Direct observation of singlet oxygen production by merocyanine 540 associated with phosphatidylcholine liposomes

The production of singlet molecular oxygen (1O2) by the photosensitizing dye merocyanine 540 (MC540) bound to phosphatidylcholine liposomes has been demonstrated by direct detection of 1O2 luminescence at 1268 nm. 1O2 phosphorescence emission was enhanced in deuterated buffer and upon saturation of the sample with oxygen and could be quenched by the addition of sodium azide to the external medium. No 1O2 luminescence was detected in nitrogen-saturated samples, in the absence of dye, or with MC540 in aqueous solution. Photobleaching of liposome-bound MC540 was also observed to be dependent on oxygen concentration. These studies are consistent with 1O2 intermediacy in the mechanism of MC540-mediated photosensitization.     

Use of Merocyanine 540 for Photodynamic Inactivation of Staphylococcus aureus Planktonic and Biofilm Cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 μg of MC 540 per ml and a light dose of 600 J/cm² or to 20 μg of MC 540 per ml and a light dose of 450 J/cm². A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 mug of MC 540 per ml and a light dose of 600 J/cm2 or to 20 mug of MC 540 per ml and a light dose of 450 J/cm2. A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

In vitro photosensitisation of a murine mammary adenocarcinoma cell line with Verteporfin.

Benzoporphyrin derivative monoacid ring A (BPD-MA) is a second generation hydrophobic photosensitiser for PDT that has been approved for ocular disease treatment. In the present paper we report the results of in vitro studies on the photosensitising activity of Verteporfin (liposomally formulated BPD-MA) using an adenocarcinoma derived cell line. Our findings show a quick and efficient uptake of Verteporfin by LM3 cells, reaching maxima concentrations after 5 hr exposure to 18 microg Verteporfin/ml. Independently on the concentration, plateau levels are attained 5 hr after exposure to Verteporfin. Exposure of the cells to the photosensitiser appears to be safe in the darkness within a broad range of concentrations. The hydroxyl radical scavenger mannitol afforded the highest protection against PDT, while L-tryptophan, a well known and efficient singlet oxygen quencher was not an effective protector at all, showing scavenging activity only when it was supplemented at concentration as high as 10 mM and when 50% of the cells were affected, showing that in addition to singlet oxygen, which is considered the primary cytotoxic agent in PDT, other interconvertible reactive oxygen specie (ROS), in particular HO are also generated. Verteporfin-PDT also induced morphological features typical of apoptotic cells. Results of the present work show that the LM3 adenocarcinoma cell can be effectively sensitised with Verteporfin-PDT.     

Antibody-targeted photolysis: in vitro immunological, photophysical, and cytotoxic properties of monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugates.

A set of anti-melanoma immunoconjugates were prepared which contained chlorin e6: antibody molar ratios of 18.9:1, 11.2:1, 6.8:1, and 1.7:1. All immunoconjugates retained antigen binding activity regardless of the chromophore:antibody substitution ratio that was attained. In contrast, the ground-state absorption spectra of the immunoconjugates showed features which appeared to be dependent on the chromophore:antibody molar ratio. In addition, the quantum yield of singlet oxygen generated by the conjugated chromophores was observed to be significantly less than that observed with the unbound dye. Time-resolved absorbance spectroscopy of the chromophore excited triplet state indicated that the loss of singlet oxygen quantum yield resulted from diminished chromophore triplet yield. Analysis of data obtained from in vitro photolysis of target melanoma cells, in combination with that obtained from the immunochemical and photochemical studies, indicates that the observed immunoconjugate phototoxicity can be reasonably quantitatively represented by (1) the ability of the immunoconjugate to bind SK-MEL-2 cell surface antigen, (2) the amount of chromophore localized at the target cells by immunoconjugate binding, (3) the delivered dose of light at 634 nm, and (4) the singlet oxygen quantum yield of the antibody-bound photosensitizer. Though these data argue strongly for photolysis by the cumulative dosage of singlet oxygen at the cell membrane, nonetheless, the concurrent photoinduced release of other cytotoxic agents should not be ruled out.     

Antioxidant enzyme inhibitors enhance singlet oxygen-induced cell death in HL-60 cells

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 μM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 μM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.     

Antioxidant enzyme inhibitors enhance singlet oxygen-induced cell death in HL-60 cells

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 μM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 μM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.     

Design, synthesis, and biological evaluation of folic acid targeted tetraphenylporphyrin as novel photosensitizers for selective photodynamic therapy

Photodynamic therapy (PDT) is a cancer treatment involving systemic administration of a tumor-localizing photosensitizer; this, when activated by the appropriate light wavelength, interacts with molecular oxygen to form a toxic, short-lived species known as singlet oxygen, which is thought to mediate cellular death. Targeted PDT offers the opportunity of enhancing photodynamic efficiency by directly targeting diseased cells and tissues. Two new conjugates of three components, folic acid/hexane-1,6-diamine/4-carboxyphenylporphyrine 1 and folic acid/2,2'-(ethylenedioxy)-bis-ethylamine/4-carboxyphenylporphyrine 2 were synthesized. The conjugates were characterized by 1H NMR, MALDI, UV-visible spectroscopy, and fluorescence quantum yield. The targeted delivery of these photoactive compounds to KB nasopharyngeal cell line, which is one of the numerous tumor cell types that overexpress folate receptors was studied. It was found that after 24 h incubation, conjugates 1 and 2 cellular uptake was on average 7-fold higher than tetraphenylporphyrin (TPP) used as reference and that 1 and 2 cellular uptake kinetics increased steadily over the 24 h period, suggesting an active transport via receptor-mediated endocytosis. In corresponding results, conjugates 1 and 2 accumulation displayed a reduction of 70% in the presence of a competitive concentration of folic acid. Survival measurements demonstrated that KB cells were significantly more sensitive to conjugated porphyrins-mediated PDT. Under the same experimental conditions and the same photosensitizer concentration, TPP displayed no photocytotoxicity while conjugates 1 and 2 showed photodynamic activity with light dose values yielding 50% growth inhibition of 22.6 and 6.7 J/cm2, respectively.     

Singlet oxygen generation by UVA light exposure of endogenous photosensitizers

UVA light (320-400 nm) has been shown to produce deleterious biological effects in tissue due to the generation of singlet oxygen by substances like flavins or urocanic acid. Riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), beta-nicotinamide adenine dinucleotide (NAD), and beta-nicotinamide adenine dinucleotide phosphate (NADP), urocanic acid, or cholesterol in solution were excited at 355 nm. Singlet oxygen was directly detected by time-resolved measurement of its luminescence at 1270 nm. NAD, NADP, and cholesterol showed no luminescence signal possibly due to the very low absorption coefficient at 355 nm. Singlet oxygen luminescence of urocanic acid was clearly detected but the signal was too weak to quantify a quantum yield. The quantum yield of singlet oxygen was precisely determined for riboflavin (PhiDelta = 0.54 +/- 0.07), FMN (PhiDelta = 0.51 +/- 0.07), and FAD (PhiDelta = 0.07 +/- 0.02). In aerated solution, riboflavin and FMN generate more singlet oxygen than exogenous photosensitizers such as Photofrin, which are applied in photodynamic therapy to kill cancer cells. With decreasing oxygen concentration, the quantum yield of singlet oxygen generation decreased, which must be considered when assessing the role of singlet oxygen at low oxygen concentrations (inside tissue).     

Photodynamic action of merocyanine 540 on erythrocyte membranes: structural perturbation of lipid and protein constituents.

erocyanine 540 (MC540) is a membrane-directed photosensitizing dye with antileukemic and antiviral properties. In this study, biophysical and biochemical techniques have been used to examine MC540-sensitized photooxidative damage in the lipid and protein compartments of a test membrane, the human erythrocyte ghost. Irradiation of MC540-sensitized ghosts with white light resulted in oxidative damage to proteins, as manifested by (i) loss of sulfhydryl groups; (ii) intermolecular cross-linking of major polypeptides; and (iii) loss of Mg(2+)-ATPase and Na+,K(+)-ATPase activities. Photooxidation also produced a rapid and progressive increase in general protein motion, as measured by electron paramagnetic resonance spectrometry (EPR) with the sulfhydryl spin label MAL-6. In addition to these effects, ghosts exposed to MC540 and light underwent lipid peroxidation. EPR with two lipophilic spin probes, 5-doxylstearate and 16-doxylstearate, showed that lipid peroxidation is accompanied by a progressive decrease in bilayer fluidity (motional freedom). At a given dye concentration, structural perturbations of proteins were detected at much lower light fluences than those of lipids. When photoreactions were carried out in the presence of ascorbate and iron, there was a strong stimulation of lipid peroxidation (attributed to free radical chain reactions), with a concomitant greater decrease in lipid mobility. Thus, the deleterious effects of photoperoxidation on lipid structure and motional freedom were greatly exacerbated by ascorbate and iron. Membrane damage similar to that described here may play a role in the phototherapeutic activity of MC540.     

Singlet oxygen production and fluorescence yields of merocyanine 540: a comparative study in solution and model membrane systems.

Singlet oxygen and fluorescence quantum yields of merocyanine 540 were measured in solution (methanol, ethanol, n-heptanol) and in model membrane systems (cationic micelles, unilamellar dimyristoyl- and dipalmitoylphosphatidylcholine vesicles). Both singlet oxygen quantum yields and fluorescence quantum yields increase with increasing viscosity/rigidity of the surrounding medium: the yield of singlet oxygen production (24 degrees C) goes from 0.002 in methanol to 0.04 in dipalmitoylphosphatidylcholine vesicles, and fluorescence yields (25 degrees C) change from 0.14 to 0.61 in the same media. The data are consistent with previous findings that photoisomerization is in direct competition with intersystem crossing and radiative relaxation. Therefore, a singlet oxygen yield close to the maximum value of 0.11 can only be achieved after both photoisomerization and internal conversion are prevented by a highly viscous environment.     

Photophysical properties and photodynamic activity in vivo of some tetrapyrroles

Some of the photophysical properties (stationary absorbance and fluorescence, fluorescence decay times and singlet oxygen quantum yields) of pheophorbide a, metal-free, ClAl-, Cu- and Mg-t-butyl-substituted phthalocyanines, metal-free, ClAl- and Cu-t-butyl-substituted naphthalocyanines and of a number of tetraphenylporphyrins (5,10,15,20-tetraphenylporphyrin, 5,10,15,20-tetra(m-hydroxyphenyl)porphyrin, 5,10,15,20-tetra(p-hydroxyphenyl)porphyrin) have been studied in comparison with hematoporphyrin IX in order to select potent photosensitizers for the photodynamic treatment of cancer. The photodynamic activity of these compounds was investigated using Lewis lung carcinoma in mice. As a consequence of the photophysical parameters (relatively short singlet state lifetimes, and high singlet oxygen quantum yields) the photodynamic activities of pheophorbide a, t-butyl-substituted ClAl-phthalocyanine and ClAl-naphthalocyanine were selected for study in greater detail. Under the conditions employed in the present study, pheophorbide a was found to be the most effective sensitizer, as judged from its strong absorption at the excitation wavelength as compared with the hematoporphyrin derivative and greater singlet oxygen quantum yield relative to the phthalocyanines and naphthalocyanines. The photodynamic activity was observed to be strongly dependent on the photophysical parameters of the compounds. The primary mechanism underlying the photodynamic activity of these sensitizers probably consists of energy transfer from the lowest triplet state of the dyes to molecular oxygen, resulting in the formation of singlet oxygen (type II of photosensitization).     

Merocyanine-sensitized photoinactivation of enveloped viruses.

A wide range of enveloped viruses, including human herpes simplex virus type 1, human cytomegalovirus, human T cell leukemia/lymphoma virus type I, human immunodeficiency virus type 1, Sindbis virus, and Friend erythroleukemia virus, are highly susceptible to merocyanine 540 (MC 540)-sensitized photoinactivation. By contrast, human pluripotent hematopoietic stem cells, red cells, factor VIII, and von Willebrand factor are much less sensitive. This suggests that MC 540 may be useful for the inactivation of enveloped viruses in blood and blood products. The dye has a low acute systemic toxicity, is rapidly eliminated from the blood stream, and has little or no mutagenic potential. The currently available data support the view that MC 540-sensitized photo-inactivation interferes with early events in the infectious process, notably the ability of the virus to adhere to and penetrate its host cell. The viral envelope is a major target of photodynamic damages which appear to be mediated at least in part by singlet molecular oxygen.     

UVB photoprotective role of mycosporines in yeast: photostability and antioxidant activity of mycosporine-glutaminol-glucoside

Several yeast species are able to synthesize and accumulate UV-radiation-absorbing mycosporine metabolites that are of unclear physiological function. In this work we analyzed the relationship between mycosporine-glutaminol glucoside (MGG) production, cell survival after UVB irradiation, and formation of cyclobutane pyrimidine dimers (CPDs). We also assessed the photostability and singlet oxygen quenching activity of MGG. A set of nine isolates of the basidiomycetous yeast Cryptococcus steppossus cultured in both dark and light conditions was used for the studies. Survival of the UVB-irradiated isolates and MGG concentration had a linear relationship when the concentration was over 2.5 mg g(-1). CPD accumulation and MGG accumulation were inversely related. MGG in aqueous solution was photostable with a photodecomposition quantum yield of 1.16 × 10(-5). MGG quenching of singlet oxygen was also observed, and the rate constant for the process in D(2)O was 5.9 × 10(7) M(-1) s(-1). Our results support the idea that MGG plays an important role as a UVB photoprotective metabolite in yeasts by protecting against direct damage on DNA and probably against indirect damage by singlet oxygen quenching.     

Photodynamic action of merocyanine 540 on carcinoma of cervix cells

Results of the studies carried out on localization and photodynamic action of merocyanine 540 (MC540) on carcinoma of cervix (HeLa) cells are presented. Fluorescence microscopic study showed that when HeLa cells were incubated with MC540 in dark, the dye localized in plasma membrane of cells. Photoirradiation of cells in presence of MC540 led to enhancement of dye uptake, intracellular localization of dye and a dose dependent decrease in cell survival. Clonogenic assay showed 96% cell killing at a light dose of 42 kJ/m2. Photosensitization of cells resulted in loss of membrane integrity, decrease in plasma membrane fluidity and reduction in mitochondrial dehydrogenase activity as measured by tetrazolium reduction (MTT) assay. At a given light dose, the relative change in plasma membrane properties was higher than the reduction in activity of mitochondrial enzyme. These results suggest plasma membrane is a primary target of photosensitization of HeLa cells by MC540.     

Cellular Defense against Singlet Oxygen-induced Oxidative Damage by Cytosolic NADP + -dependent Isocitrate Dehydrogenase

Singlet oxygen ( 1 O 2 ) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.     

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase

Singlet oxygen ( 1 O 2 ) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.     

Production of singlet oxygen-derived hydroxyl radical adducts during merocyanine-540-mediated photosensitization: analysis by ESR-spin trapping and HPLC with electrochemical detection.

Activated oxygen species produced during merocyanine 540 (MC540)-mediated photosensitization have been examined by electron spin resonance (ESR) spin trapping and by trapping reactive intermediates with salicylic acid using HPLC with electrochemical detection (HPLC-EC) for product analysis. Visible light irradiation of MC540 associated with dilauroylphosphatidylcholine liposomes in the presence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO/.OH). Addition of ethanol or methanol produced additional hyperfine splittings due to the respective hydroxyalkyl radical adducts, indicating the presence of free.OH.DMPO/.OH formation was not significantly inhibited by Desferal, catalase, or superoxide dismutase (SOD). Production of DMPO/.OH was strongly inhibited by azide and enhanced in samples prepared with deuterated phosphate buffer (PB-D2O), suggesting that singlet molecular oxygen (1O2) was an important intermediate. When MC540-treated liposomes were irradiated in the presence of salicylic acid (SA), HPLC-EC analysis indicated almost exclusive formation of 2,5-dihydroxybenzoic acid (2,5-DHBA), with production of very little 2,3-DHBA, in contrast to .OH generated by uv photolysis of H2O2, which gave nearly equimolar amounts of the two products. 2,5-DHBA production was enhanced in PB-D2O and inhibited by azide, again consistent with 1O2 intermediacy. 2,5-DHBA formation was significantly reduced in samples saturated with N2 or argon, and such samples showed no D2O enhancement. Ethanol had no effect on 2,5-DHBA production, even when present in large excess. Catalase and SOD also had no effect, and only a small inhibition was observed with Desferal. DMPO inhibited 2,5-DHBA production in a concentration-dependent fashion and enhanced formation of 2,3-DHBA. We propose that 1O2 reacts with DMPO to give an intermediate which decays to form DMPO/.OH and free.OH, and that the reaction between 1O2 and SA preferentially forms the 2,5-DHBA isomer. This latter process may provide the basis for a sensitive analytical method to detect 1O2 intermediacy. Singlet oxygen appears to be the principle activated oxygen species produced during MC540-mediated photosensitization.     

Quality Control of Photosystem II: Lipid Peroxidation Accelerates Photoinhibition under Excessive Illumination

Environmental stresses lower the efficiency of photosynthesis and sometimes cause irreversible damage to plant functions. When spinach thylakoids and Photosystem II membranes were illuminated with excessive visible light (100–1,000 µmol photons m⁻¹ s⁻¹) for 10 min at either 20°C or 30°C, the optimum quantum yield of Photosystem II decreased as the light intensity and temperature increased. Reactive oxygen species and endogenous cationic radicals produced through a photochemical reaction at and/or near the reaction center have been implicated in the damage to the D1 protein. Here we present evidence that lipid peroxidation induced by the illumination is involved in the damage to the D1 protein and the subunits of the light-harvesting complex of Photosystem II. This is reasoned from the results that considerable lipid peroxidation occurred in the thylakoids in the light, and that lipoxygenase externally added in the dark induced inhibition of Photosystem II activity in the thylakoids, production of singlet oxygen, which was monitored by electron paramagnetic resonance spin trapping, and damage to the D1 protein, in parallel with lipid peroxidation. Modification of the subunits of the light-harvesting complex of Photosystem II by malondialdehyde as well as oxidation of the subunits was also observed. We suggest that mainly singlet oxygen formed through lipid peroxidation under light stress participates in damaging the Photosystem II subunits.     

Protein damage by photoproducts of merocyanine 540.

Exposure of certain photoactive dyes to light prior to their use in biological systems (preactivation) has been shown to result in formation of long-lived cytotoxic photoproducts. The cytotoxic species responsible for the biological activity of preactivated merocyanine 540 (pMC540) appears to be a hydroperoxide generated by oxidation of ground-state dye by singlet molecular oxygen, formed via energy transfer from triplet excited-state dye to oxygen. A positive correlation (r = .93) exists between the levels of hydroperoxides and percent of tumor cells killed upon exposure to pMC540. Exposure of bovine serum albumin (BSA) (0.5 mg/mL) to pMC540 (0.2 mg/mL-1 mg/mL) results in loss of tryptophan fluorescence and 345 nm emission, suggesting a probable role of either hydroxyl (.OH) or .OH + superoxide (O2-). Polyacrylamide gel electrophoresis indicates fragmentation of treated BSA. Aggregation of pMC540-treated BSA is not detected. Bityrosine production is not observed. A dose-dependent decrease in BSA solubility is observed in treated samples, suggesting an increase in hydrophobicity. Amino acid analysis of BSA treated with pMC540 shows loss of some amino acids residues. The data presented here suggest that photoproducts of MC540 derived via the process of preactivation may mediate their effect (at least in part) by reactive oxygen species.