Singlet oxygen photosensitization by EGFP and its chromophore HBDI

The photosensitization of reactive oxygen species and, in particular, singlet oxygen by proteins from the green fluorescent protein (GFP) family influences important processes such as photobleaching and genetically targeted chromophore-assisted light inactivation. In this article, we report an investigation of singlet oxygen photoproduction by GFPs using time-resolved detection of the NIR phosphorescence of singlet oxygen at 1275 nm. We have detected singlet oxygen generated by enhanced (E)GFP, and measured a lifetime of 4 μs in deuterated solution. By comparison with the model compound of the EGFP fluorophore 4-hydroxybenzylidene-1,2-dimethylimidazoline (HBDI), our results confirm that the β-can of EGFP provides shielding of the fluorophore and reduces the production of this reactive oxygen species. In addition, our results yield new information about the triplet state of these proteins. The quantum yield for singlet oxygen photosensitization by the model chromophore HBDI is 0.004.     

L-ascorbic acid quenching of singlet delta molecular oxygen in aqueous media: generalized antioxidant property of vitamin C

L-ascorbic acid quenches singlet (1 delta g) molecular oxygen in aqueous media (pH 6.8 for [1H]H2O and pD 7.2 for [2H]D2O) as measured directly by monitoring (0,0) 1 delta g leads to 3 sigma-g emission at 1.28 micron. Singlet oxygen was generated at room temperature in the solutions via photosensitization of sodium chrysene sulfonate; this sulfonated polycyclic hydrocarbon was synthesized to provide a water soluble chromophore inert to usual dye-ascorbate photobleaching. A marked isotope effect is found; kHQ2O is 3.3 times faster than kDQ2O, suggesting ascorbic acid is chemically quenching singlet oxygen.     

Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion.

The green fluorescent protein (GFP) was used as a noninvasive probe to quantify the rheological properties of cell cytoplasm. GFP mutant S65T was purified from recombinant bacteria for solution studies, and expressed in CHO cell cytoplasm. GFP-S65T was brightly fluorescent in solution (lambda ex 492 nm, lambda em 509 nm) with a lifetime of 2.9 ns and a rotational correlation time (tc) of 20 ns. Recovery of GFP fluorescence after photobleaching was complete with a half-time (t1/2) in aqueous saline of 30 +/- 2 ms (5-micron diameter spot), giving a diffusion coefficient of 8.7 x 10(-7) cm2/s. The t1/2 was proportional to solution viscosity and was dependent on spot diameter. In contrast to fluorescein. GFP photobleaching efficiency was not affected by solution O2 content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching. The t1/2 for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), was independent of spot diameter, and was unaffected by O2 or quenchers. In cell cytoplasm, time-resolved microfluorimetry indicated a GFP lifetime of 2.6 ns and a tc of 36 +/- 3 ns, giving a relative viscosity (cytoplasm versus water) of 1.5. Photobleaching recovery of GFP in cytoplasm was 82 +/- 2% complete with a t1/2 of 83 +/- 6 ms, giving a relative viscosity of 3.2. GFP translational diffusion increased 4.7-fold as cells swelled from a relative volume of 0.5 to 2. Taken together with measurements of GFP translation and rotation in aqueous dextran solutions, the data in cytoplasm support the view that the primary barrier to GFP diffusion is collisional interactions between GFP and macromolecular solutes.     

The photodynamic inactivation of Candida guilliermondii in the presence of photodithazine

Photodithazine, a glucosamine salt of chlorin e6, enhanced the inactivation of Candida guilliermondii cells by visible light. The sensitizing effect of photodithazine was found to be related to free or cell surface-bound molecules of this dye. Sodium azide (a singlet oxygen quencher) and propyl gallate (an inhibitor of lipid peroxidation) protected yeast cells from the photodithazine-enhanced photoinactivation.     

High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy

Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.     

Photoactivation of the nematicidal compound alpha-terthienyl from roots of marigolds (Tagetes species). A possible singlet oxygen role.

The nematicidal compound alpha-terthienyl from roots of Tagetes species generates upon irradiation with near ultraviolet light reactive oxygen species on which the in vitro nematicidal activity depends. This system was studied by following the inhibition of glucose-6-phosphate dehydrogenase by photoactivated alpha-terthienyl and protection of the enzyme activity in the absence of oxygen and by various additions. Addition of mannitol, benzoate, superoxide dismutase or catalase did not have any effect nor did H2O2. This suggests that OH., O-.2, and H2O2 are not the reactive oxygen species involved. The enzyme was protected against photoactivated alpha-terthienyl in air-saturated solutions by singlet oxygen quenchers such as histidine, methionine, tryptophan, bovine serum albumin, and NaN3. Furthermore, inactivation of the enzyme was about 3.5 times faster in D2O than in H2O. When alpha-terthienyl in CH2Cl2 was irradiated in the presence of the olefin adamantylideneadamantane, a stable dioxetane was formed which decomposed to adamantanone when heated above its melting point. These results indicate a singlet oxygen-mediated process.     

Vanillin as an antioxidant in rat liver mitochondria: Inhibition of protein oxidation and lipid peroxidation induced by photosensitization

Using rat liver mitochondria, as model systems, we have examined the ability of the natural compound and the food-flavoring agent, vanillin to protect membranes against oxidative damage induced by photosensitization at concentrations normally used in food preparations. Vanillin, at a concentration of 2.5 mmol/L, has afforded significant protection against protein oxidation and lipid peroxidation in hepatic mitochondria induced by photosensitization with methylene blue plus light. The effect observed was both time- and concentration-dependent. The inhibitory effect is similar to ascorbic acid and the singlet oxygen quencher, diazabicyclo[2.2.2]octane (DABCO) but less effective than sodium azide and glutathione. Examination of possible mechanisms responsible for the observed protection, showed that vanillin has a significant ability to quench singlet oxygen (¹O₂), a reactive species responsible for damage induced during photosensitization by Type II mechanism. Hence, this flavoring compound, due to its antioxidant ability, may have potential to prevent oxidative damage to membranes in mammalian tissues and thereby the ensuing diseased states.     

Photodynamic action of bonellin, an integumentary chlorin of Bonellia viridis, Rolando (Echiura, Bonelliidae).

1. The photodynamic activity of bonellin, an integumentary chlorin of Bonellia viridis, is investigated. 2. 10(-6) M bonellin solutions haemolyze erythrocytes only in the presence of light. Previous illumination (tungsten lamp 2000-4000 lux for 1 hr) of the bonellin solutions does not affect the results. Under lowered oxygen tensions the bioactivity is depressed. Benzoquinone and singlet oxygen quenchers delay the photodynamic effect. 3. Bonellin (2 x 10(-6) M) destroys echinoid gamete function, depresses oxygen uptake of spermatozoa, and arrests development of echinoid and Bonellia eggs. These effects are produced only in the presence of light. 4. Copper bonellin is not photodynamically active. The role of copper may be to protect against photosensitization.     

Studies on the photochemical and photocytotoxic properties of the new PDT photosensitizer aluminum sulfonated phthalocyanine.

The properties of photosensitization of sulfonated aluminum phthalocyanine (ALSPC), a new photosensitizer of potential use in cancer photodynamic therapy (PDT) was studied on both the molecular and cellular levels. The mechanism of ALSPC photosensitization on the molecular level was investigated by testing its efficiency of singlet oxygen (1O2) production, using the method of tryptophan degradation and that of ESR spectroscopy and observing the enhancing effect of D2O and the quenching effect of NaN3. Results of all these experiments confirmed the important role of the Type II or 1O2 mechanism in ALSPC photosensitization. In our in-vitro experiments, ALSPC's incorporation into cells and its photocytotoxic effect were investigated on a human liver cancer cell line. The cell incorporation was illustrated by the laser-excited fluorescence spectra emitted both from cell homogenate and cell monolayers incubated with ALSPC aqueous solution. The position of fluorescence peak observed, implied that ALSPC exists in the cells mainly as monomers. The efficiency of cell killing of ALSPC photosensitization was estimated by counting surviving cells with the method of trypan blue staining and by the method of radioisotope labelling. Experiments using the latter method also showed DNA damage caused by ALSPC photosensitization.     

Influence of pH, Oxygen, and Humic Substances on Ability of Sunlight To Damage Fecal Coliforms in Waste Stabilization Pond Water

Simple beaker experiments established that light damages fecal coliforms in waste stabilization ponds by an oxygen-mediated exogenous photosensitization. Wavelengths of up to 700 nm were able to damage bacteria. The ability of wavelengths of >425 nm to damage fecal coliforms was dependent on the presence of dissolved sensitizers. The sensitizers were ubiquitous in raw sewage, unaffected by sewage treatment, not derivatives of bacteriochlorophyll or chlorophyll, absorbed well in UV light, and had a slight yellowish color; they are therefore believed to be humic substances. The ability of light to damage fecal coliforms was highly sensitive to, and completely dependent on, oxygen. Scavengers of H₂O₂ and singlet oxygen could protect the bacteria from the effects of sunlight, but scavengers of hydroxyl radicals and superoxides could not. Light-mediated damage of fecal coliforms was highly sensitive to elevated pH values, which also enabled light with wavelengths of >425 nm (in the presence of the sensitizer) to damage the bacteria. We conclude that humic substances, pH, and dissolved oxygen are important variables in the process by which light damages microorganisms in this and other environments and that these variables should be considered in future research on, and models of, the effects of light.     

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.     

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.     

Effects of Deuterium Oxide and Free Radical Scavengers on Carotenoid Photobleaching in Spinach Chloroplasts

The effect of D₂O on carotenoid photobleaching was examinedin spinach chloroplasts poisoned by carbonylcyanide m-chlorophenylhydrazone.D₂O, which prolongs a life time of singlet molecular oxygen,stimulated carotenoid photobleaching under aerobic conditions,but not under anaerobic conditions. The stimulation became smalleras the intensity of actinic light was lowered. Propyl gallateand (+)-catechin, radical scavengers, suppressed photobleaching.The suppression was greater at a low actinic light intensity.These results suggest that cartoenoid is photobleached by singletmolecular oxygen and radical chain reactions. (Received July 17, 1982; Accepted January 13, 1983)     

DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib

Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib—a BRAF inhibitor used to treat metastatic melanoma—are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage.     

Overexpression of enzymes that repair endogenous damage to DNA.

A significant contribution to human mutagenesis and carcinogenesis may come from DNA damage of endogenous, rather than exogenous, origin. Efficient repair mechanisms have evolved to cope with this. The main repair pathway involved in repair of endogenous damage is DNA base excision repair. In addition, an important contribution is given by O6-alkylguanine DNA alkyltranferase, that repairs specifically the miscoding base O6-alkylguanine. In recent years, several attempts have been carried out to enhance the efficiency of repair of endogenous damage by overexpressing in mammalian cells single enzymatic activities. In some cases (e.g. O6-alkylguanine DNA alkyltransferase or yeast AP endonuclease) this approach has been successful in improving cellular protection from endogenous and exogenous mutagens, while overexpression of other enzymatic activities (e.g. alkyl N-purine glycosylase or DNA polymerase beta) were detrimental and even produced a genome instability phenotype. The reasons for these different outcomes are analyzed and alternative enzymatic activities whose overexpression may improve the efficiency of repair of endogenous damage in human cells are proposed.     

Mechanistic aspects of Escherichia coli photodynamic inactivation by cationic tetra-meso(N-methylpyridyl)porphine.

The mechanistic aspects of Escherichia coli photodynamic inactivation (PDI) have been studied in bacteria expressing the reporter protein GFP, following transfection with wild type pGFP plasmid and treatment with the hydrophilic cationic sensitizer tetra-meso(N-methyl-4-pyridyl)porphine tetratosylate (TMPyP). Cell survival and morphology during PDI were correlated with plasmid-GFP degradation in comparison to DNA and RNA strand-breaks, while photobleaching of the GFP chromophore was used to monitor protein photodamage. Singlet oxygen generated upon TMPyP photoactivation interacted with target nucleic acid polymers in a drug-and light-dose dependent manner. The hierarchy and cascade of the photodamage was in the order: genomic-DNA > total RNA > plasmid-DNA, as revealed by specific extraction and agarose electrophoresis. The notable resistance of the plasmid DNA in comparison to genomic DNA has implications for PDI of antibiotic-resistant bacteria. Re-growth of the treated cells in fresh medium showed structural features of an SOS response. Under these conditions, DNA repair machinery was initiated by typical alignment of DNA-protein co-aggregates accompanied by lateral assembly of ribosomes, apart from damaged DNA-arrays, as depicted by electron microscopy. GFP-TMPyP interactions were demonstrated by double green and red fluorescence on electrophoresis plates analyzed by spectral imaging. Photobleaching measurements revealed specific GFP photodamage directly related to PDI of the E. coli. The kinetics of both the GFP photobleaching and the K(+) efflux, representing photodamage to cytosolic proteins and membrane damage, respectively, were found to be similar. The survival curves were correlated to chromosomal degradation and ultrastructural damage. We conclude that TMPyP-dependent PDI of E. coli is primarily dependent on genomic DNA photodamage rather than on protein or membrane malfunctions.     

Photo-oxidation of cells generates long-lived intracellular protein peroxides

Singlet oxygen is generated by several cellular, enzymatic, and chemical reactions as well as by exposure to UV or visible light in the presence of a sensitizer. Consequently, this oxidant has been proposed to be a damaging agent many pathologies. Proteins are major targets for singlet oxygen as a result of their abundance and high rate constants for reaction. In this study, we show that illumination of viable rose bengal-loaded THP-1 (human monocyte-like) cells with visible light gives rise to intracellular protein-derived peroxides. The peroxide yield increases with illumination time, requires the presence of rose bengal, is enhanced in D(2)O, and is decreased by azide, consistent with the mediation of singlet oxygen. The concentration of peroxides detected, which is not affected by glucose or ascorbate loading of the cells, corresponds to about 1.5 nmoles peroxide per 10(6) cells, or 10 nmoles/mg cell protein, and account for up to approximately 15% of the O(2) consumed by the cells. Similar peroxides have been detected on isolated cellular proteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, cellular protein peroxide levels decrease with t(1/2) about 4 h at 37 degrees C. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions gives rise to radicals as detected by EPR spin trapping. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer leads to novel long-lived, but reactive, intracellular protein peroxides via singlet oxygen-mediated reactions.     

Synthesis of β-functionalized temoporfin derivatives for an application in photodynamic therapy

The synthesis of novel β-functionalized derivatives of the clinically used photosensitizer Temoporfin has been achieved by nucleophilic addition reactions to a corresponding diketo chlorin. The β-substituted dihydroxychlorin products exhibit a strong absorption in the red spectral region, a high singlet oxygen quantum yield, and were found to be highly effective in in vitro assays against HT-29 tumor cells.