Text]
  45. Okubo, A, Rosa, RHJ, Bunce, CV, et al (1999) The relationships of age changes in retinal pigment epithelium and Bruch’s membrane Invest Ophthalmol Vis Sci 40,443-449[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 IOVSHome page
J. Liu, W. Lu, D. Reigada, J. Nguyen, A. M. Laties, and C. H. Mitchell
Restoration of Lysosomal pH in RPE Cells from Cultured Human and ABCA4-/- Mice: Pharmacologic Approaches and Functional Recovery
Invest. Ophthalmol. Vis. Sci., February 1, 2008; 49(2): 772 - 780.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. R. Kim, Y. P. Jang, S. Jockusch, N. E. Fishkin, N. J. Turro, and J. R. Sparrow
The all-trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model
PNAS, December 4, 2007; 104(49): 19273 - 19278.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Ophthalmol.Home page
M Michaelides, L L Chen, M A Brantley Jr, J L Andorf, E M Isaak, S A Jenkins, G E Holder, A C Bird, E M Stone, and A R Webster
ABCA4 mutations and discordant ABCA4 alleles in patients and siblings with bull's-eye maculopathy
Br. J. Ophthalmol., December 1, 2007; 91(12): 1650 - 1655.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
V. Justilien, J.-J. Pang, K. Renganathan, X. Zhan, J. W. Crabb, S. R. Kim, J. R. Sparrow, W. W. Hauswirth, and A. S. Lewin
SOD2 Knockdown Mouse Model of Early AMD
Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4407 - 4420.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. Abeywickrama, H. Matsuda, S. Jockusch, J. Zhou, Y. P. Jang, B.-X. Chen, Y. Itagaki, B. F. Erlanger, K. Nakanishi, N. J. Turro, et al.
Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells
PNAS, September 11, 2007; 104(37): 14610 - 14615.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
C. M. Ethen, C. Reilly, X. Feng, T. W. Olsen, and D. A. Ferrington
Age-Related Macular Degeneration and Retinal Protein Modification by 4-Hydroxy-2-nonenal
Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3469 - 3479.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
N. Zhang, J. J. Peairs, P. Yang, J. Tyrrell, J. Roberts, R. Kole, and G. J. Jaffe
The Importance of Bcl-xL in the Survival of Human RPE Cells
Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3846 - 3853.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Zhou, Y. P. Jang, S. R. Kim, and J. R. Sparrow
Complement activation by photooxidation products of A2E, a lipofuscin constituent of the retinal pigment epithelium
PNAS, October 31, 2006; 103(44): 16182 - 16187.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Ophthalmol.Home page
P Hawse
Blocking the blue.
Br. J. Ophthalmol., August 1, 2006; 90(8): 939 - 940.
[Full Text] [PDF]

Home page
 Br. J. Ophthalmol.Home page
K Hayashi and H Hayashi
Visual function in patients with yellow tinted intraocular lenses compared with vision in patients with non-tinted intraocular lenses
Br. J. Ophthalmol., August 1, 2006; 90(8): 1019 - 1023.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. P. Jang, H. Matsuda, Y. Itagaki, K. Nakanishi, and J. R. Sparrow
Characterization of Peroxy-A2E and Furan-A2E Photooxidation Products and Detection in Human and Mouse Retinal Pigment Epithelial Cell Lipofuscin
J. Biol. Chem., December 2, 2005; 280(48): 39732 - 39739.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
L. A. Voloboueva, J. Liu, J. H. Suh, B. N. Ames, and S. S. Miller
(R)-{alpha}-Lipoic Acid Protects Retinal Pigment Epithelial Cells from Oxidative Damage
Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4302 - 4310.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
O. Strauss
The Retinal Pigment Epithelium in Visual Function
Physiol Rev, July 1, 2005; 85(3): 845 - 881.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. E. Fishkin, J. R. Sparrow, R. Allikmets, and K. Nakanishi
Isolation and characterization of a retinal pigment epithelial cell fluorophore: An all-trans-retinal dimer conjugate
PNAS, May 17, 2005; 102(20): 7091 - 7096.
[Abstract] [Full Text] [PDF]

Home page
 Arch OphthalmolHome page
R. E. Braunstein and J. R. Sparrow
A Blue-Blocking Intraocular Lens Should Be Used in Cataract Surgery
Arch Ophthalmol, April 1, 2005; 123(4): 547 - 549.
[Full Text] [PDF]

Home page
 IOVSHome page
J.-H. Yang, W.-D. Le, S. F. Basinger, S. M. Wu, and C.-y. Yang
Mechanisms of Apoptosis in Human Retinal Pigment Epithelium Induced by TNF-{alpha} in Conditions of Heavy Metal Ion Deficiency
Invest. Ophthalmol. Vis. Sci., March 1, 2005; 46(3): 1039 - 1046.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
K. A. Howes, Y. Liu, J. L. Dunaief, A. Milam, J. M. Frederick, A. Marks, and W. Baehr
Receptor for Advanced Glycation End Products and Age-Related Macular Degeneration
Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3713 - 3720.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
X. Gao and P. Talalay
Induction of phase 2 genes by sulforaphane protects retinal pigment epithelial cells against photooxidative damage
PNAS, July 13, 2004; 101(28): 10446 - 10451.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
M. Rozanowska, A. Pawlak, B. Rozanowski, C. Skumatz, M. Zareba, M. E. Boulton, J. M. Burke, T. Sarna, and J. D. Simon
Age-Related Changes in the Photoreactivity of Retinal Lipofuscin Granules: Role of Chloroform-Insoluble Components
Invest. Ophthalmol. Vis. Sci., April 1, 2004; 45(4): 1052 - 1060.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Ophthalmol.Home page
M A Mainster and J R Sparrow
How much blue light should an IOL transmit?
Br. J. Ophthalmol., December 1, 2003; 87(12): 1523 - 1529.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
M Michaelides, D M Hunt, and A T Moore
The genetics of inherited macular dystrophies
J. Med. Genet., September 1, 2003; 40(9): 641 - 650.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
C. Zhang, J. Baffi, S. W. Cousins, and K. G. Csaky
Oxidant-induced cell death in retinal pigment epithelium cells mediated through the release of apoptosis-inducing factor
J. Cell Sci., May 15, 2003; 116(10): 1915 - 1923.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. R. Sparrow, H. R. Vollmer-Snarr, J. Zhou, Y. P. Jang, S. Jockusch, Y. Itagaki, and K. Nakanishi
A2E-epoxides Damage DNA in Retinal Pigment Epithelial Cells. VITAMIN E AND OTHER ANTIOXIDANTS INHIBIT A2E-EPOXIDE FORMATION
J. Biol. Chem., May 9, 2003; 278(20): 18207 - 18213.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
J. R. Sparrow, J. Zhou, and B. Cai
DNA Is a Target of the Photodynamic Effects Elicited in A2E-Laden RPE by Blue-Light Illumination
Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2245 - 2251.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. R. Sparrow
Therapy for macular degeneration: Insights from acne
PNAS, April 15, 2003; 100(8): 4353 - 4354.
[Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USA