Is There an Optimal Combination of AREDS2 Antioxidants Zeaxanthin, Vitamin E and Vitamin C on Light-Induced Toxicity of Vitamin A Aldehyde to the Retina?

Vitamins C and E and zeaxanthin are components of a supplement tested in a large clinical trial-Age-Related Eye Disease Study 2 (AREDS2)-and it has been demonstrated that they can inhibit the progression of age-related macular degeneration. The aim of this study was to determine the optimal combinations of these antioxidants to prevent the phototoxicity mediated by vitamin A aldehyde (ATR), which can accumulate in photoreceptor outer segments (POS) upon exposure to light. We used cultured retinal pigment epithelial cells ARPE-19 and liposomes containing unsaturated lipids and ATR as a model of POS. Cells and/or liposomes were enriched with lipophilic antioxidants, whereas ascorbate was added just before the exposure to light. Supplementing the cells and/or liposomes with single lipophilic antioxidants had only a minor effect on phototoxicity, but the protection substantially increased in the presence of both ways of supplementation. Combinations of zeaxanthin with α-tocopherol in liposomes and cells provided substantial protection, enhancing cell viability from ~26% in the absence of antioxidants to ~63% in the presence of 4 µM zeaxanthin and 80 µM α-tocopherol, and this protective effect was further increased to ~69% in the presence of 0.5 mM ascorbate. The protective effect of ascorbate disappeared at a concentration of 1 mM, whereas 2 mM of ascorbate exacerbated the phototoxicity. Zeaxanthin or α-tocopherol partly ameliorated the cytotoxic effects. Altogether, our results suggest that the optimal combination includes upper levels of zeaxanthin and α-tocopherol achievable by diet and/or supplementations, whereas ascorbate needs to be at a four-fold smaller concentration than that in the vitreous. The physiological relevance of the results is discussed.

Preliminary Studies of Antimicrobial Activity of New Synthesized Hybrids of 2-Thiohydantoin and 2-Quinolone Derivatives Activated with Blue Light.

Thiohydantoin and quinolone derivatives have attracted researchers' attention because of a broad spectrum of their medical applications. The aim of our research was to synthesize and analyze the antimicrobial properties of novel 2-thiohydantoin and 2-quinolone derivatives. For this purpose, two series of hybrid compounds were synthesized. Both series consisted of 2-thiohydantoin core and 2-quinolone derivative ring, however one of them was enriched with an acetic acid group at N3 atom in 2-thiohydantoin core. Antibacterial properties of these compounds were examined against bacteria: Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The antimicrobial assay was carried out using a serial dilution method to obtain the MIC. The influence of blue light irradiation on the tested compounds was investigated. The relative yield of singlet oxygen (1O2*, 1Δg) generation upon excitation with 420 nm was determined by a comparative method, employing perinaphthenone (PN) as a standard. Antimicrobial properties were also investigated after blue light irradiation of the suspensions of the hybrids and bacteria placed in microtitrate plates. Preliminary results confirmed that some of the hybrid compounds showed bacteriostatic activity to the reference Gram-positive bacterial strains and a few of them were bacteriostatic towards Gram-negative bacteria, as well. Blue light activation enhanced bacteriostatic effect of the tested compounds.

Photodegradation of Lipofuscin in Suspension and in ARPE-19 Cells and the Similarity of Fluorescence of the Photodegradation Product with Oxidized Docosahexaenoate.

Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE), where its fluorescence properties are used to assess retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in the early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE and lipofuscin-laden cells to visible light, and to determine whether an abundant component of lipofuscin, docosahexaenoate (DHA), can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible light leads to a decrease in its long-wavelength fluorescence at about 610 nm, with a concomitant increase in the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure of lipofuscin-laden cells to light leads to a loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes in fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra, together with quantitation of the intensity of long-wavelength fluorescence, can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, and hence useful for screening the retina for increased oxidative damage and early AMD-related changes.

Comparison of Antioxidant Properties of Dehydrolutein with Lutein and Zeaxanthin, and their Effects on Cultured Retinal Pigment Epithelial Cells.

Dehydrolutein accumulates in substantial concentrations in the retina. The aim of this study was to compare antioxidant properties of dehydrolutein with other retinal carotenoids, lutein, and zeaxanthin, and their effects on ARPE-19 cells. The time-resolved detection of characteristic singlet oxygen phosphorescence was used to compare the singlet oxygen quenching rate constants of dehydrolutein, lutein, and zeaxanthin. The effects of these carotenoids on photosensitized oxidation were tested in liposomes, where photo-oxidation was induced by light in the presence of photosensitizers, and monitored by oximetry. To compare the uptake of dehydrolutein, lutein, and zeaxanthin, ARPE-19 cells were incubated with carotenoids for up to 19 days, and carotenoid contents were determined by spectrophotometry in cell extracts. To investigate the effects of carotenoids on photocytotoxicity, cells were exposed to light in the presence of rose bengal or all-trans-retinal. The results demonstrate that the rate constants for singlet oxygen quenching are 0.77 × 1010, 0.55 × 1010, and 1.23 × 1010 M-1s-1 for dehydrolutein, lutein, and zeaxanthin, respectively. Overall, dehydrolutein is similar to lutein or zeaxanthin in the protection of lipids against photosensitized oxidation. ARPE-19 cells accumulate substantial amounts of both zeaxanthin and lutein, but no detectable amounts of dehydrolutein. Cells pre-incubated with carotenoids are equally susceptible to photosensitized damage as cells without carotenoids. Carotenoids provided to cells together with the extracellular photosensitizers offer partial protection against photodamage. In conclusion, the antioxidant properties of dehydrolutein are similar to lutein and zeaxanthin. The mechanism responsible for its lack of accumulation in ARPE-19 cells deserves further investigation.

Products of Docosahexaenoate Oxidation as Contributors to Photosensitising Properties of Retinal Lipofuscin.

Retinal lipofuscin which accumulates with age in the retinal pigment epithelium (RPE) is subjected to daily exposures to high fluxes of visible light and exhibits potent photosensitising properties; however, the molecules responsible for its photoreactivity remain unknown. Here, we demonstrate that autooxidation of docosahexaenoate (DHE) leads to the formation of products absorbing, in addition to UVB and UVA light, also visible light. The products of DHE oxidation exhibit potent photosensitising properties similar to photosensitising properties of lipofuscin, including generation of an excited triplet state with similar characteristics as the lipofuscin triplet state, and photosensitised formation of singlet oxygen and superoxide. The quantum yields of singlet oxygen and superoxide generation by oxidised DHE photoexcited with visible light are 2.4- and 3.6-fold higher, respectively, than for lipofuscin, which is consistent with the fact that lipofuscin contains some chromophores which do contribute to the absorption of light but not so much to its photosensitising properties. Importantly, the wavelength dependence of photooxidation induced by DHE oxidation products normalised to equal numbers of incident photons is also similar to that of lipofuscin-it steeply increases with decreasing wavelength. Altogether, our results demonstrate that products of DHE oxidation include potent photosensitiser(s) which are likely to contribute to lipofuscin photoreactivity.

The Influence of Ethanolic Extract of Brazilian Green Propolis Gel on Hygiene and Oral Microbiota in Patients after Mandible Fractures.

Maintenance of proper oral hygiene by dental plaque elimination is one of the most important factors affecting the healing process in postoperative oral wounds. Propolis is a substance produced by bees. Ethanolic extract of propolis has bactericidal, fungicidal, anti-inflammatory, and antioxidative properties. Moreover, it can scavenge free radicals. The purpose of this paper is to demonstrate the efficacy of a gel containing 3% of ethanolic extract of Brazilian green propolis (EEP-B) when used for maintaining oral hygiene in patients with postoperative oral mucosal wounds. The hygiene was assessed using API, OHI, and SBI followed by microbiological examinations. The patients were divided into two groups. Group 1 consisted of those who used a gel containing EEP-B for oral hygiene, and group 2 consisted of those who used a gel without EEP-B. Although improved oral hygiene was noted in both groups, the improvement was markedly greater in the group using gel containing EEP-B. Summing up the results of microbiological examinations, EEP-B has beneficial effect on mouth microflora in postoperative period. Propolis preparations used for oral hygiene allow eliminating microorganisms of pathogenic character and physiological flora microorganisms considered as being opportunistic, with no harmful influence on physiological microflora in oral ecosystem.

Alternative method to treat oroantral communication and fistula with autogenous bone graft and platelet rich fibrin

BACKGROUND: Removing a tooth from the jaw results in the occurrence of oroantral communication in beneficial anatomic conditions or in the case of an iatrogenic effect. Popularized treatments of the oroantral communication have numerous faults. Large bone defect eliminates the chance to introduce an implant. Purpose of this work was assessment of the usefulness of autogenous bone graft and PRF in normal bone regeneration in the site of oroantral communication. MATERIAL AND METHODS: Bone regeneration in the site of oroantral communication was assessed in 20 patients. Bone defects were supplemented autogenous bone graft from mental protuberance in 14 cases and from oblique line in 6 cases. The graft was covered with a PRF membrane. RESULTS: In the study group in all cases closure of the oroantral communication was observed. The average width of the alveolar was 13 mm and the average height was 12.5 mm. In 3 patients an average increase of alveolar height of 1.5 mm was observed. CONCLUSIONS: This method may be the best option to prepare alveolar for new implant and prosthetic solutions

Concentration dependence of vitamin C in combinations with vitamin E and zeaxanthin on light-induced toxicity to retinal pigment epithelial cells.

The purpose of this study was to determine the effects of increasing concentration of ascorbate alone and in combinations with α-tocopherol and zeaxanthin on phototoxicity to the retinal pigment epithelium. ARPE-19 cells were exposed to rose bengal and visible light in the presence and absence of antioxidants. Toxicity was quantified by an assay of cell-reductive activity. A 20 min exposure to visible light and photosensitizer decreased cell viability to ca 42%. Lipophilic antioxidants increased viabilities to ca 70%, 61% and 75% for α-tocopherol, zeaxanthin and their combination, respectively. Cell viabilities were ca 70%, 56% and 5% after exposures in the presence of 0.35, 0.7 and 1.4 mm ascorbate, respectively. A 45 min exposure increased cell death to ca 74% and >95% in the absence and presence of ascorbate, respectively. In the presence of ascorbate, zeaxanthin did not significantly affect phototoxicity. α-Tocopherol and its combination with zeaxanthin enhanced protective effects of ascorbate, but did not prevent from ascorbate-mediated deleterious effects. In conclusion, there is a narrow range of concentrations and exposure times where ascorbate exerts photoprotective effects, exceeding which leads to ascorbate-mediated increase in photocytotoxicity. Vitamin E and its combination with zeaxanthin can enhance protective effects of ascorbate, but do not ameliorate its deleterious effects.

Cytotoxicity of all-trans-retinal increases upon photodegradation.

All-trans-retinal (AtRal) can accumulate in the retina as a result of excessive exposure to light. The purpose of this study was to compare cytotoxicity of AtRal and photodegraded AtRal (dAtRal) on cultured human retinal pigment epithelial cells in dark and upon exposure to visible light. AtRal was degraded by exposure to visible light. Cytotoxicity was monitored by imaging of cell morphology, propidium iodide staining of cells with permeable plasma membrane and measurements of reductive activity of cells. Generation of singlet oxygen photosensitized by AtRal and dAtRal was monitored by time-resolved measurements of characteristic singlet oxygen phosphorescence. Photodegradation of AtRal resulted in a decrease in absorption of visible light and accumulation of the degradation products with absorption maximum at ∼330 nm. Toxicity of dAtRal was concentration-dependent and was greater during irradiation with visible light than in dark. DAtRal was more cytotoxic than AtRal both in dark and during exposure to visible light. Photochemical properties of dAtRal indicate that it may be responsible for the maximum in the action spectra of retinal photodamage recorded in animals. In conclusion, photodegradation products of AtRal may impose a significant threat to the retina and therefore their roles in retinal pathology need to be explored.

Human RPE melanosomes protect from photosensitized and iron-mediated oxidation but become pro-oxidant in the presence of iron upon photodegradation.

PURPOSE: To determine the effects of human retinal pigment epithelial (RPE) cell pigment granules on photosensitized and iron ion-mediated oxidation and the effect of the photodegradation of melanosomes on their antioxidant properties. METHODS: RPE cells were isolated from human and bovine eyes; pigmented and nonpigmented bovine retinal pigment epithelia were isolated separately. Melanosomes, melanolipofuscin, and lipofuscin granules were isolated from human RPE donors older than 60. Melanosomes were photodegraded by exposure to blue light. Oxidation of RPE cells or of linoleate was induced by iron/ascorbate in the presence and absence of pigment granules. The photosensitized oxidation of histidine was induced by blue light irradiation of cationic porphyrin. The progress of oxidation was monitored by electron spin resonance oximetry. RESULTS: Iron/ascorbate induced rapid oxidation in suspensions of nonpigmented bovine RPE cells. The rates of oxidation were diminished approximately four times in suspensions of pigmented bovine RPE cells. Adding bovine melanosomes or synthetic melanin to nonpigmented bovine RPE cells resulted in a concentration-dependent decrease in the rate of oxidation to levels similar to those of pigmented bovine retinal pigment epithelium. Human melanosomes exerted a concentration-dependent inhibitory effect on photosensitized and iron-mediated oxidation. Photodegradation of human melanosomes led to loss of the inhibitory effect on iron-mediated oxidation, whereas their ability to inhibit photosensitized oxidation was enhanced. CONCLUSIONS: Human melanosomes act as effective antioxidants by preventing iron ion-induced oxidation. Photodegradation of melanosomes results in the loss of these antioxidant properties while it preserves their ability to deactivate cationic photosensitizers.

The pro-oxidant effects of interactions of ascorbate with photoexcited melanin fade away with aging of the retina.

Photoexcited melanin from retinal pigment epithelium (RPE) has been shown to induce photo-oxidation of ascorbate with concomitant generation of hydrogen peroxide. The aim of this study was to test whether the age-related changes in melanin content and distribution in the RPE affect the susceptibility of RPE cells to ascorbate-mediated photo-oxidation. Our results demonstrate that there is an age-dependent shift in the pathways with which ascorbate interacts in human RPE. In young RPE, melanin-ascorbate interactions may lead to pro-oxidant effects, but in the aged there is no net increase in photo-oxidation in the presence of ascorbate in comparison with samples without ascorbate. However, as ascorbate undergoes light-induced depletion and photogenerates ascorbyl free radical in the old RPE cells with initial yields similar to that observed for young RPE, an influence of ascorbate on oxidation pathways is revealed in the old RPE as well. Interestingly, the pro-oxidant effects of photoexcited melanolipofuscin-ascorbate interactions are greater than for photoexcited melanosomes when normalized to the same melanin content. The pro-oxidant effects of photoexcited melanin-ascorbate interactions are strongly dependent on the irradiation wavelength, this being the greatest for the shortest wavelength studied (340 nm) and steeply decreasing with increasing wavelength but still detectable even at 600 nm.

The phototoxicity of aged human retinal melanosomes.

The purpose of this study was to determine whether an age-related increase in photoreactivity of human retinal melanosomes (MS) can cause phototoxicity to retinal pigment epithelium (RPE) cells. MS were isolated post mortem from young (20-30 years, young human melanosomes [YHMs]) and old (60-90 years, old human melanosomes [OHMs]) human eyes and from young bovine eyes (bovine melanosomes [BMs]). Confluent cultured ARPE-19 cells were fed equivalent numbers of OHMs or BMs and accumulated similar amounts of melanin as determined by electron paramagnetic resonance assay. Cells with and without MS were either maintained in the dark or exposed to blue light for up to 96 h and assessed for alterations in cell morphology, cell viability and lysosomal integrity. Incubation of cells in dark in the presence of internalized MS or irradiation of cells with blue light in the absence or presence of BMs did not significantly affect cell viability. However, exposures to blue light in the presence of OHMs resulted in abnormal cell morphology, up to approximately 75% decrease in mitochondrial activity, loss of lysosomal pH and cell death. OHMs contained significantly less melanin than YHMs, supporting the hypothesis that melanin undergoes degradation during RPE aging. Our results demonstrate that aged MS can be phototoxic to human RPE cells and support a contributing role of MS in RPE aging and in the pathogenesis of age-related macular degeneration.

The photoreactivity of ocular lipofuscin.

Lipofuscin or "age pigment" is a lipid-protein complex which accumulates in a variety of postmitotic, metabolically active cells throughout the body. These complexes, which are thought to result from the incomplete degradation of oxidised substrate, have the potential for photoreactivity. This is particularly so in the retina in which the lipofuscin not only contains retinoid metabolites but is also exposed to high oxygen and fluxes of visible light all of which provide an ideal environment for the generation of reactive oxygen species (ROS). Lipofuscin is a potent photoinducible generator of ROS with the potential to damage proteins, lipids and DNA. Retinal cell dysfunction may be strongly associated with photoreactivity of lipofuscin and may contribute to age-related disease and vision loss.

Age-related changes in the photoreactivity of retinal lipofuscin granules: role of chloroform-insoluble components.

PURPOSE: Lipofuscin accumulates in human retinal pigment epithelium (RPE) cells with age and may be the main factor responsible for the increasing susceptibility of RPE to photo-oxidation with age. As the composition, absorption, and fluorescence of lipofuscin undergo age-related changes, the purpose of this study was to determine whether photoreactivity of lipofuscin granules also changes with the donor age. METHODS: To determine whether the photoreactivity of lipofuscin itself is age related, lipofuscin granules were isolated from human RPE and pooled into age groups. Photoreactivity was assessed by measuring action spectra of photo-induced oxygen uptake and photogeneration of reactive oxygen species. Separation of chloroform-soluble (ChS) and -insoluble (ChNS) components by Folch's extraction was used to determine the factors responsible for the age-related increase in lipofuscin photoreactivity. RESULTS: The observed rates of photo-induced oxygen uptake and photo-induced accumulation of superoxide-derived spin adducts indicated that when normalized to equal numbers of lipofuscin granules, aerobic photoreactivity of lipofuscin increased with age. Both ChS and ChNS mediated photogeneration of singlet oxygen, superoxide radical anion, and photo-oxidation of added lipids and proteins. Although both ChS and ChNS exhibited substantial photoreactivities, neither exhibited significant age-related changes when normalized to equal dry mass. In contrast, ChNS contents in lipofuscin granules significantly increased with aging. CONCLUSIONS: Aerobic photoreactivity of RPE lipofuscin substantially increases with aging. This effect may be ascribed to the increased content of insoluble components.

Photoreactivity of aged human RPE melanosomes: a comparison with lipofuscin.

PURPOSE: To determine whether aging is accompanied by changes in aerobic photoreactivity of retinal pigment epithelial (RPE) melanosomes isolated from human donors of different ages, and to compare the photoreactivity of aged melanosomes with that of RPE lipofuscin. METHODS: Human RPE pigment granules were isolated from RPE cells pooled into groups according to the age of the donors. Photoreactivity was determined by blue-light-induced oxygen uptake and photogeneration of reactive oxygen species. Short-lived radical intermediates were detected by spin-trapping, hydrogen peroxide by an oxidase electrode, singlet oxygen by cholesterol assay, and lipid hydroperoxides by iodometric assay. RESULTS: Blue-light photoexcitation of melanosomes resulted in age-related increases in both oxygen uptake and the accumulation of superoxide anion spin adducts. The efficiencies of these processes, however, were still significantly lower than that induced by photoexcited lipofuscin. During irradiation of melanosomes, a substantial amount of oxygen was converted into hydrogen peroxide, whereas for lipofuscin, hydrogen peroxide accounted for not more than 3% of oxygen consumed. In contrast to lipofuscin, photoexcited melanosomes did not substantially increase the rate of oxidative reactions in the presence of polyunsaturated lipids or albumin. However, oxygen uptake was significantly elevated in the presence of ascorbate. Thus, the rate of photo-induced oxygen uptake in samples containing both ascorbate and melanosomes approached that observed in lipofuscin samples. CONCLUSIONS: Blue-light-induced photoreactivity of melanosomes increases with age, perhaps providing a source of reactive oxygen species and leading to depletion of vital cellular reductants, which, together with lipofuscin, may contribute to cellular dysfunction.