The Anthocyanins,Oenin and Callistephin,Protect RPE Cells Against Oxidative Stress

The retinal pigment epithelium (RPE) is a highly metabolic layer of postmitotic cells lining Bruch's membrane in the retina. While these cells contain endogenous photosensitizers that mediate blue light‐induced damage, it has also been shown that blue light exposure damages mitochondrial DNA in RPE cells resulting in mitochondrial dysfunction and unregulated generation of reactive oxygen species (ROS). As RPE cells are postmitotic, it is imperative to decrease oxidative stress to these cells and preserve function. Dietary plant‐derived antioxidants such as anthocyanins offer a simple and accessible solution for decreasing oxidative stress. The anthocyanins malvidin‐3‐O‐glucoside (oenin) and pelargonidin‐3‐O‐glucoside (callistephin) were tested for their ability and efficacy in decreasing ROS generation and preserving mitochondrial redox activity in blue light‐irradiated ARPE‐19 cells. A significant decrease in intracellular ROS with concurrent increase in mitochondrial redox activity was observed for tested concentrations of oenin, while callistephin was beneficial to stressed cells at higher concentrations. These findings suggest anthocyanins are effective antioxidants in blue light‐stressed RPE cells in vitro. Additionally, oxidation products of these anthocyanins were examined using LC/MS and findings suggest the possibility of multiple oxidation sites for these compounds.     

Organelle‐Targeted BODIPY Photocages: Visible‐Light‐Mediated Subcellular Photorelease

Photocaging facilitates non‐invasive and precise spatio‐temporal control over the release of biologically relevant small‐ and macro‐molecules using light. However, sub‐cellular organelles are dispersed in cells in a manner that renders selective light‐irradiation of a complete organelle impractical. Organelle‐specific photocages could provide a powerful method for releasing bioactive molecules in sub‐cellular locations. Herein, we report a general post‐synthetic method for the chemical functionalization and further conjugation of meso‐methyl BODIPY photocages and the synthesis of endoplasmic reticulum (ER)‐, lysosome‐, and mitochondria‐targeted derivatives. We also demonstrate that 2,4‐dinitrophenol, a mitochondrial uncoupler, and puromycin, a protein biosynthesis inhibitor, can be selectively photoreleased in mitochondria and ER, respectively, in live cells by using visible light. Additionally, photocaging is shown to lead to higher efficacy of the released molecules, probably owing to a localized and abrupt release.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

Effects of Narrow‐band IR‐A and of Water‐Filtered Infrared A on Fibroblasts

Exposures of the skin with electromagnetic radiation of wavelengths between 670 nm and 1400 nm are often used as a general treatment to improve wound healing and reduce pain, for example, in chronic diabetic skin lesions. We investigated the effects of water‐filtered infrared A (wIRA) and of narrow‐band IR‐A provided by a light‐emitting diode LED (LED‐IR‐A) irradiation in vitro on 3T3 fibroblast cultures under defined conditions with and without glyoxal administration. Glyoxal triggers the formation of advanced glycation end products, thereby mimicking a diabetic metabolic state. Cell viability and apoptotic changes were determined by flow cytometry after vital staining with Annexin V, YO‐PRO‐1 and propidium iodide (PI), and by SubG1 assay. Mitochondrial function and oxidative stress were examined by vital staining for radical production, mitochondrial membrane potential (MMP) and the ratio of reduced‐to‐oxidized glutathione (GSH/GSSG). The metabolic state was monitored by a resazurin conversion assay. The numbers of apoptotic cells were reduced in cultures irradiated with wIRA or LED‐IR‐A. More mitochondria showed a well‐polarized MMP after wIRA irradiation in glyoxal damaged cells. LED‐IR‐A treatment specifically restored the GSH/GSSG ratio. The immediate positive effects of wIRA and LED‐IR‐A observed in living cells, particularly on mitochondria, reflect the therapeutic benefits of wIRA and LED‐IR‐A.     

Primary photodamage sites and mitochondrial events after Foscan photosensitization of MCF-7 human breast cancer cells

To determine the initial photodamage sites of Foscan-mediated photodynamic treatment, we evaluated the enzymatic activities in selected organelles immediately after light exposure of MCF-7 cells. The measurements indicated that the enzymes located in the Golgi apparatus (uridine 5'-diphosphate galactosyl transferase) and in the endoplasmic reticulum (ER) (nicotinamide adenine dinucleotide [reduced] [NADH] cytochrome c [cyt c] reductase) are inactivated by the treatment, whereas mitochondrial marker enzymes (cyt c oxidase and dehydrogenases) were unaffected. This indicates that the ER and the Golgi apparatus are the primary intracellular sites damaged by Foscan-mediated PDT in MCF-7 cells. We further investigated whether the specific mitochondria events could be associated with Foscan photoinduced cell death. The dose response profiles of mitochondrial depolarization and cytochrome c release immediately after Foscan-based PDT were very different from that of overall cell death. By 24 h post-PDT the fluence dependency was strikingly similar for both mitochondrial alterations and cell death. Therefore, although mitochondria are not directly affected by the treatment, they can be strongly implicated in Foscan-mediated MCF-7 cell death by late and indirect mechanism.     

Mechanism and efficiency of cell death of type II photosensitizers: effect of zinc chelation

A series of meso-substituted tetra-cationic porphyrins, which have methyl and octyl substituents, was studied in order to understand the effect of zinc chelation and photosensitizer subcellular localization in the mechanism of cell death. Zinc chelation does not change the photophysical properties of the photosensitizers (all molecules studied are type II photosensitizers) but affects considerably the interaction of the porphyrins with membranes, reducing mitochondrial accumulation. The total amount of intracellular reactive species induced by treating cells with photosensitizer and light is similar for zinc-chelated and free-base porphyrins that have the same alkyl substituent. Zinc-chelated porphyrins, which are poorly accumulated in mitochondria, show higher efficiency of cell death with features of apoptosis (higher MTT response compared with trypan blue staining, specific acridine orange/ethidium bromide staining, loss of mitochondrial transmembrane potential, stronger cytochrome c release and larger sub-G1 cell population), whereas nonchelated porphyrins, which are considerably more concentrated in mitochondria, triggered mainly necrotic cell death. We hypothesized that zinc-chelation protects the photoinduced properties of the porphyrins in the mitochondrial environment.     

RED LIGHT-INDUCED REDUCTION OF A PARTICLE-ASSOCIATED b-TYPE CYTOCHROME FROM CORN IN THE PRESENCE OF METHYLENE BLUE*

Abstract— In the presence of methylene blue, red light causes the reduction of a h-type cytochrome in particulate fractions from corn coleoptiles. Two types of difference spectra for the cytochromes in these fractions are presented: (a) red light-minus-dark in the presence of methylene blue, and (b) dithionite-reduced-minus-oxidized. Comparison of these spectra shows that photoexcited methylene blue selectively reduces a b-type cytochrome which constitutes at most only 30% of the total dithionite-reducible cytochrome present in the most active fractions. The photoreducible cytochrome has an alpha band at room temperature near 557 nm. Bleaching of methylene blue precedes cytochrome reduction under appropriate conditions, suggesting that the photoreduced dye is donating an electron to the cytochrome. This electron transfer does not involve a flavin, at least as judged by the absence of light-induced spectral changes attributable to flavins. Preliminary kinetic studies suggest that EDTA provides the pool of electrons for the reaction. The cytochrome cannot be assigned exclusively either to mitochondria or to endoplasmic reticulum, as judged by its sedimentation properties. These results and the current literature are discussed in the context of the hypothesis that this b-type cytochrome may be involved in the photoreception mechanism for blue and uv light in vivo.     

Ultrafluorogenic Coumarin–Tetrazine Probes for Real‐Time Biological Imaging

We have developed a series of new ultrafluorogenic probes in the blue‐green region of the visible‐light spectrum that display fluorescence enhancement exceeding 11 000‐fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans‐cyclooctene(TCO)–tetrazine chemistry platform. By exploiting highly efficient through‐bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No‐wash, fluorogenic imaging of diverse targets including cell‐surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.     

Effects of Light‐emitting Diode Radiations on Human Retinal Pigment Epithelial Cells In Vitro

Human visual system is exposed to high levels of natural and artificial lights of different spectra and intensities along lifetime. Light‐emitting diodes (LEDs) are the basic lighting components in screens of PCs, phones and TV sets; hence it is so important to know the implications of LED radiations on the human visual system. The aim of this study was to investigate the effect of LEDs radiations on human retinal pigment epithelial cells (HRPEpiC). They were exposed to three light–darkness (12 h/12 h) cycles, using blue‐468 nm, green‐525 nm, red‐616 nm and white light. Cellular viability of HRPEpiC was evaluated by labeling all nuclei with DAPI; Production of reactive oxygen species (ROS) was determined by H2DCFDA staining; mitochondrial membrane potential was quantified by TMRM staining; DNA damage was determined by H2AX histone activation, and apoptosis was evaluated by caspases‐3,‐7 activation. It is shown that LED radiations decrease 75–99% cellular viability, and increase 66–89% cellular apoptosis. They also increase ROS production and DNA damage. Fluorescence intensity of apoptosis was 3.7% in nonirradiated cells and 88.8%, 86.1%, 83.9% and 65.5% in cells exposed to white, blue, green or red light, respectively. This study indicates three light–darkness (12 h/12 h) cycles of exposure to LED lighting affect in vitro HRPEpiC.     

PHOTOREGULATION OF NITRATE UTILIZATION IN GREEN ALGAE AND HIGHER PLANTS*

Abstract— Nitrate reductase from eukaryotes can be reversibly inactivated, blue light being an effective activating agent both in vitro and in vivo. Hydroxylamine proved to be a powerful inactivating agent of Ankistrodesmus braunii nitrate reductase. Irradiation with blue light of NH₂OH-inactivated nitrate reductase, specially in the presence of μM amounts of FAD, promoted the recovery of the enzyme activity. Similarly, photoexcited methylene blue reactivated spinach nitrate reductase. On the other hand, in vitro nitrate reductase is highly susceptible to photodynamic inactivation caused by singlet O₂. Aerobic incubation of the active spinach enzyme with either FMN or methylene blue under either blue or red light respectively led to its irreversible inactivation. Irradiation of frozen and thawed spinach leaf discs also promoted, in situ, an irreversible inactivation of nitrate reductase, provided that 6₂ was present in the incubation mixture. Thus, either in vitro or in situ, light can cause two quite different responses of nitrate reductase, its blue light-dependent photoactivation in a flavin sensitized reaction and its photodynamic inactivation in a singlet O₂-dependent process.     

Characterization of Retinal Pigment Epithelial Melanin and Degraded Synthetic Melanin Using Mass Spectrometry and In Vitro Biochemical Diagnostics

With increasing age, there is an observable loss of melanin in retinal pigment epithelial (RPE) cells. It is possible that degradation of the pigment contributes to the pathogenesis of retinal disease, as the cellular antioxidant material is depleted. Functionally, intact melanin maintains protective qualities, while oxidative degradation of melanin promotes reactive oxygen species (ROS) generation and formation of metabolic byproducts, such as melanolipofuscin. Understanding the structural and functional changes to RPE melanin with increasing age may contribute to a better understanding of disease progression and risk factors for conditions such as age‐related macular degeneration (AMD). In this study, human donor RPE melanin is characterized using MALDI mass spectrometry to follow melanin degradation trends. In vitro models using ARPE‐19 cells are used to assess photo‐reactivity in repigmented cells. Significant protection against intracellular ROS produced by blue light is observed in calf melanin‐pigmented cells versus unpigmented and black latex bead controls (P < 0.0001). UV‐B exposure to aged human melanin‐pigmented cells results in a significant increase in nitric oxide production versus control cells (P < 0.001). Peroxide‐treated synthetic melanin is characterized to elucidate degradation products that may contribute to RPE cell damage.     

Melatonin Mediates Monochromatic Light‐induced Insulin‐like Growth Factor 1 Secretion of Chick Liver: Involvement of Membrane Receptors

Monochromatic lights influenced the proliferation and differentiation of skeletal satellite cells in broilers by the enhancement of insulin‐like growth factor 1 (IGF‐1) secretion. However, whether melatonin (MEL)‐mediated monochromatic lights influenced the IGF‐1 secretion remains unclear. Newly hatched broilers, including intact, sham operation and pinealectomy groups, were exposed to blue (BL), green (GL), red (RL) and white light (WL) from a light‐emitting diode system for 14 days. The results showed that GL effectively promoted the secretion of MEL and IGF‐1, the expression of proliferating cell nuclear antigen and MEL receptor subtypes Mel1a, Mel1b and Mel1c in the liver compared to BL and RL in vivo. Moreover, those was a positive correlation between MEL and IGF‐1 (r = 0.834). After pinealectomy, however, these parameters declined, and there were no differences between GL and other monochromatic light treatments. In vitro, exogenous MEL increased hepatocyte proliferation and IGF‐1 secretion. Meanwhile, the MEL enhancements were suppressed by prazosin (selective Mel1c antagonist), followed by luzindole (nonselective Mel1a/Mel1b antagonist), but not suppressed by 4‐phenyl‐2‐propionamideotetralin (selective Mel1b antagonist). These findings demonstrated that MEL mediated the monochromatic light‐induced secretion of IGF‐1 in chicks’ livers by Mel1c and that Mel1a may be involved in this process.     

Activation of the General Stress Response of Bacillus subtilis by Visible Light

A key challenge for microbiology is to understand how evolution has shaped the wiring of regulatory networks. This is amplified by the paucity of information of power‐spectra of physicochemical stimuli to which microorganisms are exposed. Future studies of genome evolution, driven by altered stimulus regimes, will therefore require a versatile signal transduction system that allows accurate signal dosing. Here, we review the general stress response of Bacillus subtilis, and its upstream signal transduction network, as a candidate system. It can be activated by red and blue light, and by many additional stimuli. Signal integration therefore is an intricate function of this system. The blue‐light response is elicited via the photoreceptor YtvA, which forms an integral part of stressosomes, to activate expression of the stress regulon of B. subtilis. Signal transfer through this network can be assayed with reporter enzymes, while intermediate steps can be studied with live‐cell imaging of fluorescently tagged proteins. Different parts of this system have been studied in vitro, such that its computational modeling has made significant progress. One can directly relate the microscopic characteristics of YtvA with activation of the general stress regulon, making this system a very well‐suited system for network evolution studies.     

In vitro photodynamic activity of a series of methylene blue analogues

We have synthesized a series of symmetrical phenothiazines in which the methyl groups of methylene blue have been substituted by longer alkyl chains. Intrinsic photosensitizing ability was not altered by increasing the chain length. However, in vitro phototoxicity after 2 h incubation of RIF-1 murine fibrosarcoma cells followed the order n-propyl > n-pentyl > n-butyl > n-hexyl > ethyl > methyl, with ethyl and n-propyl analogues being 14- and 130-fold more phototoxic than methylene blue, respectively. All analogues also had an improved ratio of phototoxicity: dark toxicity (4:1 to 27:1) compared with methylene blue (3:1). Phototoxicity did not correlate with cellular phenothiazine levels, suggesting that the site of subcellular localization may be more important. After 2 h incubation of RIF-1 cells with the phototoxicity LD50 concentration, methylene blue and all analogues were observed to be localized in the lysosomes by fluorescence microscopy. On exposure to light, methylene blue relocalized to the nucleus, the ethyl analogue did not relocalize, whereas the more phototoxic n-propyl - n-hexyl analogues relocalized to the mitochondria. Relocalization to the mitochondria was associated with an octanol: buffer partition coefficient > or = 1. Therefore, the longer-chain analogues of methylene blue show significantly improved phototoxicity in vitro and, in addition, are expected to avoid the problems of mutagenicity associated with the nuclear localization of methylene blue.     

Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light‐dependent repair of ultraviolet (UV)‐induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6‐4 photolyase, which repairs 6‐4 pyrimidine–pyrimidone photoproducts (6‐4PPs). Although the Cry‐DASH protein is classified as a cryptochrome, it also has light‐dependent DNA repair activity. To determine the significance of the three light‐dependent repair enzymes in recovering from solar UV‐induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light‐dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light‐dependent repair of CPD was lost in the CPD photolyase‐deficient mutant, whereas weak repair activity against 6‐4PPs persisted in the 6‐4 photolyase‐deficient mutant. These results suggest the existence of a heretofore unknown 6‐4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates.     

Iridium(III) Anthraquinone Complexes as Two‐Photon Phosphorescence Probes for Mitochondria Imaging and Tracking under Hypoxia

In the present study, four mitochondria‐specific and two‐photon phosphorescence iridium(III) complexes, Ir1 – Ir4 , were developed for mitochondria imaging in hypoxic tumor cells. The iridium(III) complex has two anthraquinone groups that are hypoxia‐sensitive moieties. The phosphorescence of the iridium(III) complex was quenched by the functions of the intramolecular quinone unit, and it was restored through two‐electron bioreduction under hypoxia. When the probes were reduced by reductase to hydroquinone derivative products under hypoxia, a significant enhancement in phosphorescence intensity was observed under one‐ (λ=405 nm) and two‐photon (λ=720 nm) excitation, with a two‐photon absorption cross section of 76–153 GM at λ=720 nm. More importantly, these probes possessed excellent specificity for mitochondria, which allowed imaging and tracking of the mitochondrial morphological changes in a hypoxic environment over a long period of time. Moreover, the probes can visualize hypoxic mitochondria in 3D multicellular spheroids and living zebrafish through two‐photon phosphorescence imaging.     

DFT/TDDFT Studies on the Electronic Structures and Spectral Properties of Carbazole‐based Blue Light‐emitting Dendrimers

The purpose of this paper is to provide an in‐depth investigation of the electronic and optical properties of two series of carbazole‐based blue light‐emitting dendrimers, including 1 – 6 six oligomers. These materials show great potential for application in organic light‐emitting diodes as efficient blue‐light and red‐light emitting materials due to the tuning of the optical and electronic properties by the use of different electron donors (D) and electron acceptors (A). The geometric and electronic structures of these compounds in the ground state are calculated using density functional theory (DFT) and the ab initio HF, whereas the lowest singlet excited states were optimized by ab initio single excitation configuration interaction (CIS). All DFT calculations are performed using the B3LYP functional on 6‐31G* basis set. The outcomes show that the highest occupied molecular orbitals (HOMOs), lowest occupied molecular orbitals (LUMOs), energies gaps, ionization potentials, electron affinities and reorganization energies of each molecular are affected by different D and A moieties and different substitute positions.     

MITOCHONDRIAL PHOTOSENSITIZATION BY PHOTOFRIN II

Abstract V-79 Chinese hamster cells grown as monolayers or as multicell spheroids were treated with Photofrin II (10 μ.g m⁻¹ for 16 h) and various doses of red light irradiation. The resulting biochemical and functional damage to cell mitochondria was studied. The activities of both succinic dehydrogenese and cytochrome c oxidase were found to decrease in a light dose-dependent manner. The respiratory control quotient (RC) decreased in parallel with a decrease in the activities of the respiratory chain proteins. Our data also showed a distinct temporal difference in the relative progression of mitochondrial damage and cell death as assessed by loss of discrete Rhodamine-123 (Rh-123) localization and trypan blue infiltration, respectively. Mitochondrial damage was detected immediately, as seen by derealization of Rh-123 resulting from dissipation of the electrochemical gradient in damaged mitochondria. Trypan blue infiltration occurs with a distinct time lag. These findings are consistent with the hypothesis that, at least for long Photofrin II incubation times, the mitochondrion is a primary target of photosensitization. The subsequent changes in cell membrane permeability may be a delayed result of decreased bioenergetics of the Photofrin II photosensitized cell.     

Reversible Effects of Photodamage Directed Toward Mitochondria

When the initial effect of photodynamic therapy (PDT) involves mitochondrial photodamage, an early effect is loss of the mitochondrial membrane potential (ΔΨ_m). Using murine hepatoma 1c1c7 cells and a photosensitizing agent known to target mitochondria, we examined loss of ΔΨ_m, initiation of apoptosis and loss of viability as a function of time and light dose. There was a correlation between loss of viability and the rapid disappearance of ΔΨ_m, as detected by the potential‐sensitive probe Mitotracker Orange (MTO). Loss of ΔΨ_m was, however, reversible even with a substantial loss of viability. Unless there was a supralethal level of photodamage, 1c1c7 cells recovered their mitochondrial membrane potential, even if the cell population was on the pathway to apoptosis and cell death. These results indicate that when mitochondria are the initial PDT target, a qualitative estimate of photokilling can be provided by assessing the initial loss of ΔΨ_m.