Blue Light Induced A2E Oxidation in Rat’s Eyes - Experimental Animal Model of Dry AMD

Abstract

Previous studies have shown that short-wavelength blue visible light induces retinal injury and may be a risk factor for age related macular degeneration. A2E is a blue light absorbing retinal chromophore that accumulates with age. Our previous in vitro studies have determined that although A2E itself has a low phototoxic efficiency, the oxidation products of A2E that are formed in the presence of visible light can contribute to observed retinal pigment epithelial photodamage. The purpose of this study was to investigate the effects of blue light on retinal phototoxicity and its relationship to A2E, oxidized A2E and its isomers. Sprague-Dawley albino rats were dark adapted for 24 hours. Control rats remained in the dark while experimental rats were exposed to blue light (λ = 450 nm, 3.1 mW/cm²) for 6 hours. Isolated retinas were homogenized in Folch extraction mixture and then in chloroform. The dried extracts were reconstituted and divided for determination of organic soluble compound. Esters of fatty acids were determined with GC-MS, A2E and other chromophores using HPLC, and A2E oxidation products with LC-MS. Exposure of rat eyes to blue light did not significantly change the fatty acid composition of the retina. The A2E concentration (normalized to fatty acid content) in blue light exposed animals was found to be lower than the A2E concentration in control rats. The concentrations of all-trans-retinal-ethanolamine adduct and iso-A2E a precursor and an isomer of A2E respectively, were also lower after blue-light exposure than in the retinas of rats housed in the dark. On the other hand, the amount of oxidized forms of A2E was higher in the animals exposed to blue light. We conclude that in the rat eye, blue-light exposure promotes the oxidation of A2E and iso-A2E to the products that are toxic to retinal tissue. Although high concentrations of A2E may be cytotoxic to the retina, the phototoxicity associated with blue light damage to the retina is in part a result of the formation of toxic A2E oxides. This effect may partially explain the association between blue light induced retinal injury and macular degeneration.

Introduction

Age-related macular degeneration (AMD) is the principal cause of blindness among the elderly in the United States and in European and Australian (Oceanic, Pacific) nations.^1–4 Little is known about its etiology; however, recent studies have uncovered several risk factors, namely: aging, smoking, a high fat diet, genetics, and gender.⁵ Sunlight is an environmental factor that has been identified⁶ but epidemiologic studies assessing the association between light exposure and AMD have provided inconsistent results. Two cohort based studies^7,8 showed an association while case control studies^9,10 failed to confirm this relationship, although the Age-Related Eye Disease Study (AREDS)¹¹ did suggest an association between increased light exposure and AMD.

Retinal light damage in rats results in a retinal degeneration exhibiting features of atrophic age-related macular degeneration, including photoreceptor and retinal pigment epithelium (RPE) degeneration, choriocapillaris atrophy, and retinal remodeling.¹² Short-wavelength blue light has the greatest potential for phototoxicity.¹³ Blue light can induce generation of reactive oxygen species by mitochondrial cytochromes,¹⁴ inhibit cytochrome oxidase,^15,16 and lead to calcium accumulation¹⁷ and apoptosis.¹⁸ Damage may be mediated through rhodopsin, as blue light causes photoreversal of bleaching.¹⁹

The first clinical markers for macular degeneration are the accumulation of a fluorescent material (lipofuscin) in the retinal pigment epithelium^20,21 and deposits of lipids and lipoproteins (drusen and basal deposits) between the RPE and the basement membrane and within Bruch’s membrane.^22,23 The lipofuscin accumulated in the lysosomes can act as a photooxidizing agent in the presence of short wavelength (400–430 nm) blue light.^24,25 The resultant lipid oxidation damages the lysosomal membrane and promotes hydrolytic enzyme leakage and cellular damage.²⁶ Damage to RPE cells can disrupt the daily removal of damaged photoreceptor outer segments (disc shedding through RPE phagocytosis)²⁷ and contribute to the pathogenesis of AMD. In addition to lipofuscin there may be other potential blue light-absorbing endogenous components i.e. all-trans-retinal and protoporphyrin that may cause retinal phototoxicity.^25,28–39

A major fluorophore of RPE lipofuscin is A2E, a bis-retinaldehyde-phosphatidylethanolamine,⁴⁰ which increases with age.^20,41 It was first isolated by Eldred⁴² and the correct structure determined by Sakai.⁴³ A2E has been shown to be cytotoxic through several mechanisms.^27,44 Vives-Bauza et al.²⁷ have shown that A2E impairs phagocytosis. A2E also detaches cytochrome c from mitochondria and induces apoptosis in RPE cells.⁴⁵ A2E-loaded RPE cells fail to completely digest phospholipids and block late endosomal cholesterol efflux, leading to an accumulation of cholesteryl esters,⁴⁶ which leads to a buildup of cholesterol and oxidized cholesterol. Destruction of post-mitotic RPE and/or buildup of undigested material in RPE could contribute to the development of age-related macular degeneration.

We have previously shown that although A2E is cytotoxic to human pigmented RPE,⁴⁷ it has little photoreactivity.⁴⁸ It was determined that A2E can photogenerate singlet oxygen but the quantum yield of that process is less than 1%.^48–50 A2E does not efficiently produce a long-lived triplet excited state, an intermediate that is essential for the generation of singlet oxygen or other reactive oxygen species.^47,51 A2E is far less photoreactive than liposfuscin,^29,52,34 all-trans-retinal,³⁹ protoporphyrin⁵³ or other endogenous blue light chromophores in the retina.^54,55

Blue light induced A2E oxidation leads to the formation of A2E oxidation products, whose exact structure(s) have yet to be determined.^56–59 Once formed, oxidized A2E products are highly toxic to RPE cells.^60,61 The biosynthesis of A2E and its conversion to its oxidized form(s) has been found to be greatly accelerated by light in the abcr−/− transgenic mouse model for Stargardt’s macular degeneration.^62,63

We report here a study of the in vivo effects of blue light phototoxicity toward the retina in the rat eye and its relationship to the formation of A2E and oxidized A2E.

Materials and Methods

Reagents

Ethanolamine, hexane and toluene (both HPLC grade) were purchased from Aldrich (Milwaukee, WI, USA). Methanol (HPLC grade) was from Anachemia Science (Sparks, NV, USA). 1-Palmytolyl-2-hydroxy-sn-glycero-3-phosphocholine (PHGPc) was from Avanti Polar Lipids (Alabaster, AL, USA). Acetic acid (glacial) was from Caledon Laboratories (Georgetown, ON, Canada). Glutaraldehyde and paraformaldehyde were from Electron Microscopy Sciences (Hatfield, PA, USA). Heptadecanoic acid was from Fluka (St. Louis, MO, USA). Hydrochloric acid, 2-propanol, sodium chloride and water (HPLC grade) were from Mallinckrodt Baker (Phillipsburg, NJ, USA). Ethanol was from Pharmco-AAPER (Charlotte, NC, USA). Butylated hydroxytoluene (BHT), chloroform (HPLC grade) and all-trans-retinal were from Sigma (St. Louis, MO, USA). Boron trifluoride (BF₃) in methanol was from Supelco (Bellefonte, PA, USA). Sterile phosphate buffered saline without Ca²⁺ and Mg²⁺ (PBS) was prepared at Media and Glassware Units at NIEHS (RTP, NC, USA). All reagents used for experiments were at least analytical grade.

Animal treatments

Male Sprague-Dawley rats (300–450 grams) were randomly assigned to control (N=15) or light-exposed (N=15) groups. Rats were housed under broad-band fluorescent [Sylvania Cool White (Danvers, MA, USA), 484 lux] cyclic light (12 light: 12 dark). Food and water were available ad lib. All experimental procedures, as well as animal care and handling, adhered to the guidelines outlined in the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. All experimental procedures were also reviewed and approved by Institutional Animal Care and Use Committee at Alcon.

Induction of photochemical lesions

Control and light-exposed rats were dark adapted for 24 hours. Photooxidatively induced lesions were generated in light-exposed rats after dark adaptation by exposure to blue light [3.1 mW/cm², Philips fluorescent lamps (F40/BB)] for 6 hours. Rats were single housed in clear polycarbonate cages with minimal bedding to prevent burrowing. Movement of rats within the cage was unrestricted during this light exposure. Rats were sacrificed immediately after light exposure along with dark-adapted control rats. Eyes were enucleated under dim red light; one eye was frozen in liquid nitrogen for biochemical analysis and one eye was used for histology (Fig. 1).

Figure 1.

The protocol of blue light exposure of the animals.

Tissue preparation for histology

Ocular tissues from control and light-exposed rats were obtained under deep anesthesia. Orientation of the eye was maintained by placement of a marker suture at the 12-o’clock position prior to ocular enucleation. The cornea and lens were removed and posterior poles were fixed by immersion in a mixture of paraformaldehyde (2%) and glutaraldehyde (2%) in (0.1 M) phosphate buffer (pH 7.4). Tissues were washed, dehydrated in an ascending ethanol series and embedded in JB-4 plastic resin. Thick sections (1 to 1.5 μm) were cut and stained (Multiple Stain, Polysciences, Washington, PA, USA) and analyzed using a quantitative computer image analysis system attached to the microscope. Retinal Pigment Epithelium (RPE), Outer Nuclear Layer (ONL) and Inner Nuclear Layer (INL) thickness as well as inner segments (IS) and outer segments (OS) lengths were measured to assess retinal lesions. As the INL is not significantly affected by light exposure, this layer served as an additional control measurement. Retinal measurements were made at three nasal and three temporal retinal locations separated by 200 μm. For each eye, a minimum of four sections were randomly selected including one through the optic nerve and three superior retina positions.

Tissue preparation for chemical analysis

All following procedures were performed on ice and in red light. A group of 4–6 eyeballs was hemisected. The retinas and RPE were isolated, homogenized in a Dounce glass-glass homogenizer in a cold mixture of 3.5 ml phosphate buffer (0.1 M, pH 7.4), 3.2 ml deoxygenated chloroform and 1.6 ml BHT (50 μM) in methanol. The chloroform-soluble components of the tissue homogenate were extracted by 1-min intensive shaking. Phases were separated by centrifugation at 2500 g for 5 min at 4°C. The lower organic phase was transferred to an empty vial and placed on ice. The remaining sample was diluted with 12 ml water and 24 ml deoxygenated chloroform was added. The leftover phospholipids and organic soluble chromophores were extracted to chloroform as previously. Both organic solutions were combined and evaporated in vacuum using a rotovap. Each dried sample was reconstituted in a small volume of chloroform/methanol (2:1) mixture, centrifuged at 16000 g and 4°C for 5 min and divided for determination of A2E, its derivatives, and fatty acids, and each sample was evaporated in a nitrogen stream.

Synthesis of A2E

A2E was synthesized as described previously.⁴⁰ Briefly, a solution of all-trans-retinal (117 mM) and ethanolamine (52 mM) in a deoxygenated ethanol and acetic acid mixture (650:2, v/v) was stirred at room temperature in the dark for 2 days. The post-synthesis mixture was purified by silica gel column chromatography and then by HPLC. The purity of A2E was checked using HPLC and TLC methods. A2E concentration was determined by absorption spectroscopy⁴⁰ using a Hewlett Packard diode array 8453 spectrophotometer (Hewlett Packard GmbH, Waldbronn, Germany). Dried A2E samples were stored under argon at −80°C for further use.

A2E analysis

The content of A2E and other retinoids was determined using HPLC. The mobile phase consisted of 48% hexane and 52% a mixture of 2-propanol / ethanol / water / acetic acid (376:100:50:0.275). The extracts from the rat retinas and a standard of synthetic A2E were reconstituted in 50 μl of the mobile phase directly before injection to a Partisil SilicaMicrosorb 5 μ, 250×4.6 mm HPLC column (Alltech) equipped with Safeguard 4.3 mm Intersil 5 μ Si (MetaChem). The samples were eluted at a flow rate of 1.5 ml/min.

Detection of A2E oxidation products

Each dried sample of the retinal extract and A2E standard was reconstituted in 25 μl of a solution of PHGPc (100 μM) as an internal standard in a deoxygenated chloroform / methanol / water (33:66:1) mixture. Products of A2E oxidation were detected in a LCQ^DUO mass spectrometer (Thermo Finnigan, San Jose, CA) with at the following parameters: ion charge – positive, sheath gas flow rate – 40 au, electrospray ionization – 5 kV, spray current – 0.44 μA, capillary temperature - 200°C, capillary voltage - +15 V, tube lens offset – 55 V, and collision energy – 30 V.

Acidic hydrolysis of retinal lipids and derivatization of fatty acids

Each dried retinal extract was reconstituted in 350 μl of a deoxygenated mixture of chloroform / methanol (2:1) containing 20 nmol heptadecanoic acid as an internal standard and evaporated in a nitrogen stream. Then, each sample was dissolved in 400 μl of a deoxygenated mixture of methanol / chloroform / HCl (10:1:1) and hydrolyzed overnight at 60°C. After cooling, 400 μl NaCl (2%) water solution, 400 μl toluene, and 400 μl hexane were added to each vial. The samples were shaken intensively for 1 min and centrifuged at 1000 g and 22°C for 5 min. The upper organic phase of each sample was transferred to an empty vial and evaporated in a nitrogen stream. In the meantime, 800 μl hexane was added to each lower water phase, and extracted as above. The organic phase was transferred to the vial containing the corresponding dried extract and evaporated.

Free fatty acids were derivatized to methyl esters by incubation in 60 μl boron trifluoride (10%) in methanol at 60°C for 20 min. The samples were cooled down to room temperature and diluted with 600 μl hexane and 200 μl aqueous saturated NaCl. The samples were shaken and centrifuged as previously. The lower water layer was removed and washing in saturated NaCl solution was repeated. The upper hexane phase of each sample was transferred to an empty vial and evaporated in a nitrogen stream. Fatty acids remaining in the water phase were extracted with an additional 500 μl hexane and combined with the organic phase collected previously. Hexane solutions of fatty acids were evaporated completely and dried samples were stored at −80°C until mass spectrometric analysis.

Fatty acid and cholesterol analysis

Gas chromatography / mass spectrometry (GC/MS) analyses were performed in triplicate on a Thermo Finnigan Trace GC 2000 and Trace MS Plus equipped with an AS2000 autosampler and a cold-on-column injector. Either an SPB-5 (Supelco, Bellefonte, PA) or a ZB-5ms (Phenomenex, Torrance, CA) column with dimensions 0.32 mm × 30 m and a film thickness of 0.25 μm, preceded by a 0.53 mm × 5 m deactivated fused silica guard column, was used for the chromatographic separations. The initial oven temperature was 50°C and was held for 2 min. The temperature was ramped at 10°C/min to 130°C, then continued at 7.5°C/min to 300°C, which was maintained for 4.5 min before equilibration at 50°C for the next injection. Helium was used as the carrier gas at a flow rate of 1.9 ml/min. Positive ions were produced via electron impact (EI+) at an energy of 70 eV. A mass to charge (m/z) range of 50–800 was scanned every 0.5 s from 2 min to 35 min post injection. The source temperature was 225°C and the GC interface was held at 280°C. Fatty acid methyl ester (FAME) peaks were identified by comparison of spectra with those in the NIST mass spectral library and by comparison to spectra and retention times of Supelco 37 Component FAME Mix (Sigma-Aldrich). Relative abundances were determined from the total ion chromatogram. A correction factor was applied to convert peak areas to weight percentages based on results obtained from analysis of the Supelco FAME mix.

Statistical analysis

Data in graphs in figures 2–7 are presented as mean ± SD of 3 to 8 experiments; p values were calculated using the t- test.

Figure 2.

(A) Retinal layer thickness measured in control dark-adapted rat retinas (N=6) and in the retinas obtained from rats immediately after a 6-hour blue light exposure (N=8). No significant changes in RPE or ONL layer thickness were detected. Length of photoreceptor inner and outer segments was significantly reduced by 12% in light exposed retinas compared to controls (t-test). (B) Cross section images of the retinas isolated from the rats exposed to blue light or housed in the dark for 6 hours. The retinas were evaluated immediately after light exposure. Pyknotic nuclei in the ONL and disorganization of photoreceptor inner and outer segments were the earliest retinal changes that were observed (Calibration bar = 10 μm)

Results

In control rats, average RPE thickness was 2.8 μm [Standard Error of the Mean (S.E.M.) ± 0.08 μm], average ONL thickness was 34.4 μm (S.E.M. ± 0.8 μm) and average photoreceptor segment length was 23.4 μm (S.E.M. ± 0.4 μm). Evaluation of retinal lesions immediately after a 6-hour blue-light exposure demonstrated a significant (ANOVA, p = 0.002) shortening of the photoreceptor inner and outer segment lengths in light-exposed rats. Photoreceptor segment lengths were 12% shorter in light-exposed retinas compared to normal (Fig. 2A). Blue-light exposure for 6 hours did not result in significant thinning of the INL or ONL nor did we see flattening of the RPE. Immediately after light exposure 40 to 50% of the photoreceptor nuclei were pyknotic with condensation of chromatin and dark staining nuclei (Fig. 2B). It has been previously observed that when retinas are evaluated 4 weeks after this blue-light exposure, RPE thickness, ONL thickness and photoreceptor segment length are reduced 60%, 61% and 67%, respectively.⁶⁴

The most abundant derivatized fatty acids (FA-s) extracted from the rat retina possessed chains of 16 – 22 carbon atoms (Fig. 3). In rats kept in the dark, the most abundant saturated FA-s in retinal extracts were palmitic and stearic acids, making up 22.1 and 13.0 % respectively, while the main unsaturated retinal fatty acids were palmitoleic, arachidonic, and docosahexaenoic acids, with abundances of 3.7, 5.2, and 14.9 % respectively. A large group of unsaturated retinal FA-s consisted of a mixture of molecules containing 18-carbon chains, including elaidic, oleic, linoleaidic, linoleic and linolenic acids. Generally, the concentration of unsaturated fatty acids in the rat retina was almost twice as high as the saturated ones (Fig. 4).

Figure 3.

Fatty acid (FA) composition expressed as FA mole percent in retinas isolated from rats exposed to blue light or housed in the dark. Plain and striped bars represent saturated and unsaturated FA, respectively. Phospholipids present in the retinal extracts were subjected to acidic hydrolysis. Free FA-s were derivatized to methyl esters and analyzed by GC-MS with heptadecanoic acid (C17:0) as an internal standard. The numbers x, y and z in the FA formulas (Cx:ynz) mean: the number of carbon atoms in a FA molecule, the number of carbon-carbon double bonds (C=C) in a FA molecule and the number of a carbon atom, counted starting the methyl end of the FA molecule at which the first double bond C=C is placed, respectively; mix. – a mixture of FA isomers; C18:unsat. mix – a mixture of polyunsaturated FA-s containing 18 carbon atoms in their molecules. Bars represent the average of 3 GC-MS replicates ± SD.

Figure 4.

Global abundance of saturated and unsaturated fatty acids expressed as fatty acid mole percent in the retinas of the rats exposed to blue light or housed in the dark. Bars represent the average of 3 GC-MS replicates ± SD.

Exposure of the rats to blue light caused a slight decrease in linoleaidic, linoleic, and docosahexaenoic acid concentrations and a small rise in the saturated FA abundance (Fig. 3). The differences, however, were not statistically significant, suggesting that oxidation of unsaturated fatty acids was not observed immediately after the blue-light exposure used in this study.

There was little or no change in the total molecular percentage of saturated and unsaturated fatty acids after irradiation (Fig. 4). Thus, fatty acids were used as a normalization factor for other photoxidation markers such as retinal chromophores.

A2E, one of the lipofuscin chromophores, was detected in the retinas of the rats used in our study at the level of 6 pmol/μmol FA. Blue light illumination caused a significant six-fold decrease in the total amount of A2E in the retina in comparison to animals housed in the dark (Fig 5A). The concentration of the A2E isomer (iso-A2E) in the rats’ retinas decreased about four-fold during blue light exposure (Fig. 5B). An additional chromophore - a precursor of A2E - was previously observed in extracts from photoreceptor outer segments of wild and abcr −/− mice⁶² and later described^65,66 as the imine form of retinal and ethanolamine. We detected such a chromophore in the extracts from the rat eyes. Its HPLC peak was observed at a retention time of ~3.8 min, while A2E peaked at ~6.3 min (Fig. 5C). The absorption spectrum of the additional chromophore had a characteristic single band with maximal absorbance at 438 nm (Fig. 5D). A2E, however, was characterized by 2 bands with local maximal absorbance – one at the same wavelength as the additional chromophore (438 nm) and another at 334 nm. In a trend similar to that of the A2E isomers, the level of the precursor decreased substantially in the retinas of animals exposed to intense blue light (Fig. 5E).

Figure 5.

(A), iso-A2E (B) and all-trans-retinal-ethanolamine (atRal-EA) adduct (E) amounts detected in the extract from the retinas of rats illuminated with blue light or housed in the dark. Quantitative analysis of the retinoids was performed using HPLC with synthetic A2E as a standard. A2E amounts were normalized to fatty acid (FA) content. Each bar represents an average of 5 measurements ± SD. (C) HPLC chromatogram of the extract from the rat retinas. The inset contains a part of the chromatogram that was zoomed to present peaks of atRal-EA and A2E. (D) Absorption spectra of A2E and atRal-EA obtained during HPLC analysis of an extract from retinas of 4 rats illuminated with blue light.

We monitored A2E and its oxidized products using mass spectrometry. Molecules were detected at m/z (mass to charge ratio) of 608, 624 and 640, which are products of addition of 1, 2 and 3 oxygen atoms to the A2E molecule (M = 592), respectively. We also observed such A2E and iso-A2E oxidation products in rat retina extracts (Fig. 6). The abundance of the product at m/z = 624 was twice as high as either of the other two forms. Blue light exposure enhanced A2E and iso-A2E oxidation by a factor of 2.5 in the rat retina. Although blue light exposure increases the total amount of oxidized A2E, the ratio of mono-, di- and tri-oxidized A2E remains the same, whether in the dark or light.

Figure 6.

Blue light induced oxidation of A2E in the rat retina. The abundance of the oxidized form of A2E was determined using ESI-MS. The bars represent the abundance of A2E oxidative forms with molecular masses higher than A2E (M = 592) by 16, 32, and 48 daltons normalized to the internal standard and total fatty acid content in the samples. Each bar symbolizes an average of 5 measurements ± SD.

Discussion

Short-wavelength blue visible light (430 nm) is thought to be a risk factor for AMD, but the mechanism for the damage is still to be determined. The acute blue light exposure used in this model results in severe, irreversible, retinal lesions.⁶⁴ Five days after light exposure the electroretinogram (ERG) was reduced approximately 69% and when re-evaluated 3 weeks later, recovery of ERG response amplitudes were not measured. RPE cells were flattened, photoreceptor cell loss was obvious and photoreceptor inner and outer segment lengths were shortened. The mechanism of blue light damage may be inactivation of cytochrome oxidase leading to ATP depletion¹⁶ generation of reactive oxygen species by mitochondrial cytochromes¹⁴ and/ or photoreversal of rhodopsin bleaching and increased generation of toxic rhodopsin intermediates.¹⁹ Blue light exposure has also been shown to result in complement activation. Photooxidation of A2E in human ARPE-19 cells results in complement activation and C3a formation.⁶⁷ In the blue light damage model used in this investigation, we have also shown deposition of C3, Factor B, Factor H and membrane attack complex (MAC).⁶⁸ Changes in the ageing human retina may result from photo-oxidative stress to outer segments enriched in polyunsaturated fatty acids and under high oxygen tension provided by the choroid, along with outer retinal cells rich in photosensitizers such as A2E, lipofuscin and cytochrome c oxidase. This provides an environment ripe for oxidative insult and carboxyethylpyrrole (CEP)-adduct formation that can initiate cellular damage and maculopathy.^69,37

We have used this in vivo blue light damage model to investigate another mechanism for the induction of AMD; namely the photooxidation of retinal components by blue-light absorbing chromophores, which accumulate with age in the retina. Lipofuscin accumulates with age, produces singlet oxygen and other reactive oxygen species very efficiently in the presence of blue light in vivo and in vitro and subsequently damages the retina. One of the fluorescent components of lipofuscin is A2E. The question has been raised whether A2E is the primary photooxidizing agent for blue light damage to the retina. It has been reported that A2E is the source of blue light-mediated reactive oxygen species that can lead to RPE cell apoptosis.^70–72 However, A2E has been found to be very inefficient at producing reactive oxygen species, especially compared to other blue light chromophores in the aged retina.^48–50 A2E does not efficiently produce a long-lived triplet excited state, an intermediate that is essential for the generation of singlet oxygen or other reactive oxygen species.^47,51

However, oxidized A2E is certainly cytotoxic. While it has been suggested that A2E photooxidizes itself to form oxidized A2E⁶⁰ it is much more likely that other endogenous photoactive components (lipofuscin, protoporphyrin) oxidize A2E in vivo.^25,28–36

In this study we investigated the effects of blue light on the in vivo phototoxicity toward the retina in the rat eye and its relationship to A2E and oxidized A2E and its isomers. It appears that blue light exposure promotes the oxidation of A2E and iso-A2E in the rat eyes. As the A2E oxides are toxic to retinal tissue, this may partially explain blue light-induced retinal injury. A2E may not be photooxidizing itself; however, there are sufficient phototoxic chromophores including lipofuscin that may easily photooxidize A2E to the mono, di- and tri-oxidation products of A2E leading to extensive damage to the retina. These studies suggest that similar phototoxic effects may also take place in human eyes.